CN101415829A - Plants having enhanced yield-related traits and a method for making the same - Google Patents

Abstract

The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield-related traits in plants, in particular for increasing plant yield and/or early vigour, relative to control plants. More specifically, the present invention concerns a method for enhancing yield-related traits comprising modifying the expression of a nucleic acid encoding a HAL3 polypeptide, MADS15 polypeptide, PLT transcription factor polypeptide, basic/helix-loop-helix (bHLH) transcription factor, or SPL15 transcription factor. The invention also provides constructs useful in the methods of the invention.

Description

Plant and the method that is used to produce this plant with enhanced yield correlated character

Relate generally to biology field of the present invention also relates to the method that is used for improving in the plant expression by the nucleic acid of regulating coding GRP (growth associated protein) the various plants growth characteristics.The invention still further relates to the plant of being regulated expression of the nucleic acid with coding GRP, wherein said plant has the growth characteristics of improvement with respect to corresponding wild-type plant or other control plant.The present invention also is provided for the construct in the inventive method.

The world population of sustainable growth is supplied the research that atrophy has stimulated relevant increase farm efficiency with agricultural with the arable land.The plant that conventional crop and the utilization of Horticulture improved means select breeding technique to have welcome characteristic with evaluation.Yet this type of selects breeding technique to have several defectives, and promptly these technology generally expend a lot of work and produce such plant, and it often contains the heterology hereditary component, and it may always not cause the desired proterties transmitted from the parental generation plant.Recent advances in molecular biology has allowed the germplasm of human improvement animal and plant.The genetic engineering of plant makes and can separate and operate genetic material (generally being in DNA or rna form) and import this genetic material subsequently to plant.This type of technology has generation and possesses diversified economy, agronomy or the crop of Horticulture improvement proterties or the ability of plant.

Proterties with special economic meaning is the output that increases.But output is normally defined the measuring result from the economic worth of crop.This result can define with regard to quantity and/or quality aspect.Output directly depends on Several Factors, for example the number of organ and size, plant structure (for example Zhi number), seed generation, leaf aging etc.The important factor that root development, nutrient intake, stress tolerance and early stage vigor (earlyvigor) also can be decision output.Optimize aforementioned factor thereby can contribution be arranged increasing crop yield.

Seed production is the proterties of particularly important, because the seed of numerous plants is important to people and Animal nutrition.Crop such as corn, rice, wheat, rape and soybean account for above the human total heat of half and take in, no matter by direct consumption seed itself or by consuming the meat product that produces based on the seed of processing.Crop also is the source of used numerous type metabolites in sugar, oil and the industrial processes.Seed contains embryo (origin of new talent and Xin Gen) and endosperm (source of nutrition that is used for embryonic development during duration of germination and the seedling early growth).Seed development relates to several genes and needs metabolite to be transferred to the seed of growing from root, leaf and stem.Endosperm especially assimilates carbohydrate, oil and proteinic metabolic precursor thereof and they is synthesized the storage macromole to fill seed.

Another important character for numerous crops is early stage vigor.Improving early stage vigor is the important goal of modern rice breeding plan on temperate zone and tropical rice varieties.It is important that long root is planted in the rice for correct soil fixing at water.In that direct sowing is to the situation that is submerged the field with rice, and under the situation that plant must emerge rapidly from water, long seedling is relevant with vigor.Under the situation of implementing drilling, long mesocotyl and coleoptile are important for well emerging.With early stage vigor artificial reconstructed will be extremely important in agricultural to endophytic ability.For example, bad early stage vigor has limited based on corn (the Zea mayes L.) hybrid of Corn Belt germplasm (Corn Belt germplasm) and has introduced a fine variety European Atlantic ocean region.

Another important character is improved abiotic stress tolerance.Abiotic stress is the major cause of world wide crop loss, reduces mean yield and surpass 50% (Wang etc., Planta (2003) 218:1-14) for most of staple crop plants.Abiotic stress can be caused by arid, salinity, extreme temperature, chemical toxicity and oxidative stress.Improving plant will be very big economic advantages to the peasant and can allow during unfavourable condition and in arable farming otherwise be impossible land raise crop the ability of abiotic stress tolerance at world wide.

Crop yield thereby can increase by optimizing one of aforementioned factor.

Depend on end-use, may have precedence over other yield traits the improvement of some yield traits.For example for use as feed or timber production or biofuel resource for, increasing the plant nutrition body portion may wish, and for use as flour, starch or oil production for, increase is planted a subparameter and may especially be wished.Even if in the middle of kind of subparameter, some parameter can be more preferably in other parameter, and this depends on application.Number of mechanisms can have contribution to increasing seed production, and no matter form is the seed size of increase or the number seeds of increase.

A kind of method that increases output (seed production and/or biomass) in the plant can be the multiple signal pathway by the inherent growth mechanism of regulating plant such as cell cycle or involved in plant growth or participation defense mechanism.

Have now found that and in plant, to express the multiple growth characteristics that improves in the plant by the nucleic acid of regulating the coding GRP (growth associated protein) in the plant.GRP can be one of following albumen: MADS box transcription factor (OsMADS15), PLT transcription factor, bHLH transcription factor, SPL15 transcription factor and salt tolerant protein (HAL3).

Background

Salt tolerant protein

Numerous enzyme processs in the cell need the participation of coenzyme A (CoA or CoASH).Coenzyme A (CoASH) itself is a height polar molecule, is made of adenosine 3 ', the 5 '-bisphosphate that is connected with 4-phosphoric acid pantothenic acid (4-phosphopantethenicacid) (vitamin B5) and therefore is connected with Mercamine Cysteamine.Free SH base can be esterified, and this depends on the process that CoA participates in.In bacterium, animal and plant, illustrated CoA synthetic approach.In plant, CoA precursor 4 '-phosphopan tetheine halfcystine (PPC) to 4 '-conversion of phosphopantetheine by flavoprotein 4 '-phosphopantothenoylcysteine decarboxylase (PPCDC or HAL3, J.Biol.Chem.276 such as Kupke, 19190-19196,2001) catalysis.The proteinic gene of coding HAL3 can be the part of a minigene family: Arabidopis thaliana (Arabidopsis) genome comprises 2 isotypes (AtHAL3a and AtHAL3b; Plant such as Espinoza-Ruiz J.20,529-539,1999), in tobacco, have 3 kinds of HAL3 genes J.Exp.Bot.55 such as (, 387-395,2004) Yonamine, and HAL3 is a single copy gene in rice.Someone proposes AtHAL3 and other HAL3 protein and plays a role as tripolymer, and each monomer has bonded vitamin B2 phosphate (FMN) Structure 8 such as (, 961-969,2000) Albert.Infer the FMN cofactor producing 4 '-play a role in the redox reaction of phosphopantetheine.

Yet the proteinic molecular function of HAL3 is not fully set forth.HAL3 protein shows and the yeast SIS2 protein (Ferrando etc., Mol.Cell.Biol.15, homology 5470-5481) that relate to salt tolerance.The plant of ectopic expression HAL3 or vegetable cell show improved salt, osmotic pressure or lithium stress tolerance (Espinoza-Ruiz etc., 1999; Yonamine etc., 2004).Report that the tobacco HAL3a overexpression in the BY2 cell causes proline content increase in the born of the same parents, this may have contribution (Yonamine etc., 2004) to the salt tolerant phenotype.In addition, the arabidopsis thaliana of overexpression AtHAL3a shows growth velocity (Espinoza-Ruiz etc., 1999) faster than wild-type plant.Confirm that AtHAL3b protein and cyclin CDKB 1:1 interact (WO 00/36124), thereby infer that AtHAL3b is useful for giving the plant salt stress tolerance and increasing plant growth rate down for condition of salt stress.The nucleic acid expression in the expansion tissue that now has been surprised to find preferential increase coding HAL3 polypeptide has produced the plant that has the output of increase with respect to control plant, and it is 10% at least that wherein said output increase is compared with control plant.

Transcription factor

Transcription factor be normally defined with cis regulatory elements (for example enhanser, TATA box) in conjunction with and combine down the protein that can activate and/or suppress to transcribe with RNA polymerase.At least 1533 kinds of transcriptional of arabidopsis gene group coding, this account for its estimate gene number～5.9% (Riechmann etc., 2000 (Science the 290th volumes, 2105-2109)).

MADS15

MADS box gene constitutes the big gene family of the eukaryotic transcription instrumentality of the many aspects that participate in yeast, plant and animal growth.MADS box genes encoding be responsible for its target gene regulatory region in the strong conservative MADS structural domain of DNA keying action of particular cartridge.This gene family can be divided into two main systems, I type and II type.II type gene has another name called MIKC type protein, refers to following 4 functional structure territories (Fig. 5, (Jack, Plant Mol.Biol.46,515-520,2001)) that they have:

Be used for DNA bonded MADS, about 60 amino acid (high conservative) are positioned at proteinic N-terminal;

Interleave structural domain I (conservative property is lower), participate in the dimeric selectivity of MADS and form;

Keratin sulfate structural domain K (very conservative) is responsible for dimerization;

Carboxyl petiolarea C (sequence and variable-length) participates in transcriptional activation or participates in forming poly transcription factor complex body.

In Arabidopis thaliana, identified and surpassed 100 MADS box genes, and they are classified into 12 kinds of clade with system's occurring mode, every kind of clade has (the J Mol.Evol.43 such as Thiessen of specific departing from the MADS consensus sequence, 484-516,1996) .OsMADS15 belongs to SQUA clade (with regard to SQUAMOSA, it is from Common Snapdragon (Antirrhinummajus)).(Nature 353,31-7) gene of SQUA clade classified as A function organ identity gene with reference to the ABC floral organ identity specialization model that was proposed in 1991 by Coen and Meyerowitz.Except that OsMADS15, rice OsMADS14, OsMADS18 and OsMADS20 also are the parts of SQUA clade.

The SQUA clade is subdivided into 2 subgroups in dicotyledons, i.e. the inferior clade (OsMADS15 clusters therein) of AP1 and FUL.These inferior clade are divergent (Litt and Irish, Genetics 165,821-833,2003) on the specific amino acids motif at their proteinic separately carboxyl terminal places basically.Except having specific AP1 amino acid motif, dicotyledons AP1 clade dependency protein comprises the farnesylation motif at its carboxyl terminal usually, and (this motif is CAAX, wherein C is a halfcystine, and A normally aliphatic amino acid and X is methionine(Met), glutamine, Serine, halfcystine or L-Ala).In monocotyledons, SQUA clade protein is subdivided into two main groups, and it can be distinguished based on the conservative carboxyl terminal motif in last 15 the amino acid scopes of this protein: LPPWMLS (for example OsMADS15, SEQ ID NO:117) and LPPWMLR (for example OsMADS18).Opposite with the dicotyledons sequence of SQUA clade, the monocotyledons sequence of SQUA clade does not have the farnesylation motif at its carboxyl terminal.

Infer that compound the playing a role of OsMADS15 and other protein forms with the control organ.Yet, do not identify mutant so far with visible phenotypic; Remove outside the data that provide among the WO 01/14559 (EP1209232, US6,995,302), the ectopic expression that relates to OsMADS15 is not provided or reduces the experimental data of expressing.In the disclosure, transgenosis buckwheat (Fagopyrum esculentum) plant of expressing OsMADS15 with sense orientation shows the branch that increases, and the transgenic plant of antisence construct have the growth and the downtrod branch of minimizing.With the Da Ye that has adopted construct to transform take root (

Daigremontiana) have leaf development around root, this is regarded as the index that branch increases.The author claims with regard to the structure of flower development and these flowers and does not observe variation.OsMADS15 is no APETALA1 (AP1) to homologue directly in Arabidopis thaliana.The ectopic expression of AP1 causes flowering time to reduce, and the blooming and have unusual colored of AP1 mutant display delay.

PLT

AP2 (apetala2)/ERF (ethylene response response element binding factor) family has comprised the transcription factor with at least one high conservative DNA binding domains-AP2 structural domain.The AP2 structural domain at first APETALA2 (AP2) (a kind of participate in that development program such as meristematic tissue identity are regulated and the arabidopsis thaliana protein of floral organ specialization (Jofuku etc., (1994) Plant Cell 6,1211-1225)) in description.Whether contain one (ERF subfamily) or 2 (AP2 subfamily) DNA binding domainss in them and be divided into subfamily (Figure 12) AP2/ERF is protein-based.In Arabidopis thaliana (Arabidopsisthaliana) genome, identified above 140 kinds of AP2/ERF genes (Reichmann etc. (2000) Science 290:2105-2110), wherein belonged to AP2 subfamily (Kim etc. (2006) Mol Bio Evol 23 (1): 107-120) up to 18 kinds of AP2/ERF genes.

Verified two kinds of Arabidopis thaliana AP2 subfamily transcription factor polypeptide by Plethora 1 (PLT1) and Plethora 2 (PLT2) gene (being referred to as the PLT gene) coding are for the stem cell specialization in the root meristematic tissue especially and to keep be essential.In RT-PCR analyzed, the PLT transcript mainly detected in root, and it is closely related to show that PLT expresses with the root identity.Atopy PLT induced expression top territory homology brick shape in the Arabidopis thaliana embryo changes root stem cell, root or hypocotyl (Aida etc. (2004) Cell 119:109-120) into.

U.S. Patent application 2004/0045049 and International Patent Application WO 03/013227 provide the nucleotide sequence of coding Arabidopis thaliana PLT1 transcription factor (being called G1793 in this application).Compare with the wild-type contrast, report that the overexpression of G1793 (using the CaMV 35S promoter) in Arabidopis thaliana causes the change of cotyledon form, the slight minimizing of plant overall dimensions and thin inflorescence (may have unusual colored).G1793 overexpression person compares according to plant and produces more seed oil.Provide coding potential soybean (Glycine max) directly to homologue, rice (Oryza sativa) directly to homologue and Zea mays (Zea mays) directly to the nucleotide sequence of homologue.

International Patent Application WO 03/002751 provides the soybean nucleotide sequence of coding PLT polypeptide, and it has sequence similarity with the corn nucleotide sequence of summarizing analysis (by dna microarray) evaluation by gene.

International Patent Application WO 05/075655 provides rice and the Zea mays nucleotide sequence that has sequence similarity with Arabidopis thaliana PLT gene.

bHLH

Alkalescence/helix-loop-helix (bHLH) protein is the transcription factor superfamily that combines and fully be characterized by important adjusting composition in the regulate several biological processes as dimer and specific DNA target site in the non-plant eukaryote.The distinctive feature of bHLH family is the bipartite texture territory that is made of about 60 amino acid.This bipartite texture territory is made of in conjunction with the elementary zone with two alpha-helixs that separated out by variable loop the DNA that combines with the Hexanucleotide E box that has.Described two alpha-helixs promote dimerization, allow to form between different family members homodimer and heterodimer.Although the bHLH structural domain is an evolution conservative, yet except that this structural domain, there is seldom sequence similarity between the clade.

Bailey etc., 2003 (The Plant Cell, the 15th volume 2497-2501) is reported in that detected bHLH gene number is 162 kinds in the Arabidopis thaliana, and this makes the bHLH gene become transcription factor family maximum in the Arabidopis thaliana; Report that the rice genome contains 131 kinds of bHLH genes (Buck and Atchley, 2003 (J.Mol.E the 56th volumes: 742-750)).Heim etc., 2003 (Mol.Biol.E the 20th volumes (5): 735-747) from Arabidopis thaliana, identified 12 subfamilies of bHLH gene based on structural similarity.

Report that the bHLH protein from plant that has characterized has effect (Buck and Atchley, 2003) in anthocyanin biosynthesizing, the effect of phytochrome signal, sphaeroprotein expression, cracking of fruit, carpel and epidermis are grown.

SPL

The conjugated protein sample of squamosa promotor (SPL) transcription factor polypeptide is various protein (Klein etc. (1996) the Mol Gen Genet 259:7-16 of structure of the high conservative DNA binding domains (DBD) of total about 80 amino-acid residues of length; Cardon etc. (1999) Gene 237:91-104).SPL transcription factor DNA consensus sequence binding site in the target gene promoters is 5 '-TNCGTACAA-3 ', and wherein N represents any base.At inner 10 conservative halfcystines (Cys) or Histidine (His) residue (seeing Figure 23) of existing of SPL DBD, wherein 8 residues are in conjunction with referring to that for SPL specificity zinc tertiary structure forms the zinc coordination residue of necessary 2 zine ions (Yamasaki etc. (2004) J Mol Biol 337:49-63).Second conservative property feature in the SPL DBD is two fens nuclear localization signals.Outside DBD, in whole plants circle, find a microRNA (miRNA) target motif (miR156) (in the coding region or) (Rhoades etc. (2002) Cell 110:513-520) in the nucleotide sequence of great majority coding SPL transcription factor polypeptide at 3 ' UTR.MiRNA suppresses to regulate and control SPL genetic expression to transcribe the back mode by the mRNA degraded of guiding coding SPL or by translation.

(Science 290:2105-2109 such as Riechmann, 2000) 16 kinds of SPL transcription factor polypeptide in the Arabidopis thaliana have been reported, sequence similarity very low (except aforementioned feature) between they self, the size of the SPL polypeptide of derivation is a 131-927 amino acid.But, in the SPL of this kind of plant family, detect the SPL transcription factor polypeptide of total higher sequence homology to (Cardon etc. (1999)).

The SPL transcription factor polypeptide (existing only in the plant) of verified so far sign is at development of plants, especially play a role in flower development.Report that the transgenic plant of overexpression SPL3 transcription factor polypeptide bloom more morning (Cardon etc. (1997) Plant J 12:367-377).

In European patent application EP 1033405, the nucleotide sequence of SPL15 transcription factor polypeptide and the peptide sequence of derivation are provided.

In International Patent Application WO 03013227, provide nucleotide sequence (with the peptide sequence of deriving; Internal reference G2346), the part of its coding SPL15 transcription factor polypeptide, yet apart from 38 amino acid of C-terminal of SPL15 transcription factor.The transgenic arabidopsis plant of SPL15 transcription factor (or G2346) polypeptide that the composing type overexpression is modified has the cotyledon of slight expansion.Growing late period, reporting that this kind of plant does not have to show the difference consistent with control plant.

The invention summary

The nucleic acid that now has been surprised to find preferential increase coding HAL3 polypeptide is expressed in the expansion tissue and has been produced the plant that has the seed production of enhanced yield correlated character, the early stage vigor that especially increases and increase with respect to control plant, and wherein seed production increases that to compare with control plant be 10% at least.

According to another embodiment of the invention, with respect to control plant enhancing output correlated character, especially the method that increases the seed production of early stage vigor of plant and increase plant is provided, and it comprises the expression of nucleic acid in plant expansion tissue of preferential increase coding HAL3 polypeptide.

Now be surprised to find the expression of nucleic acids of regulating coding MADS15 polypeptide produced with respect to control plant have the enhanced yield correlated character, the plant of the output that especially increases.

According to an embodiment, the method that increases plant biomass with respect to control plant is provided, it nucleic acid that comprises increase coding MADS15 polypeptide is expressed in plant.The output that increases comprises the trophicity biomass of increase, but does not comprise the seed production of increase.

According to another embodiment, the invention provides the method that increases plant biomass with respect to control plant, comprise the level and/or the activity that reduce endogenous MADS15 polypeptide.The output that increases comprises higher seed production, but does not comprise the trophicity biomass of increase.

Now be surprised to find the expression of nucleic acid that increases coding PLT transcription factor polypeptide produced with respect to control plant have the enhanced yield correlated character, the plant of the output that especially increases.

According to another embodiment, the method that increases plant biomass with respect to control plant is provided, comprise the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide.

The expression of nucleic acid in plant that now has been surprised to find the bHLH transcription factor of regulating the coding particular category produced the plant that has the enhanced yield correlated character with respect to control plant.The bHLH transcription factor that is suitable for strengthening the certain kinds of output correlated character in the plant is described in detail hereinafter.

According to another embodiment, the invention provides the method that strengthens output correlated character in the plant with respect to control plant, comprise the expression of nucleic acid in plant of the bHLH transcription factor of regulating the coding certain kinds.

Now be surprised to find the expression of nucleic acid in plant that increases coding SPL15 transcription factor polypeptide produced with respect to control plant have the enhanced yield correlated character, the plant of the output that especially increases.

According to another embodiment, the invention provides the method that increases output in the plant with respect to control plant, comprise the expression of nucleic acid in plant that increases coding SPL15 transcription factor polypeptide.

Definition

Polypeptides

Term " polypeptide " and " protein " are used interchangeably in this article and refer to be in amino acid in the random length polymerized form.

Polynucleotide/nucleic acid/nucleotide sequence/nucleotide sequence

Term ＂ polynucleotide ＂, ＂ nucleotide sequence ＂, ＂ nucleotide sequence ＂, " nucleic acid " are used interchangeably in this article and refer to be in Nucleotide in the random length polymerized form, i.e. ribonucleotide or deoxyribonucleotide or these two combination.

Control plant

To select suitable control plant be the habitual part that is provided with of experiment and can comprise the corresponding wild-type plant or not have the corresponding plant of goal gene.Control plant generally is plant species or or even the identical mutation identical with plant to be assessed.Control plant also can be the inefficacy zygote of plant to be assessed." control plant " not only refers to whole strain plant as used in this article, also refers to plant part, comprises seed and plants subdivision.

Homologue

Proteinic " homologue " comprises such peptide, oligopeptides, polypeptide, protein and enzyme, and they have amino-acid substitution, disappearance and/or insertion and have similar biologic activity and functionally active to the non-modifying protein of described peptide, oligopeptides, polypeptide, protein and enzyme source with respect to the above-mentioned protein of non-modification.

Disappearance refers to remove one or more amino acid from protein.

Insertion refers to the importing in the predetermined site in protein of one or more amino-acid residues.Insertion can comprise single or multiple amino acid whose aminoterminals fusions and/or carboxyl terminal merges and the interior insertion of sequence.Usually, littler in the insertion meeting of aminoacid sequence inside than aminoterminal fusion or carboxyl terminal fusion, about 1-10 residue rank.The example of aminoterminal or carboxyl terminal fusion rotein or fusogenic peptide comprise as the binding domains of used transcriptional activator in the yeast two-hybrid system or activation structure territory, bacteriophage coat protein, (Histidine)-6-label, glutathione S-transferase-label, albumin A, maltose binding protein, Tetrahydrofolate dehydrogenase, Tag100 epi-position, c-myc epi-position,

Epi-position, lacZ, CMP (calmodulin binding peptide), HA epi-position, PROTEIN C epi-position and VSV epi-position.

Displacement refers to replace proteinic amino acid with other amino acid with similar characteristics (as the tendency of similar hydrophobicity, wetting ability, antigenicity, formation or destruction α-Luo Xuanjiegou or beta sheet structure).Amino-acid substitution generally is single residue, but can be a bunch collection property, and this depends on the functional constraint that places polypeptide; Inserting can be about 1-10 amino-acid residue rank usually.Preferably conservative amino acid displacement of amino-acid substitution.The preservative replacement table is (seeing for example Creighton (1984) Proteins.W.H.Freeman and Company and following table 1) well-known in the art.

Table 1: conservative amino acid metathetical example

Residue	Preservative replacement	Residue	Preservative replacement
				Ala	Ser	Leu	Ile；Val
Arg	Lys	Lys	Arg；Gln
				Asn	Gln；His	Met	Leu；Ile

Asp	Glu	Phe	Met；Leu；Tyr
				Gln	Asn	Ser	Thr；Gly
Cys	Ser	Thr	Ser；Val
				Glu	Asp	Trp	Tyr
Gly	Pro	Tyr	Trp；Phe
				His	Asn；Gln	Val	Ile；Leu
Ile	Leu，Val

Amino-acid substitution, disappearance and/or insert and to use the peptide synthetic technology well-known in the art such as the solid phase method of peptide synthesis etc. or by the recombinant DNA operation and carry out easily.Being used to operate dna sequence dna is well-known in the art with the method that produces proteinic displacement, insertion or disappearance variant.For example, the technology that is used for producing at the predetermined site place of DNA replacement mutation is that those skilled in the art are well-known and comprise M13 mutagenesis, T7-Gen vitro mutagenesis method (USB, Clevelaand, OH), the site-directed mutagenesis (Stratagene of QuickChange, San Diego, CA), site-directed mutagenesis or other site-directed mutagenesis of PCR-mediation.

Derivative

" derivative " comprises such peptide, oligopeptides, polypeptide, wherein compare with the aminoacid sequence of the protein (as target protein) of natural existence form, they comprise the interpolation of the amino-acid residue that the amino-acid residue that exists with non-natural exists amino acid whose displacement or non-natural.Proteinic " derivative " also comprises such peptide, oligopeptides, polypeptide; wherein compare with the aminoacid sequence of the natural existence form of polypeptide, they comprise naturally occurring change (glycosylation, acidylate, isoprenylation, phosphorylation, Semen Myristicae acidylate, sulphating etc.) amino-acid residue or non-natural change amino-acid residue.Compare with the aminoacid sequence that derivative is originated, this derivative can also comprise and covalently or non-covalently one or more non-aminoacid replacement bases of bonded or the interpolation (for example reporter molecule or other part) of described aminoacid sequence, as amino-acid residue for promote to detect this derivative bonded reporter molecule and exist with non-natural that naturally occurring proteinic aminoacid sequence compares.

Directly to homologue/collateral line homologue

Directly comprise the evolution notion that is used for describing the gene ancestral relationship to homologue and collateral line homologue.The collateral line homologue is the gene of same species endogenous origin in my late grandfather's gene replication, and is from the different biological genes that originate from species formation to homologue directly.

Structural domain

Term " structural domain ＂ refers to along the sequence alignment result of evolution related protein and at one group of conservative amino acid of specific location.Although the amino acid in other position can change between homologue, yet may be essential amino acid in proteinic structure, stability or function aspects in the amino acid indication of the high conservative of specific location.Structural domain is because of being identified by the conservative degree of the height in the aligned sequences of protein homology thing family, and they can be as identifying that thing is to determine whether the polypeptide of being discussed belongs to the peptide family of before having identified arbitrarily.

Motif/consensus sequence/label

Term " motif " or " consensus sequence " or " label " refer to short conserved regions in the sequence of evolution related protein.Motif is the high conservative part of structural domain often, but also can only comprise the part of structural domain, maybe can be positioned at (if whole amino acid of motif are positioned at outside the structural domain of definition) outside the conserved domain.

Hybridization

Term as defined herein " hybridization ＂ is the process of the mutual renaturation of homologous complementary nucleotide sequence basically wherein.Crossover process can be carried out in solution fully, and promptly two kinds of complementary nucleic acid all are in the solution.Crossover process also can take place under one of complementary nucleic acid is fixed to the situation of matrix such as magnetic bead, agarose (Sepharose) pearl or any other resin.Crossover process also can be fixed on solid support such as nitrocellulose filter or the nylon membrane or be fixed to by for example photolithography under the situation on the silicate glasses upholder (latter is called nucleic acid array or microarray or is called nucleic acid chip) for example at one of complementary nucleic acid carries out.For hybridization is taken place, usually with nucleic acid molecule thermally denature or chemical modification so that double-stranded unwinding become two strands and/or remove hair clip or other secondary structure from single-chain nucleic acid.

Term " severity " refer to the condition of hybridizing therein.The severity of hybridization is formed by condition such as temperature, salt concn, ionic strength and hybridization buffer to be influenced.Usually, low stringency is chosen as when ionic strength of determining and pH, is lower than about 30 ℃ of particular sequence pyrolysis chain temperature (Tm).Medium stringency be this moment temperature be lower than Tm about 20 ℃ and high stringency be this moment temperature be lower than about 10 ℃ of Tm.High stringency hybridization condition generally is used to separate the hybridization sequences that has high sequence similarity with target nucleic acid sequence.Yet nucleic acid can depart from sequence and because of the degeneracy of the genetic codon substantially the same polypeptide of still encoding.Thereby sometimes may need medium stringency hybridization condition to identify this type of nucleic acid molecule.

Tm is the temperature when ionic strength of determining and pH, 50% target sequence and the probe hybridization that mates fully under described temperature.Tm depends on the based composition and the length of solution condition and probe.For example, long sequence is hybridized under comparatively high temps specifically.Obtain maximum hybridization speed until 32 ℃ for about 16 ℃ from being lower than Tm.The existence of monovalent cation in solution reduced the Coulomb repulsion of two nucleic acid interchain, thereby promotes hybrid molecule to form; This effect is tangible (for greater concn, this effect can be ignored) for the na concn up to 0.4M.Methane amide reduces the melting temperature(Tm) of DNA-DNA and DNA-RNA duplex, and every percentage ratio methane amide reduces 0.6-0.7 ℃, and adds 50% methane amide and allow to hybridize at 30-45 ℃, though hybridization speed can reduce.Base-pair mismatch has reduced the thermostability of hybridization speed and duplex.On average and for big probe, every % base mispairing Tm descends about 1 ℃.The type that depends on hybrid molecule, Tm can use following equation to calculate:

1) DNA-DNA hybrid molecule (Meinkoth and Wahl, Anal.Biochem., 138:267-284,1984):

Tm=81.5 ℃+16.6xlog10[Na+] a+0.41x%[G/Cb]-500x[Lc]-the 1-0.61x% methane amide

2) DNA-RNA or RNA-RNA hybrid molecule

Tm＝79.8+18.5(log10[Na+]a)+0.58(％G/Cb)+11.8(％G/Cb)2-820/Lc

3) few DNA or few RNAd hybrid molecule:

For＜20 Nucleotide: Tm=2 (ln)

For 20-35 Nucleotide: Tm=22+1.46 (ln)

A or for other monovalent cation, but only be accurate in the 0.01-0.4M scope.

B is accurate in the 30%-75% scope for %GC only.

The length (in base pair) of c L=duplex.

D Oligo, oligonucleotide; Ln, the useful length of primer=2 * (G/C number)+(A/T number).

Any control non-specific binding that can numerous known technologies, for example with proteinaceous solution closed film, add heterology RNA, heterology DNA and SDS handles to hybridization buffer and with the RNA enzyme.For the non-homology probe, a series of hybridization can be undertaken by changing one of following condition: (i) reduce renaturation temperature (for example from 68 ℃ to 42 ℃) progressively or (ii) reduce methane amide concentration (for example from 50% to 0%) progressively.The technician understands during the hybridization can change and will keep or change the multiple parameter of stringency.

Except that the hybridization condition, the hybridization specificity generally also depends on the function of post-hybridization washing.For removing because of the background due to the non-specific hybridization, sample is with the salts solution washing of dilution.The key factor of this type of washing comprises the ionic strength and the temperature of final washing soln: salt concn is low more and wash temperature is high more, and then Xi Di severity is high more.Wash conditions is generally on the hybridization severity or be lower than hybridization severity and carrying out.Positive hybridization produces the signal that doubles background signal at least.Usually, the suitable stringency that is used for nucleic acid hybridization analysis method or gene amplification detection method as mentioned above.Also can select stricter or more undemanding condition.The technician understands during the washing can change and will keep or change the multiple parameter of stringency.

For example, be used for length and be included in 65 ℃ greater than the common high stringency hybridization condition of the DNA hybrid molecule of 50 Nucleotide and in 1 * SSC and 50% methane amide, hybridize, wash in 0.3 * SSC at 65 ℃ subsequently in 1 * SSC or at 42 ℃.Be used for length and be included in 55 ℃ greater than the example of the medium stringency hybridization condition of the DNA hybrid molecule of 50 Nucleotide and in 6 * SSC and 50% methane amide, hybridize, wash in 2 * SSC at 50 ℃ subsequently in 4 * SSC or at 40 ℃.The length of hybrid molecule is the expection length of hybrid nucleic acid.When the known nucleic acid hybridization of sequence, can determine hybrid molecule length herein by aligned sequences and the described conserved regions of evaluation.1 * SSC is 0.15MNaCl and 15mM Trisodium Citrate; Hybridization solution and washing soln can comprise 5 * Denhardt reagent, 0.5-1.0% SDS, the fragmentation salmon sperm DNA of 100 μ g/ml sex change, 0.5% trisodium phosphate extraly.

In order to define the purpose of severity level, can be with reference to (2001) MolecularCloning:a laboratory manual such as Sambrook, third edition Cold Spring Harbor LaboratoryPress, CSH, New York or with reference to Current Protocols in Molecular Biology, John Wiley ﹠amp; Sons, N.Y. (1989 and annual upgrade version).

Splice variant

Term as used in this article " splice variant " comprise wherein excise, replace, be shifted or add selected intron and/or exon or wherein intron shortened or the variant of the nucleotide sequence that extends.This type of variant will be a kind of variant that has wherein kept proteinic biologic activity basically; This can realize by the proteinic functional fragment of selective retention.This type of splice variant can find or can manually make at occurring in nature.Being used to predict with the method for separating this type of splice variant is well-known in the artly (to see for example Foissac and Schiex, BMC Bioinformatics.2005; 6:25).

Allelic variant

Allelotrope or allelic variant are the alternative forms of given gene, are positioned at identical chromosome position.Allelic variant comprises single nucleotide polymorphism (SNP) and little insertion/deletion polymorphism (INDEL).The size of INDEL is usually less than 100bp.SNP and INDEL are formed on the maximum set of sequence variants in the most of biological natural existence polymorphism strain system.

Gene reorganization/orthogenesis

Consisting of of gene reorganization or orthogenesis: DNA reorganization repeatedly, suitably screening and/or selection have the proteinic nucleic acid of improvement biologic activity or variant (Castle etc., (2004) Science 304 (5674): 1151-4 of its part to produce coding subsequently; United States Patent (USP) 5,811,238 and 6,395,547).

Regulatory element/regulating and controlling sequence/promotor

Term " regulatory element ", " regulating and controlling sequence " and " promotor " all are used interchangeably and mean in a broad sense the modulability nucleotide sequence that can realize that the sequence that is attached thereto is expressed in this article.Term " promotor " refer generally to be positioned at genetic transcription starting point upstream and participate in identification and in conjunction with RNA polymerase and other protein, thereby instruct the nucleic acid regulating and controlling sequence of the transcribed nucleic acid that effectively connects.Aforementioned term comprises from typical eukaryotic gene group gene and (comprising for the required TATA box of accurate transcripting starting, have or do not have CCAAT box sequence) in deutero-transcriptional regulatory sequences and replying grow stimulation and/or outside stimulus or with the tissue specificity mode change genetic expression the additional adjustment element (as, upstream activating sequence, enhanser and silencer).This term also comprises the transcriptional regulatory sequences of typical prokaryotic gene, and it can comprise-35 box sequences and/or-10 box transcriptional regulatory sequences in the case.Term " regulatory element " also comprises gives, activates or strengthen synthetic fusion molecule or the derivative that nucleic acid molecule is expressed in cell, tissue or organ.

" plant promoter " comprises the regulatory element that mediation encoding sequence section is expressed in vegetable cell.Therefore, plant promoter needs not be plant origin, but can be derived from virus or microorganism, for example from the virus of invasion and attack vegetable cell." plant promoter " also can plant-derived cell, for example comes to use by oneself to treat the nucleotide sequence institute plant transformed expressing and describe in this article in the inventive method.This also is applicable to other " plant " modulability signal, as " plant " terminator.The promotor upstream that is used for the nucleotide sequence of the inventive method can be modified by one or more nucleotide subsitutions, insertion and/or disappearance, but do not disturb promotor, open reading-frame (ORF) (ORF) or 3 ' regulatory region such as terminator or functional or active away from other 3 ' regulatory region of ORF.The activity of promotor also might be because of the sequence of modifying this promotor or by more active promotor even thoroughly replace this promotor from the promotor of allos biology and increase.For expressing in plant, as mentioned above, nucleic acid molecule must effectively be connected to or comprise suitable promotor, and wherein said promotor is on orthochronous point and with needed space expression pattern expressing gene.

For identifying functional equivalent promotor, the promotor intensity of candidate's promotor and/or expression pattern can be by effectively being connected this promotor with reporter gene and analyzing this report gene and analyze in the expression level and the pattern of the multiple tissue of plant.Suitable known reporter gene comprises for example β-glucuronidase or beta-galactosidase enzymes.Promoter activity is analyzed by the enzymic activity of measuring β-glucuronidase or beta-galactosidase enzymes.Promotor intensity and/or expression pattern can compare with the promotor intensity and/or the expression pattern of reference promotor (as a kind of promotor used in the inventive method) subsequently.Alternatively, promotor intensity can be used the densitometric analysis method of means known in the art such as Northern blotting and autoradiogram(ARGM), quantitative PCR in real time or RT-PCR (Heid etc., 1996GenomeMethods 6:986-994), by quantitative mRNA or by the mRNA level of used nucleic acid in the inventive method and the mRNA level comparison of housekeeping gene (as 18S rRNA) are analyzed.Usually " weak promoter " means and drives encoding sequence expression promoter on low-level." low-level " means at about 1/10,000 transcript of each cell to about 1/100,000 transcript, to the level of about 1/500,0000 transcript.On the contrary, " strong promoter " drive encoding sequence high level or at about 1/10 transcript of each cell to about 1/100 transcript, to about 1/1,000 transcript, express.

Effectively connect

Term as used in this article " effectively connect " refer to functionally be connected between promoter sequence and the goal gene, to such an extent as to can starting goal gene, promoter sequence transcribes.

Constitutive promoter

" constitutive promoter " refers in the major part of g and D but all during the stage and in the promotor that transcriptional activity is arranged at least one cell, tissue or organ under most of envrionment conditions.Following table 2 provides the example of constitutive promoter.

Table 2: the example of constitutive promoter

Gene source	Reference
		Actin muscle	McElroy etc., Plant Cell, 2:163-171,1990
HMGB	WO 2004/070039
		CAMV35S	Odell etc., Nature, 313:810-812,1985
CaMV19S	Nilsson etc., Physiol.Plant.100:456-462,1997
		GOS2	De Pater etc., Plant J Nov; 2 (6): 837-44,1992, WO 2004/065596
Ubiquitin	Christensen etc., Plant Mol.Biol.18:675-689,1992
		The rice cyclophilin	Buchholz etc., Plant Mol Biol.25 (5): 837-43,1994
Corn H3 histone	Lepetit etc., Mol.Gen.Genet.231:276-285,1992
		Clover H3 histone	Plant Mol.Biol.11:641-649 such as Wu, 1988
Actin muscle 2	An etc., Plant are (1) J.10; 107-121,1996
		34S FMV	Sanger etc., Plant.Mol.Biol., 14,1990:433-443
The Rubisco small subunit	US 4,962,028
		OCS	Leisner(1988)Proc Natl Acad Sci USA 85(5)：2553
SAD1	Jain etc., Crop Science, 39 (6), 1999:1696
		SAD2	Jain etc., Crop Science, 39 (6), 1999:1696
nos	Shaw etc. (1984) Nucleic Acids Res.12 (20): 7831-7846
		The V-ATP enzyme	WO 01/14572

Super promotor	WO 95/14098
		G box protein matter	WO 94/12015

The omnipresence promotor

Institute is in a organized way or activity arranged in the cell basically at biology for the omnipresence promotor.

Grow the modulability promotor

Grow the modulability promotor and during certain growth period or in experience is grown the plant part that changes activity is being arranged.

Inducible promoter

(summary is seen Gatz 1997 to inducible promoter replying chemical, Annu.Rev.PlantPhysiol.Plant Mol.Biol., 48:89-108), the transcripting starting that has induced or increase when environmental stimulus or physical stimulation, maybe can be " stress-inducing ", promptly when being exposed to multiple stress conditions, plant activated, or " pathogen-inducible ", promptly when being exposed to multiple pathogenic agent, plant activated.

Organ specificity/tissue-specific promoter

Organ specificity or tissue-specific promoter can preferentially start the promotor of transcribing some organ official or in organizing as leaf, root, seed tissue etc.For example, " root-specific promoter " is that advantage ground has the promotor of transcriptional activity in roots of plants, and essentially no activity in any other parts of plant is although allow any leakage to express in these other parts of plant.Can only in some cell, start the promotor of transcribing and be called " cell-specific " in this article.

Chlorenchyma specificity promoter as defined herein is mainly to have the promotor of transcriptional activity in chlorenchyma, and essentially no activity in any other parts of plant is although allow any leakage to express in these other parts of plant.

The example that can be used for implementing the chlorenchyma specificity promoter of the inventive method shows in following table 3.

Table 3: the example that the chlorenchyma specificity starts

Gene	Express	Reference
			The corn orthophosphate dikinase	The leaf specificity	Fukavama etc., 2001

(orthophosphate dikinase)
			The corn phosphoric acid enol pyruvic acid carboxylase	The leaf specificity	Kausch etc., 2001
The rice phosphoric acid enol pyruvic acid carboxylase	The leaf specificity	Liu etc., 2003
			Rice Rubisco small subunit	The leaf specificity	Nomura etc., 2000
Rice β expansion albumen EXBP9	The seedling specificity	WO 2004/070039
			Pigeonpea (Pigeonpea) Rubisco small subunit	The leaf specificity	Panguluri etc., 2005
Pea RBCS3A	The leaf specificity

Another example of tissue-specific promoter is the meristematic tissue specificity promoter, it mainly has transcriptional activity in the merism tissue, essentially no activity in any other parts of plant is although allow any leakage to express in these other parts of plant.

Terminator

Term " terminator " comprise such regulating and controlling sequence, it is the dna sequence dna at transcription unit's end, sends primary transcript is carried out the signal that 3 ' processing and poly-adenosine and termination are transcribed.Terminator can be derived from natural gene, from multiple other plant gene or from T-DNA.Terminator to be added can be from for example nopaline synthase or octopine synthase gene or alternatively from other plant gene or more preferably from any other eukaryotic gene.

Regulate

Term " adjusting " with regard to expression or genetic expression, mean such process, wherein expression level is compared with control plant because of described expression of gene changes, and preferably, expression level increases.Any kind that original expression of being regulated can be structure RNA (rRNA, tRNA) or mRNA is expressed, and is translation subsequently.Term " adjusting is active " should mean any variation of nucleotide sequence of the present invention or coded protein expression, and this causes the output of plant increase and/or the growth of increase.

Expression/the overexpression that increases

Term as used in this article " expression that increases " or " overexpression " to mean for original wild-type expression level be extra any formal representation.

In this area write up be used to increase the method for gene or gene product expression and they for example comprise, by the overexpression of suitable promoters driven, use transcriptional enhancer or translational enhancer.Can in the suitable location (generally being the upstream) of the polynucleotide of non-allos form, import isolating nucleic acid, so that go up the expression of nucleic acids of tone coded desired polypeptides as promotor or enhancer element.For example, the endogenous promotor can change in vivo by sudden change, disappearance and/or displacement and (sees Kmiec, U.S. Patent number 5,565,350; Zarling etc. PCT/US93/03868), maybe can import vegetable cell with correct direction and distance with respect to gene of the present invention with isolating promotor, so that controlling gene is expressed.

If need expression of polypeptides, wish that usually 3 ' end in the polynucleotide encoding district comprises the poly-adenosine district.The poly-adenosine district can be from natural gene, from multiple other plant gene or from T-DNA.3 ' end sequence to be added can be from for example nopaline synthase or octopine synthase gene or alternatively from another plant gene or more not preferably from any other eukaryotic gene.

Intron sequences also can be added on the encoding sequence of 5 ' non-translational region (UTR) or part coding property sequence, to be increased in the amount of the ripe information that accumulates in the endochylema.But verified montage intron being included in the transcription unit in expression of plants construct and animal expression construct increases genetic expression to reaching 1000 times of (Buchman and Berg (1988) Mol.Cell biol.8:4395-4405 on mRNA level and the protein level; Callis etc. (1987) Gens Dev 1:1183-1200).This type of intron enhancement of genetic expression is the strongest generally near being positioned at transcription unit 5 ' end the time.It is known in the art using corn intron A dh1- S introne 1,2 and 6, Bronze-1 intron.For general information, see: " corn handbook, the 116th chapter, editor Freeling and Walbot, Springer, N.Y. (1994).

Native gene

" endogenous " gene of mentioning herein not only refers to the gene of being discussed that exists with its natural form (promptly without any the mankind intervene) as in plant, and also refers to be in the homologous genes (or homologous nucleic acid/gene) basically of (again) the subsequently importing plant (transgenosis) of unpack format.For example, contain this genetically modified transgenic plant and can meet with the significantly reduction that transgene expression significantly reduces and/or native gene is expressed.

The expression that reduces

" reducing or basic the removal " of mentioning herein means native gene expression and/or polypeptide level and/or the polypeptide active reduction with respect to control plant.Compare with control plant, reducing or removing to increase progressively preferred sequence substantially is at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduction.

In order to reduce or to remove the expression of native gene in plant substantially, need the Nucleotide of successive basically of the sufficient length of nucleotide sequence.In order to carry out gene silencing, this length can be few to 20,19,18,17,16,15,14,13,12,11,10 or still less Nucleotide, and perhaps this length can the whole gene of as many as (comprising 5 ' and/or 3 ' UTR, part or all).Basically the successive nucleotide fragments can come the own coding target protein nucleic acid (target gene) or from the target protein of can encoding directly to any nucleic acid of homologue, collateral line homologue or homologue.Preferably, basically the successive nucleotide fragments can form hydrogen bond with target gene (sense strand or antisense strand), more preferably, the successive nucleotide fragments has 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to increase progressively preferred sequence and target gene (sense strand or antisense strand) basically.The nucleotide sequence of coding (functional) polypeptide is not that discussed herein to be used to reduce or to remove substantially the several different methods that native gene expresses required.

This reduction of expressing or basic removal can use conventional instrument and technology to finish.Being used for reducing or removing substantially the preferred method that native gene expresses is to import and express such genetic constructs plant, its amplifying nucleic acid (be from goal gene or any nucleic acid one section successive nucleotide sequence basically in the case, wherein said any nucleic acid can encode any target protein directly to homologue, collateral line homologue or homologue) be cloned in the described genetic constructs as (partially or completely) inverted repeats that separates by transcribed spacer (non-coding DNA).

In this preferable methods, use nucleic acid or its part (be in the case from goal gene or from any nucleic acid one section of deutero-successive nucleotide sequence basically, wherein said any nucleic acid can encode target protein directly to homologue, collateral line homologue or homologue) inverted repeats (preferably can form hairpin structure), the expression that the silence effect by RNA mediation reduces or remove basically native gene.Inverted repeats is cloned in comprising the expression vector of regulating and controlling sequence.Non-coding DNA nucleotide sequence (intervening sequence, for example matrix attachment regions fragment (MAR), intron, polylinker etc.) is between two reverse nucleic acid that form inverted repeats.After inverted repeats is transcribed, form chimeric RNA with (partially or completely) self complementary structure.This double-stranded RNA structure is called hairpin RNA (hpRNA).HpRNA is processed into siRNA by plant, and it is impregnated in the reticent mixture of RNA inducibility (RISC).RISC further cuts the mRNA transcript, thereby reduces the number of the mRNA transcript of one-tenth polypeptide to be translated significantly.For other general details, see for example (1998) WO 98/53083 such as Grierson; Waterhouse etc. (1999) WO 99/53050).

The enforcement of the inventive method does not rely in the plant to import and to express and wherein is cloned into the genetic constructs of nucleic acid as inverted repeats, but several known " gene silencing " method any or multiplely can be used for realizing identical effect.

A kind ofly be used to reduce that native gene expresses is the genetic expression silence (downward modulation) of RNA mediation as this method.Silence acts in this case and is triggered in plant by similar to the endogenous target gene basically double-stranded RNA sequence (dsRNA).This dsRNA is arrived about 26 Nucleotide by the further processing of plant into about 20, is called short interferential RNA (siRNA).SiRNA is impregnated in the reticent mixture of RNA inducibility (RISC), and wherein said RISC further cuts the mRNA transcript of endogenous target gene, thereby reduces the number of the mRNA transcript of one-tenth polypeptide to be translated significantly.Preferably, the double-stranded RNA sequence is corresponding to target gene.

Another example of RNA silencing methods comprise with sense orientation import nucleotide sequence or its part (be in the case from goal gene or from any nucleic acid one section of deutero-successive Nucleotide basically, wherein said any nucleic acid can encode target protein directly to homologue, collateral line homologue or homologue) to plant." sense orientation " refers to and its mRNA transcript homologous dna sequence dna.Thereby will in plant, import at least one copy of this nucleotide sequence.This extra nucleotide sequence can reduce native gene expresses, and produces and is known as inhibiting altogether phenomenon.When several additional copies of nucleotide sequence import plant, the reduction of genetic expression will be more obvious, because have positive correlation between the inhibiting together triggering of high transcript level.

Another example of RNA silencing methods comprises the use anti sense nucleotide sequence.The ＂ that ＂ antisense ＂ nucleotide sequence comprises with coded protein has adopted ＂ nucleic acid array complementation, promptly with the coding strand complementation of double-stranded cDNA molecule, or with mRNA transcript sequence complementary nucleotide sequence.Anti sense nucleotide sequence preferably with treat reticent native gene complementation.Complementary " coding region " that can be positioned at gene and/or " non-coding region ".Term " coding region ＂ refers to comprise the nucleotide sequence district of the codon that is translated into amino-acid residue.Term " non-coding region ＂ refers to be distributed in being transcribed of both sides, coding region but do not translate into amino acid whose 5 ' and 3 ' sequence (be also referred to as 5 ' and 3 ' non-translational region).

Anti sense nucleotide sequence can be according to Watson and the design of Crick base pairing rules.Anti sense nucleotide sequence can with whole nucleic acid array complementation (be in the case from goal gene or from any nucleic acid one section of deutero-successive Nucleotide basically, wherein said any nucleic acid can encode target protein directly to homologue, collateral line homologue or homologue), but also can be only with the oligonucleotide of a part (comprising mRNA5 ' and the 3 ' UTR) antisense of nucleotide sequence.For example, Antisensedigonucleotsequence sequence can with the regional complementarity around the translation starting point of the mRNA transcript of coded polypeptide.The length of suitable Antisensedigonucleotsequence sequence is known in the art and can be from about 50,45,40,35,30,25,20,15 or 10 Nucleotide of length or Nucleotide still less.Anti sense nucleotide sequence of the present invention can utilize means known in the art, uses chemosynthesis and enzyme ligation and makes up.For example, anti sense nucleotide sequence (for example Antisensedigonucleotsequence sequence) can use the Nucleotide of naturally occurring Nucleotide or multiple modification to synthesize chemically, the Nucleotide of wherein said modification is designed to be intended to increase the biological stability of molecule or increases anti sense nucleotide sequence and the physical stability of the duplex that forms between the phosphorothioate odn sequence is arranged, for example, the Nucleotide that can use phosphorothioate derivative and acridine to replace.The example that can be used for producing the modified nucleotide of anti sense nucleotide sequence is well-known in the art.Known nucleotide modification comprise methylate, cyclisation and ' add cap ' and replace one or more naturally occurring Nucleotide with analogue (as inosine).Other nucleotide modification is well-known in the art.

This anti sense nucleotide sequence can use nucleotide sequence wherein with antisense orientation in addition the expression vector of subclone (promptly the RNA that transcribes from the nucleic acid that inserts will be antisense orientation with the purpose target nucleic acid) produce in the biology mode.Preferably, the generation of anti sense nucleotide sequence in plant undertaken by the nucleic acid construct of stable integration, antisense oligonucleotide and terminator that wherein said nucleic acid construct comprises promotor, effectively connects.

The nucleic acid molecule (no matter import in plant or (in situ) produce) in position that is used for the reticent effect of the inventive method is with the mRNA transcript and/or the genomic dna hybridization of coded polypeptide or combine, so that for example by suppressing to transcribe and/or translation and the expression of arrestin matter.Hybridization can be passed through to form due to the conventional Nucleotide complementarity of stablizing duplex, or under the situation of the anti sense nucleotide sequence that is incorporated into DNA duplex, due to the interaction of duplex major groove internal specific.Anti sense nucleotide sequence can be by transforming or importing plant at particular organization's position direct injection.Alternatively, anti sense nucleotide sequence can be modified for the selected cell of target and systemic administration subsequently.For example, for systemic administration, anti sense nucleotide sequence can be modified so that their specific combination are expressed in acceptor or the antigen on the selected cell surface, for example by connect anti sense nucleotide sequence to cell surface receptor or antigen bonded peptide or antibody.Anti sense nucleotide sequence also can use described carrier to send herein and pass to cell.

According to another aspect, anti sense nucleotide sequence is a α-different nucleotide sequence.Different nucleotide sequence of α and complementary RNA form specific double-stranded hybrid molecule, and be wherein opposite with usual b-unit, described chain be parallel to each other (Gaultier etc. (1987) Nucl Ac Res 15:6625-6641).Anti sense nucleotide sequence also can comprise 2 '-the o-methyl ribonucleotides (Inoue etc. (1987) Nucl Ac Res 15,6131-6148) or chimeric RNA-DNA analogue (Inoue etc. (1987) FEBS Lett.215,327-330).

Reduction that native gene is expressed or basic removal also can be used ribozyme and carry out.Ribozyme is the catalytic RNA molecule with ribonuclease activity, can cut the single-chain nucleic acid sequence that has complementary region with it, as mRNA.Therefore, (for example hammerhead ribozyme is (at Haselhoff and Gerlach (1988) Nature 334 for ribozyme, describe among the 585-591) can be used for the mRNA transcript of catalytic ground cutting coded polypeptide, thereby reduce the number of the mRNA transcript of one-tenth polypeptide to be translated significantly.Can design the specific ribozyme of nucleotide sequence tool (is for example seen: U.S. Patent numbers such as Cech 4,987,071; With U.S. Patent numbers 5,116,742 such as Cech).Alternatively, corresponding to the mRNA transcript of nucleotide sequence can be used for from the RNA library of molecules, selecting catalytic RNA with specific ribonucleic acid enzymic activity (Bartel and Szostak (1993) Science 261,1411-1418).The purposes that ribozyme is used for the plant gene silencing is ((1994) WO94/00012 such as Atkins for example known in the art; Lenne etc. (1995) WO 95/03404; Lutziger etc. (2000) WO 00/00619; (1997) WO 97/38116 such as Prinsen etc. (1997) WO 97/13865 and Scott).

Gene silencing also can be by inserting mutagenesis (for example T-DNA inserts or transposon inserts) or by ((1999) Plant is (3) J.20: 357-62), the strategy of (Amplicon VIGSWO 98/36083) or Baulcombe (WO 99/15682) and other people description realizes as Angell and Baulcombe.

When having sudden change on the native gene and/or have sudden change on importing isolating gene/nucleic acid of plant subsequently, gene silencing also can take place.Reduction or basic removal can be caused by non-functional polypeptide.For example, polypeptide can with multiple interaction protein bound; One or more sudden changes and/or brachymemma thereby can provide still can binding interactions protein (as receptor protein) but can not show the polypeptide (as playing the part of signal effect) of its normal function.

The method of another kind of gene silencing is the triple-helix structure that target is fixed and generegulation district (for example promotor and/or enhanser) complementary nucleotide sequence stops gene to be transcribed in target cell with formation.See Helene, C., Anticancer Drug Res.6,569-84,1991; Helene etc., Ann.N.Y.Acad.Sci.660,27-361992 and Maher, L.J.Bioassays 14,807-15,1992.

Other method, as using at the antibody of endogenous polypeptide suppressing the function of this polypeptide in plant, or the signal pathway that disturbs described polypeptide to participate in, will be well-known for the technician.Especially, what can conceive is the biological function that artificial molecule can be used to suppress the target polypeptide, or is used to the signal pathway that disturbs the target polypeptide to participate.

Alternatively, can set up screening procedure to identify the natural variant of gene in plant population, wherein said variant coding has the active polypeptide of reduction.This type of natural variant also can be used for for example carrying out homologous recombination.

Artificial and/or natural microRNA (miRNA) can be used for knocking out genetic expression and/or mRNA translation.Endogenous miRNA is the little RNA of strand of a common 19-24 length of nucleotides.Their major function is that regulatory gene is expressed and/or the mRNA translation.Most plant micrornas (miRNA) has completely with its target sequence or is approaching complementary completely.Yet, exist to have the nearly natural target of 5 mispairing.They by the double-stranded specific RNA enzyme of cutting enzyme family from having the characteristic processing the long non-coding RNA of structure of turning back.Adding man-hour, they are by mixing this complex body with the main component Argonaute protein bound of the reticent mixture of RNA inducibility (RISC).MiRNA serves as the specific component of RISC, so target nucleic acid (the being mRNA mostly) base pairing in they and the tenuigenin.Follow-up adjusting incident comprises the said target mrna cutting and destroys and/or the translation inhibition.Therefore the effect of miRNA overexpression obtains reflection on the mRNA level that target gene reduces.

The artificial microRNA (amiRNAs) of common 21 length of nucleotides can genetic modification with the negative genetic expression of regulating single or multiple goal gene specifically.The determinative of the selection of plant micrornas target is well-known in the art.The empirical parameter that is used for target identification has been determined and can be used for the specific amiRNA of aided design, Schwab R, 2005.The convenient tool that is used to design and produce amiRNA and precursor thereof also is that the public is obtainable, Schwab etc., 2006.

Be optimum performance, be used for reducing gene silent technology that native gene expresses plant and need use from monocotyledonous nucleotide sequence with transforming monocots with use nucleotide sequence from dicotyledons to transform dicotyledons.Preferably, will import from the nucleotide sequence of any given plant species in the same species.For example, will be converted into rice plant from the nucleotide sequence of rice.Yet, be not the identical plant species of plant that definitely requires nucleotide sequence to be imported to originate from will to import with this nucleotide sequence.As long as exist sizable homology just enough between endogenous target gene and the nucleic acid to be imported.

Above-described is the example that is used for reducing or removes substantially the several different methods that native gene expresses plant.To such an extent as to those skilled in the art can adjust easily and aforementionedly be used for reticent method for example by utilizing suitable promotor to realize to reduce native gene whole strain plant or in the expression of its part.

Selective marker (gene)/reporter gene

＂ selective marker ＂, ＂ selectable marker gene ＂ or " reporter gene " comprise any gene from phenotype to cell that give, wherein at the described gene of described cell inner expression promote to identify and/or to select with nucleic acid construct institute's transfection of the present invention or cell transformed.These marker gene can be identified the successful transfer of nucleic acid molecule by a series of different principle.Suitable mark can be selected from the mark of giving antibiotic resistance or Herbicid resistant, the new metabolism proterties of importing or allowing visual selection.The example of selectable marker gene comprise the gene of giving antibiotic resistance (as make the nptII of Xin Meisu and kantlex phosphorylation or make the hpt of Totomycin phosphorylation or give to for example bleomycin, Streptomycin sulphate, tsiklomitsin, paraxin, penbritin, gentamicin, Geneticin (Geneticin) (G418), the gene of the resistance of spectinomycin or blasticidin), the gene of conferring herbicide resistance (for example provides

The bar of resistance; The aroA or the gox of glyphosate resistance be provided or give for example gene of the resistance of imidazolone, phosphinothricin or sulfourea) or provide the metabolism proterties gene (as allow plant use seminose as the manA of sole carbon source or utilize xylose isomerase or the anti-nutrition mark such as the 2-deoxyglucose resistance of wood sugar).The expression of visual marker gene causes forming color (for example β-glucuronidase, GUS or beta-galactosidase enzymes substrate coloured with it for example X-Gal), luminous (as luciferin/luciferase system) or fluorescence (green fluorescent protein GFP and derivative thereof).This list is only represented the possible mark of minority.The technician is familiar with this type of mark.Depend on biology and system of selection, preferred different mark.

Known to nucleic acid stability or integration,temporal during to vegetable cell, the cellular uptake foreign DNA of small portion and as required it is integrated into cellular genome only, this depends on the rotaring dyeing technology of used expression vector and use.For identifying and select these integrons, the gene of the selective marker of will encoding usually one of (as indicated above) imports host cell together with goal gene.These marks therein these genes because of using in the non-functional mutant of disappearance due to the ordinary method for example.In addition, the nucleic acid molecule of coding selective marker can import in the host cell, with the sequence of used polypeptide in comprising code book invention polypeptide or the inventive method on identical carrier, or on independent carrier.Can be by having selected to identify (for example having the cell survival of selective marker of integration and other necrocytosis) with the nucleic acid stability cells transfected that imports.

Because in case successfully imported nucleic acid, then no longer need in the genetically modified host cell or do not wish underlined gene, especially antibiotic resistance gene and herbicide resistance gene, the inventive method that therefore is used to import nucleic acid is advantageously used the technology that can remove or excise these marker gene.A kind ofly be called the cotransformation method as this method.The cotransformation method is used two kinds of carriers being used to simultaneously transform, and a kind of carrier carries nucleic acid of the present invention and another kind of carrier carries marker gene.A high proportion of transformant is accepted, or under the situation of plant, comprise (up to 40% or more transformant) these two kinds of carriers.Under situation about transforming with Agrobacterium (Agrobacterium), transformant is only accepted the part of carrier usually, and promptly flank has the sequence of T-DNA, and it represents expression cassette usually.Marker gene can be removed from plant transformed by hybridizing subsequently.In another approach, the marker gene that is integrated into transposon is used for transforming (being called the Ac/Ds technology) with the nucleic acid of wanting.Transformant can be instantaneous or stably transform with the nucleic acid construct that causes transposase to be expressed with originate plant hybridization or transformant of transposase.(about 10%) in some cases, transposon is jumped out the genome of host cell and is lost when successfully taking place to transform.Under other more susceptible condition, transposon skips to different positions.In these cases, marker gene must be removed by hybridizing.In microbiology, developed the technology that realizes or promote to detect this class incident.Another advantageous method depends on recombination system; The advantage of this method is and needn't removes by hybridization.The most well-known system of the type is called the Cre/lox system.Cre1 is the recombinase that removes sequence between the loxP sequence.If marker gene is integrated between the loxP sequence, when then having expressed successfully generation conversion by recombinase, marker gene is removed.Other recombination system is HIN/HIX, FLP/FRT and REP/STB system (Tribble etc., J.Biol.Chem., 275,2000:22255-22267; Velmurugan etc., J.Cell Biol., 149,2000:553-566).Nucleotide sequence of the present invention might be integrated into Plant Genome in the locus specificity mode.These methods also can be applied to microorganism such as yeast, fungi or bacterium naturally.

Genetically modified/transgenosis/reorganization

Be the object of the invention, the genetically modified ＂ of ＂, " transgenosis " or ＂ reorganization ＂ means the expression cassette, gene construct or the carrier that comprise this nucleotide sequence with regard to nucleotide sequence or with the biology of nucleotide sequence of the present invention, expression cassette or carrier conversion, these make up all and produce by recombination method, wherein

Coding is used for the nucleic acid sequences to proteins of the inventive method, or

The genetic regulation sequence that effectively is connected with nucleotide sequence of the present invention, promotor for example, or

A) and b)

Be not in its natural genotypic environment or modify by genetic manipulation method, be modified with may for example adopt replace, add, disappearance, inversion or insert the form of one or more nucleotide residues.Natural genotypic environment be interpreted as mean the source in the plant natural gene group locus or chromogene seat or in genomic library, exist.Under the situation of genomic library, the natural genotypic environment of nucleotide sequence preferably obtains keeping, and is kept at least in part.This environment is distributed at least one side of nucleotide sequence and has 50bp at least, preferred 500bp at least, especially preferred 1000bp at least, the most preferably sequence length of 5000bp at least.The naturally occurring combination of the corresponding nucleotide sequence of used polypeptide in the natural promoter of the nucleotide sequence of naturally occurring expression cassette-for example and the code book inventive method, as hereinbefore defined-when being subjected to modifying, become transgene expression cassette by non-natural synthetic (" manually ") method (as mutagenic treatment) at this expression cassette.Appropriate method is for example at US 5,565,350 or WO00/15815 in describe.

Be the object of the invention, therefore transgenic plant are interpreted as above and mean the natural gene seat that nucleic acid used in the inventive method is not arranged in described this nucleic acid of Plant Genome that described nucleic acid might homology or the expression of allos ground.Yet as mentioned, although transgenosis also means nucleic acid of the present invention or used in the methods of the invention nucleic acid is in the natural place of this nucleic acid in the Plant Genome, yet its sequence is modified for native sequences, and/or the adjusting sequence of described native sequences is modified.Transgenosis is interpreted as preferably to mean in the non-natural locus of nucleic acid of the present invention in genome and expresses that the homology that nucleic acid promptly takes place is expressed or preferred heterogenous expression.Preferred transgenic plant have been mentioned in this article.

Transform

Term " importing " or " conversion " comprise that exogenous polynucleotide are transferred in the host cell as mentioned in this article, and what the method that no matter is used to transform is.Can follow-up clone's property propagation the plant tissue of (no matter take place or the embryo is taken place) by organ can transform and the whole strain plant that can therefrom regenerate with genetic constructs of the present invention.The concrete tissue of selecting will depend on clone's property proliferating system of the concrete species that can be used for and be suitable for just transforming most.The example organization target comprises leaf dish, pollen, embryo, cotyledon, hypocotyl, megagametophyte, callus, existing meristematic tissue (for example apical meristem, axillalry bud and root meristematic tissue) and inductive meristematic tissue (for example cotyledon meristematic tissue and hypocotyl meristematic tissue).Polynucleotide can instantaneous or stably import host cell and can keep to nonconformity, for example as plasmid.Alternatively, polynucleotide can be integrated in the host genome.The transformed plant cells that produces can be used for regenerating in the manner known to persons skilled in the art the conversion plant subsequently.

Alien gene is converted into and is called conversion in the Plant Genome.The conversion of plant species is quite conventional technology now.Advantageously, the arbitrary method in several method for transformation can be used for goal gene is imported suitable ancester cell.Be used for transforming and the described method of the plant that regenerates can be used for instantaneous conversion or be used for stable conversion from plant tissue or vegetable cell.Method for transformation comprises that the chemical, the dna direct that use liposome, electroporation, increase dissociative DNA to take in are injected to the conversion method and the micro-projective method (microprojection) of plant, particle gun blast technique, use virus or pollen.Method for transformation can be selected from calcium/polyoxyethylene glycol method (Krens, F.A. etc., (1982) Nature296, the 72-74 that is used for protoplastis; (1987) Plant Mol Biol 8:363-373 such as Negrutiu I); The electroporation of protoplastis ((1985) Bio/Technol 3 such as Shillito R.D., 1099-1102); Micro-injection (Crossway A etc., (1986) Mol.Gen Genet 202:179-185) to vegetable material; The particle bombardment method (Klein TM etc., (1987) Nature 327:70) of DNA or RNA coating, (nonconformity) virus infection method etc.Transgenic plant comprise the genetically modified crops plant, preferably produce by agriculture bacillus mediated conversion method.Favourable method for transformation is the conversion method of in plant (in planta).For this purpose, for example might make Agrobacterium act on the meristematic tissue that plant seed maybe might be inoculated plant with Agrobacterium.To act on complete plant or act on flower primordium at least be particularly advantageous to the verified Agrobacterium suspension that makes conversion according to the present invention.Plant continues subsequently to cultivate that (Clough and Bent, Plant J. (1998) 16,735-743) until the seed that obtains the plant of handling.Be used for method that agriculture bacillus mediated rice transforms and comprise and be used for the known method that rice transforms, as those methods of in arbitrary following document, describing: European patent application EP 1198985 A1, Aldemita and Hodges (Planta199:612-617,1996); Chan etc. (Plant Mol Biol 22 (3): 491-506,1993), Hiei etc. (Plant J 6 (2): 271-282,1994), its disclosure is incorporated herein by reference in this article, as providing fully.Under the situation that corn transforms, (Nat.Biotechnol 14 (6): 745-50 for preferable methods such as Ishida etc., 1996) or Frame etc. (Plant Physiol 129 (1): 13-22,2002) describe, its disclosure is incorporated herein by reference as fully in this article.Described method by way of example mode further by B.Jenes etc., Techniques for Gene Transfer,: Transgenic Plants, the 1st volume, Engineering and Utilization, editor S.D.Kung and R.Wu, Academic Press (1993) 128-143 and at Potrykus Annu.Rev.PlantPhysiol.Plant Molec.Biol.42 (1991) 205-225) in the description.Nucleic acid to be expressed or construct preferably are cloned into the carrier that is suitable for transforming agrobacterium tumefaciens (Agrobacterium tumefaciens), for example pBin19 (Bevan etc., Nucl.Acids Res.12 (1984) 8711).The Agrobacterium that is transformed by this carrier can be used to transform plant according to known way subsequently, the plant of using for example as model, (Arabidopsis is in scope of the present invention as Arabidopis thaliana, be not considered as crop plants) or crop plants, for example tobacco plant is also cultivated them subsequently by the leaf that soaks abrasive leaf or chopping in Agrobacterium solution in suitable medium.The conversion of plant by agrobacterium tumefaciens for example by

With Willmitzer at Nucl.Acid Res. (1988) 16, Vectors for Gene Transfer in Higher Plants is described in 9877 or especially from F.F.White; At Transgenic Plants, the 1st volume, Engineering and Utilization, editor S.D.Kung and R.Wu, AcademicPress is known in 1993, the 15-38 pages or leaves.

Except transformant cell (its necessary subsequently complete plant of regeneration), also might transform the merismatic cell of plant and special those cells that develop into gamete that transform.In this case, the gamete of conversion is followed natural development of plants process, produces transgenic plant.Therefore, for example the Arabidopis thaliana seed is handled with Agrobacterium and obtain seed from is grown plant, and wherein a certain proportion of described plant is transformed and is genetically modified [Feldman, KA and Marks MD (1987) Mol GenGenet 208:274-289 therefore; Feldmann K (1992).: editor C Koncz, N-H Chua and J Shell, Methods in Arabidopsis Research.W ord Scientific, Singapore, 274-289 page or leaf].Alternative method based on remove inflorescence repeatedly and make in the rosette in the heart the excision position and the Agrobacterium incubation of conversion, thereby the seed that transforms can obtain at later time point equally, and (Chang (1994) Plant is J.5:551-558; Katavic (1994) Mol Gen Genet, 245:363-370).Yet especially effective means is the vacuum infiltration method of improvement, as " flower is contaminated " method.Under the situation of Arabidopis thaliana vacuum infiltration method, complete plant is under reduced pressure handled [Bechthold, N (1993) with the Agrobacterium suspension.C R Acad Sci Paris Life Sci, 316:1194-1199], and under the situation of " flower dip method ", of short duration incubation [the Clough of Agrobacterium suspension that the flower tissue and the tensio-active agent of growing handled, SJ and Bent, AF (1998) The Plant J.16,735-743].Gathered in the crops a certain proportion of transgenic seed in both cases, and these seeds can be distinguished by under aforesaid selection condition, cultivating with the non-transgenic seed.In addition, the stable conversion of plastid is favourable because plastid in most of crop with the heredity of parent mode, reduce or eliminated transgenosis through the pollen flow risk.The conversion of chloroplast gene group generally by at Klaus etc., 2004[Nature Biotechnology 22 (2), 225-229] in the exemplary method realization of being showed.In brief, sequence to be transformed be cloned into together with selectable marker gene and chloroplast gene group homologous flanking sequence between.These homologous flanking sequences instruct locus specificity to be integrated in the plastom(e).Numerous different plant species having been described plastid transforms and summarizes and can come from Bock (2001) transgenosis plastid (Transgenic plastids in basicresearch and plant biotechnology) .J Mol Biol.2001 September 21 in fundamental research and Plant Biotechnology; 312 (3): 425-38 or Maliga, P (2003) plastid transformation technology commercialization progress (Progress towardscommercialization of plastid transformation technology) .TrendsBiotechnol.21,20-28.Further the biotechnology progress has been made report with the form of unmarked plastid transformant recently, described unmarked plastid transformant can produce (Klaus etc. by the instantaneous marker gene of integrating altogether, 2004, Nature Biotechnology 22 (2), 225-229).

T-DNA activates labelization

T-DNA activates labelization Science (1992) 1350-1353 such as () Hayashi and relates in the genome area of goal gene or gene coding region upstream or downstream 10kb sentence structure like this and insert T-DNA (containing promotor (also can be translational enhancer or intron) usually), makes promotor instruct and is decided expression of gene by target.Usually, the promotor control that the natural promoter of deciding gene by target regulating effect that described target is decided genetic expression is destroyed and this gene is in new importing down.Promotor generally is embedded among the T-DNA.This T-DNA inserts Plant Genome randomly, for example by agroinfection, and causes near the improvement of the gene insertion T-DNA to be expressed.Cause is expressed near the improvement of the gene of the promotor that imports, the transgenic plant performance dominant phenotype of generation.

TILLING

TILLING (genome interior orientation inductive local damage) is used for producing and/or identifying the nucleic acid induced-mutation technique, and wherein said nucleic acid encoding has to modify expresses and/or active protein.TILLING also allows to select to carry the plant of this type of mutation variants.These mutation variants may be displayed on the intensity aspect or aspect the position or in the expression (if for example sudden change influence promotor) of improvement aspect the time.These mutation variants can show than showed active higher activity by the gene that is in its natural form.TILLING is with high-density mutagenesis and high-throughput screening method combination.The general step of following in TILLING is: (Redei GP and Koncz C (1992) are at Methods inArabidopsis Research in (a) EMS mutagenesis, Koncz C, Chua NH, Schell J, Singapore edits, World Scientific Publishing Co, the 16-82 page or leaf; Feldmann etc., at Meyerowitz EM, Somerville CR edits (1994), Arabidopsis.Cold Spring HarborLaboratory Press, Cold Spring Harbor, NY, 137-172 page or leaf; Lightner J and Caspar T (1998) be at J Martinez-Zapater, J Salinas editor, Methods onMolecular Biology the 82nd volume .Humana Press, Totowa, NJ, 91-104 page or leaf); (b) DNA of individual prepares and compiles; (c) pcr amplification purpose district; (d) sex change and renaturation are to allow to form heteroduplex; (e) DHPLC, wherein with heteroduplex whether the existence in compiling thing detect and be an extra peak in the color atlas; (f) identify mutated individual; (g) to the order-checking of sudden change PCR product.The method that is used for TILLING is (McCallum etc., (2000) NatBiotechnol 18:455-457 well-known in the art; Summary is seen Stemple (2004) Nat Rev Genet 5 (2): 145-50).

Homologous recombination

The nucleic acid that homologous recombination allows to select imports in the selected position of determining in genome.Homologous recombination is the standard technique that is used for unicellular lower eukaryote such as yeast or liver moss sword-like leave moss (Physcomitrella) in bio-science routinely.The method that is used for carrying out homologous recombination plant is not only to model plant (Offringa etc. (1990) EMBO J 9 (10): 3077-84) but also to crop plants rice (Terada etc. (2002) Nat Biotech 20 (10): 1030-4 for example; Iida and Terada (2004) Curr Opin Biotech 15 (2): 132-8) be described.

Output

Term " output " but mean the measuring result of economic worth usually, general with specify crop, and area and relevant with the time period.Single plant part based on they number, size and/or weight and directly output is had contribution, or actual output is the every acre yield for certain crop and a year, and this determines divided by the acreage of plantation by ultimate production (comprise results with output assessment).

Early stage vigor

Early stage vigor (especially during plant-growth is early stage active, healthy, well balanced growth) can produce because of plant adaptability increases, and its reason is that for example plant adapts to its environment (promptly optimizing the use of the energy and the distribution between the Miao Yugen) better.Plant with early stage vigor also shows the seedling survival of increase and better crop foundation, this often causes field piece (crop fitly grows, and promptly most of plants reach each stage of growth on the substantially the same time) and often better and higher output highly uniformly.Thereby early stage vigor can be by the multiple factor such as thousand seed weight, sprout percentage ratio, the percentage ratio of emerging, growth of seedling, seedling height, root length, root and seedling biomass and numerous other factors are determined.

Increase/improvement/enhancing

Term " increase ", " improvement " or " enhancing " be interchangeable and should on the application's implication, refer to compare at least 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more output and/or growth with control plant as defined herein.

Seed production

The seed production itself that increases can show as following one or more indexs: a) seed biomass (seed gross weight) increases, and this can be based on single seed and/or every strain plant and/or per hectare or every acre; B) every strain plant increases spends number; C) (full) seed number of Zeng Jiaing; D) the full rate of the seed of Zeng Jiaing (it is expressed as the ratio between full seed number and the seed sum); E) harvest index of Zeng Jiaing, it is expressed as the ratio that can gather in the crops part (as seed) output and total biomass; And f) thousand seed weight of Zeng Jiaing (TKW), this is from the full seed number and the gross weight extrapolation thereof of counting.The TKW that increases can be because of due to the seed size and/or seed weight that increase, and also can be because of due to the increase of embryo and/or endosperm size.

The increase of seed production also can show as the increase of seed size and/or seed volume.In addition, the increase of seed production itself can self-expression be the increase of seed area and/or seed length and/or seed width and/or seed girth also.The output that increases also can produce the structure of improvement, or can occur because of the structure of improvement.

Plant

Term as used in this article " plant " comprise ancestors and offspring and the plant part of whole strain plant, plant, comprise seed, branch, stem, leaf, root (comprising stem tuber), flower and tissue and organ, wherein every kind of mentioned object comprises goal gene/nucleic acid.Term " plant " also comprise vegetable cell, suspension culture, callus, embryo, meristem zone, gametophyte, sporophyte, pollen and sporule, same every kind of object of mentioning comprises goal gene/nucleic acid.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia (Viridiplantae) superfamily, especially monocotyledons and dicotyledons comprise being selected from following feeding or feed beans, ornamental plant, food crop, tree or shrub: maple species (Acer spp.), Actinidia species (Actinidia spp.), Abelmoschus species (Abelmoschus spp.), sisal hemp (Agavesisalana), Agropyron species (Agropyron spp.), the bent grass (Agrostisstolonifera) of crawling, allium species (Allium spp.), Amaranthus species (Amaranthus spp.), Europe beach grass (Ammophila arenaria), pineapple (Ananas comosus), Anona species (Annona spp.), celery (Apium graveolens), Arachis species (Arachis spp.), Artocarpus Forst species (Artocarpus spp.), officinalis (Asparagus officinalis), Avena species (Avena spp.) (oat (Avena sativa) for example, wild avena sativa (Avena fatua), than praising oat (Avena byzantina), Avena fatua var.sativa, hybrid oat (Avena hybrida), carambola (Averrhoa carambola), Ce Sinobambusa (Bambusa sp.), wax gourd (Benincasahispida), Brazil's chestnut (Bertholletia excelsea), beet (Beta vulgaris), Btassica species (Brassica spp.) (colea (Brassica napus) for example, overgrown with weeds blue or green species (Brassica rapassp.) [canola oil dish, rape (oilseed rape), turnip (turnip rape)]), Cadabafarinosa, tea (Camellia sinensis), Canna generalis Bailey (Canna indica), hemp (Cannabissativa), Capsicum species (Capsicum spp.), Carex elata, papaya (Carica papaya), carissa macrocarpa (Carissa macrocarpa), hickory species (Carya spp.), safflower (Carthamus tinctorius), Castanea species (Castanea spp.), America kapok (Ceibapentandra), hare's-lettuce (Cichorium endivia), Cinnamomum species (Cinnamomum spp.), watermelon (Citrullus lanatus), both citrus species (Citrus spp.), cocoanut species (Cocos spp.), Coffea species (Coffea spp.), taro (Colocasia esculenta), Africa Firmiana species (Colaspp.), Corchorus (Corchorus sp.), coriander (Coriandrum sativum), Corylus species (Corylus spp.), hawthorn species (Crataegus spp.), Stigma Croci (Crocus sativus), Cucurbita species (Cucurbita spp.), Cucumis species (Cucumis spp.), cynara scolymus belongs to (Cynara spp. species), Radix Dauci Sativae (Daucus carota), acutifoliate podocarpium herb species (Desmodium spp.), longan (Dimocarpus longan), Wild yam species (Dioscorea spp.), Diospyros species (Diospyrosspp.), Echinochloa species (Echinochloa spp.), oil palm belongs to (Elaeis) (oil palm (Elaeisguineensis) for example, America oil palm Elaeis (oleifera)) Finger-millet (Eleusine coracana), Plumegrass (Erianthus sp.), loquat (Eriobotrya japonica), eucalyptus belongs to (Eucalyptus sp.), red young fruit (Eugenia uniflora), Fagopyrum species (Fagopyrum spp.), Fagus species (Fagusspp.), alta fascue (Festuca arundinacea), Fructus Fici (Ficus carica), cumquat species (Fortunella spp.), Fragaria species (Fragaria spp.), ginkgo (Ginkgo biloba), Glycine (Glycine spp.) (soybean for example, soybean (Soja hispida) or soybean (Soja max)), upland cotton (Gossypium hirstum), Helianthus (Helianthus spp.) (for example Sunflower Receptacle (Helianthusannuus)), long tube tawny daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), Hordeum (Hordeum spp.) (for example barley (Hordeum vulgare)), sweet potato (Ipomoeabatatas), Juglans species (Juglans spp.), lettuce (Lactuca sativa), Lathyrus species (Lathyrus spp.), Lens culinaris (Lens culinaris), flax (Linum usitatissimum), lichee (Litchi chinensis), Lotus species (Lotus spp.), patola (Luffa acutangula), lupinus species (Lupinus spp.), Luzula sylvatica, tomato belongs to (Lycopersicon spp.) (tomato (Lycopersicon esculentum for example, Lycopersicon lycopersicum, Lycopersicon pyriforme)), sclerderm Macroptilium species (Macrotyloma spp.), Malus species (Malus spp.), recessed edge Malpighia coccigera (Malpighia emarginata), shea (Mammeaamericana), mango (Mangifera indica), cassava species (Manihot spp.), sapota (Manilkara zapota), clover (Medicago sativa), Melilotus suaveolens Ledeb. species (Melilotus spp.), Mentha species (Mentha spp.), awns (Miscanthus sinensis), Momordica species (Momordica spp.), black mulberry (Morus nigra), Musa species (Musa spp.), Nicotiana species (Nicotiana spp.), Olea species (Olea spp.), Opuntia species (Opuntiaspp.), bird foot Macroptilium species (Ornithopus spp.), Oryza (Oryza spp.) (rice for example, broad-leaved rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), Purple Granadilla (Passiflora edulis), Selinum pastinaca (Pastinaca sativa), Pennisetum species (Pennisetum sp.), Persea species (Persea spp.), celery (Petroselinum crispum), Phalaris grass (Phalaris arundinacea), Phaseolus species (Phaseolus spp.), timothy grass (Phleumpratense), thorn certain herbaceous plants with big flowers species (Phoenix spp.), south reed (Phragmites australis), Physalis species (Physalis spp.), Pinus species (Pinus spp.), Pistacia vera (Pistacia vera), Pisum species (Pisum spp.), annual bluegrass species (Poa spp.), Populus species (Populusspp.), mesquite grass species (Prosopis spp.), Prunus species (Prunus spp.), Psidium species (Psidium spp.), pomegranate (Punica granatum), European pear (Pyrus communis), oak species (Quercus spp.), radish (Raphanus sativus), rheum rhabarbarum (Rheumrhabarbarum), currant species (Ribes spp.), castor-oil plant (Ricinus communis), rubus species (Rubus spp.), saccharum species (Saccharum spp.), Salix species (Salixsp.), Sambucus species (Sambucus spp.), rye (Secale cereale), flax species (Sesamum spp.), sinapsis alba species (Sinapis sp.), Solanum (Solanum spp.) (potato (Solanum tuberosum) for example, red eggplant (Solanum integrifolium) or tomato (Solanumlycopersicum)), dichromatism chinese sorghum (Sorghum bicolor), spinach species (Spinacia spp.), Syzygium species (Syzygium spp.), Tagetes species (Tagetes spp.), tamarind (Tamarindus indica), cocoa tree (Theobroma cacao), Clover species (Trifoliumspp.), Triticosecale rimpaui, Triticum (Triticum spp.) (common wheat (Triticum aestivum) for example, durum wheat (Triticum durum), cylinder wheat (Triticumturgidum), Triticum hybernum, Macha wheat (Triticum macha) (Triticum macha), common wheat (Triticum sativum) or common wheat (Triticum vulgare)), little Flower of Chinese Globeflower (Tropaeolumminus), Flower of Chinese Globeflower (Tropaeolum majus), genus vaccinium species (Vaccinium spp.), tare species (Vicia spp.), Vigna species (Vigna spp.), sweet violet (Viola odorata), Vitis species (Vitis spp.), Zea mays, Zizania palustris, zizyphus species (Ziziphus spp.) or the like.

Detailed Description Of The Invention

HAL3 describes in detail

According to first embodiment of the present invention, the method that increases plant biomass with respect to control plant is provided, comprise the expression of nucleic acid in plant expansion tissue of preferential increase coding HAL3 polypeptide.

Any HAL3 polypeptide that means as defined herein of quoting of " protein that is used for the inventive method " after this.Any nucleic acid that means the HAL3 polypeptide like this of to encode of quoting of " nucleic acid that is used for the inventive method " after this.The nucleic acid of plant to be imported (and thereby be used for implementing the inventive method) is the proteinic arbitrary nucleic acid of the present described type of coding, hereinafter is called " HAL3 nucleic acid " or " HAL3 gene " again.

" reference ", " with reference to plant ", " contrast ", " control plant ", " wild-type " or " wild-type plant " especially such cell, tissue, organ, plant or its part, it does not produce according to the inventive method.Therefore, term " wild-type ", " contrast " or " reference " be the cell or the part that can exchange mutually and can be the plant of not modified or handling according to described the inventive method herein, as organoid or tissue or plant.Therefore, as the cell of the plant of wild-type, contrast or reference or part as organoid or plant as much as possible corresponding to cell, plant or its part, and identical as far as possible with theme of the present invention on other any characteristic except that the result of the inventive method.Therefore, wild-type, contrast or reference are handled in the same manner or as far as possible in the same manner, also promptly only so the conditioned disjunction characteristic can be different, and wherein said conditioned disjunction characteristic does not influence the quality of institute's test characteristic.In other words, this meaning wild-type refers to such plant (1), and it carries gene or allelic change or does not adjust form, or refers to that (2) are used to obtain parent material/plant of plant that the inventive method produces.

Preferably, under similar condition, carry out to any comparison between the plant that produces by the inventive method at wild-type plant.Term " similar condition " mean full terms, for example culture condition or growth conditions, analysis condition (as damping fluid composition, temperature, substrate, pathogenic agent strain system, concentration etc.) keep identical between experiment to be compared.

" reference ", " contrast " or " wild-type " preferably such object, for example organoid, cell, tissue, plant, its do not adjusted, modified according to described the inventive method herein or processing and what its characteristic in office on similar to theme of the present invention as far as possible.Reference, contrast or wild-type are similar to theme of the present invention as far as possible on its genome, transcript group, protein group or metabolite group.Preferably, term " with reference to-", the organoid of " contrast-" or " wild-type-", cell, tissue or plant relate to such organoid, cell, tissue or plant, itself and organoid of the present invention, cell, in tissue or plant or its part heredity much at one, preferred 95%, more preferably 98%, even more preferably 99.00%, especially 99.10%, 99.30%, 99.50%, 99.70%, 99.90%, 99.99%, 99.999% or higher, most preferably, " reference ", " contrast " or " wild-type " preferably such object, organoid for example, cell, tissue or plant, itself and the plant of using according to the inventive method, cell, organoid is identical in heredity, removes nucleic acid molecule or is changed according to the inventive method by the gene product of described nucleic acid molecule encoding, outside adjusting or modifying.

When contrast, with reference to or wild-type only because of not being that the theme of the inventive method is different from theme of the present invention, so can not provide under the situation of contrast, reference or wild-type, contrast, with reference to or wild-type can be such plant, wherein the reason that activity is regulated is given as described metabolite under at embodiment increases.

The increase of the activity that relates to polypeptide in cell, tissue, organoid, organ or biological or its part compared with contrast, reference or wild-type and preferably reached at least 5%, preferably at least 10% or at least 15%, especially preferably at least 20%, 25%, 30% or higher, extremely especially preferably at least 40%, 50% or 60%, most preferably be at least 70% or higher.

The biomass (weight) that term " output of increase " means one or more parts of plant increases, and described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.Preferably, the increase of generation is the output at least 10% that surpasses corresponding wild-type plant.

Especially, this type of can gather in the crops part is seed, and the enforcement of the inventive method produces the plant that has the seed production of increase with respect to the seed production of control plant.

The phenotypic character that term " expression " or " genetic expression " mean due to one or more genetic transcriptions of Yin Teding occurs.Term " expression " or " genetic expression " especially mean one or more genetic transcriptions and become structure RNA (rRNA, tRNA) or mRNA, and mRNA translates into protein subsequently.This process comprises that DNA transcribes, processes the mRNA product and the mRNA product that obtain and translates into activated protein.

The enforcement of the inventive method produces the plant with enhanced yield correlated character.Especially the enforcement of the inventive method produces with respect to control plant the plant of the seed production that has the output of increase, especially increases.In " definition " part in this article term " output " and " seed production " have been described in more detail.

In this article the reference of enhanced yield correlated character is meant biomass (weight) increase of one or more parts of plant, described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.Especially, this type of can gather in the crops part is seed, and the enforcement of the inventive method produces the plant that has the seed production of increase with respect to the seed production of suitable control plant.

With the corn is example, and the output increase can show as following one or more indexs: the increase of the increase of the per hectare or the increase of acre plant number, every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

According to preferred feature of the present invention, the enforcement of the inventive method produces the plant that has the seed production of increase with respect to control plant.Thereby, providing the method that increases seed production in the plant with respect to the seed production of control plant according to the present invention, the nucleic acid that described method comprises preferential increase coding HAL3 polypeptide is in seedling tissue, the preferably expression in the expansion tissue plantling.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of control plant, these plants might show on the corresponding stage in its life cycle increase growth velocity (its life cycle during the small part).

The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Plant with growth velocity of increase can possess short life cycle.The life cycle of plant can be considered as meaning from dry mature seed and grow to the needed time in stage that plant has produced the dry mature seed similar to parent material.This life cycle can be influenced by following factors, as early stage vigor, growth velocity, green degree index, flowering time and seed maturity speed.The increase of growth velocity can take place during life cycle on the one or more stages in life cycle or in whole plants basically plant.The growth velocity that increases during plant early stage in life cycle can reflect the enhanced vigor.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this can not (similar effect can obtain with flowering time early).If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

According to preferred feature of the present invention, the enforcement of the inventive method produces the plant that has the growth velocity of increase with respect to control plant.Thereby according to the present invention, provide with respect to the method that increases plant growth rate, described method comprises regulates the expression of nucleic acid in plant of expressing, preferably increase defined coding HAL3 polypeptide herein.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term as used in this article " non-coercing " condition is the envrionment conditions that allows the plant optimum growh.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method is with respect to the appropriate control plant of cultivating under comparable conditions, gives under the non-stress conditions or the output that increases of the plant of cultivating under slight drought condition.Thereby according to the present invention, be provided under the non-stress conditions or increase the method for output in the plant of under slight drought condition, cultivating, described method comprises that the nucleic acid that increases coding HAL3 polypeptide expresses in plant.

The enforcement of the inventive method produces with respect to the appropriate control plant of cultivating under comparable conditions, the plant of under the nutritive deficiency condition, especially cultivating under nitrogen shortage condition that increases output that has.Thereby, increasing the method for output in the plant that is provided under the nutritive deficiency condition, cultivating according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding HAL3 polypeptide.Nutritive deficiency can because of nutraceutical shortage or excessive due to, described nutrition for example is nitrogen, phosphoric acid salt and other P contained compound, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.

According to second preferred feature of the present invention, the enforcement of the inventive method produces the plant that has the plant germination gesture of increase with respect to control plant, especially early stage at development of plants (generally is to sprout 3 weeks of back, 4 weeks in the situation of rice and corn, these can be different between species and species), cause early stage vigor.Thereby, being provided for increasing the method for the early stage vigor of plant according to the present invention, described method comprises adjusting, the preferred expression of nucleic acid in plant that increases coding HAL3 polypeptide.Preferably, the increase of seedling vigor realizes by the nucleic acid that is expressed in the coding HAL3 polypeptide under the control of seedling specificity promoter.Also be provided for producing with respect to control plant and have the method for the plant of early stage vigor, described method comprises adjusting, the preferred expression of nucleic acid in plant that increases coding HAL3 polypeptide.

Early stage vigor also can produce because of plant adaptability increases, and its reason is that for example plant adapts to its environment (promptly optimizing the use of the energy and the distribution between the Miao Yugen) better.Plant with early stage vigor also shows the seedling survival of increase and better crop foundation, this often causes field piece (crop fitly grows, and promptly most of plants reach each stage of growth on the substantially the same time) and often better and higher output highly uniformly.Thereby early stage vigor can be by the multiple factor such as thousand seed weight, sprout percentage ratio, the percentage ratio of emerging, growth of seedling, seedling height, root length, root and seedling biomass and numerous other factors are determined.

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

Other favourable plant is selected from composite family (Asteraceae) as Helianthus (Helianthus), Tagetes is species Sunflower Receptacle [Sunflower Receptacle (sunflower)] for example, spiceleaf Flower of Aztec Marigold (Tagetes lucida), Flower of Aztec Marigold (Tagetes erecta) or Tagetes signata (Tagetes tenuifolia) [Marigold], Cruciferae (Brassicaceae) is as Btassica (Brassica), Arabidopsis (Arabadopsis) is the species colea for example, overgrown with weeds blue or green [canola oil dish, rape, turnip] or Arabidopis thaliana.Pulse family (Fabaceae) is as Glycine for example species soybean, soybean (Soja hispida) or soybean (Soja max) [soybean].Flax family (Linaceae) (Linaceae) is as linum (Linum) species flax [flax (flax, linseed)] for example; Gramineae such as Hordeum (Hordeum), Secale (Secale), Avena (Avena), sorghum (Sorghum), Oryza (Oryza), Zea (Zea), Triticum (Triticum) be species barley [barley] for example; Rye [rye], oat, wild avena sativa, than praising oat, Avena fatua var.sativa, hybrid oat [oat], dichromatism chinese sorghum [Chinese sorghum, grain], rice, broad-leaved rice [rice], Zea mays [corn, corn], common wheat, durum wheat, cylinder wheat, Triticum hybernum, Macha wheat (Triticum macha), common wheat (Triticumsativum) or common wheat (Triticum vulgare) [wheat, bread wheat, common wheat]; Solanaceae (Solanaceae) belongs to (Lycopersicon) for example potato [potato], tomato (Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersiconpyriforme, Solanum integrifolium or Solanum lycopersicum) [tomato (tomato)] as Solanum (Solanum), tomato.

Term " HAL3 polypeptide " refers to belong to superfamily HFCD (the homology oligomer contains the Cys decarboxylase of flavine as defined herein; Kupke J.Biol.Chem.276,27597-27604,2001) flavoprotein.These protein have flavine binding motif, conservative reactive site residue, and are tripolymer enzyme or ten dimer enzymes.HAL3 protein preferably comprises (i) substrate in conjunction with spiral, the (ii) property inserted His motif, (iii) PXMNXXMW motif and (iv) substrate identification folder (Fig. 1) from aminoterminal to carboxyl terminal.Predict that these four structural domains participate in the substrate combination, the property inserted His motif comprises the conservative His residue that participates in reactive site, and the interior sequence of substrate identification folder can be to a certain degree variable aspect sequence and length (Blaesse etc., EMBO are J.19,6299-6310,2000).Constitutional features (i)-is (iv) also described in (2001) such as Kupke, and its disclosure is incorporated herein by reference in this article.Generally, the HAL3 polypeptide can be in conjunction with the FMN cofactor.

Substrate generally is contained in the have following consensus sequence sequence motifs 1 of (SEQ ID NO:6) in conjunction with spiral:

(k/R)pr(v/I)(I/l)laa(s/T)gsva(a/S)(i/M/V)kf(g/E/A/S)(n/S/I)l(c/V/A)(h/R/G)(c/S/I)(f/L)(t/S/C)(e/D/Q)wa(e/D)v(r/K)av(v/A/S)。

The property inserted His motif, PXMNXXMW motif and substrate identification folder are the parts with sequence motifs 2 of following consensus sequence (SEQ ID NO:7):

vlhielr(r/K/Q)wad(v/I/A)(l/M)(v/I)iaplsantl(g/A)kiagg(l/M)cdnlltc(i/V)(i/V)rawd(y/F)(t/S/N/D/K)kp(l/F/M/I)f(v/A)apamnt(l/F)mw(n/s/T)npft(e/S/Q/A)(r/k)h(l/F/I)(x1)(X2)(l/i/m)(d/n/s)(e/l/q)(l/m)g(i/v/L)(t/s/a/i)l(i/v)pp(i/v/t)(k/t/s)k(r/t/k)lacgd(y/h)g(n/t)gam(a/s)e

Wherein X1 can be arbitrary amino acid, and preferably X1 is one of l, V, E, H, D, M, I or Q, and wherein X2 can be arbitrary amino acid, and preferred X2 is one of S, L, T, A or V.

These motifs form the part of a bigger conserved regions in the protein, and as an example, the conserved regions of Arabidopis thaliana HAL3a provides as SEQ ID NO:8.The HAL3 polypeptide that is used for the inventive method has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to increase progressively preferred sequence with the conserved regions of being represented by SEQ ID NO:8.

Comprise the flavoprotein structural domain that can use special database to be identified as in SEQ ID NO:8, exemplifying and comprising feature mentioned above (i) conserved regions extremely (iv) in the HAL3 protein, described database is for example SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95,5857-5864; Letunic etc. (2002) Nucleic Acids Res 30,242-244), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and the function of explaining in the automatization sequence thereof. (and) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002)).This FMN binding domains (Pfam clauses and subclauses PF02441, InterPro clauses and subclauses IPR003382) generally is present in the flavin protease.Term " structural domain " and " motif " definition in " definition " part above.

Also can use as above-described herein as the conserved regions of SEQ ID NO:8 representative among the SEQ ID NO:2 and be used for the method that aligned sequences is used for comparison and identify at other HAL3 protein.In some cases, can adjust default parameters to regulate the severity of search.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.By this way, can identify almost accurate short coupling, comprise conservative motif 1 and 2 (SEQ ID NO:6 and 7), or have the coupling that one or more conservative propertys change on any position.

HAL3 polypeptide (at least with its natural form) and their the common catalysis 4 of homologue '-decarboxylation of phosphopan tetheine halfcystine is 4 '-phosphopantetheine, this is a step in the coenzyme A biosynthesizing, and this reaction can be tested in biochemical analysis; Alternatively, the HAL3 activity can (Kupke etc. 2001 by measuring with the complementary assay of dfp mutant Escherichia coli (E.coli) bacterial strain; Yonamine etc. 2004).This enzyme also can make the pantothenylcysteine decarboxylation become pantoyl cysteamine (pantothenoylcysteamine).In addition, this protein participates in giving salt stress tolerance and osmotic stress tolerance to plant, and wherein said characteristic can be used for the bioassay method of HAL3.

(by SEQ ID NO:1 coding) SEQ ID NO:2 is the example of HAL3 polypeptide, and wherein said HAL3 polypeptide comprises following feature from aminoterminal to carboxyl terminal: (i) substrate is in conjunction with spiral, the (ii) property inserted His motif, (iii) PXMNXXMW motif and (iv) substrate identification folder; And has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with the conserved regions that increases progressively preferred sequence and SEQ ID NO:8 representative.Other example that comprises feature (i) HAL3 polypeptide extremely (iv) provides in the embodiment Table A partly hereinafter.

The homologue of HAL3 polypeptide also can be used for implementing method of the present invention.Homologue (or homologous protein) can use conventional techniques well-known in the art as identifying easily by sequence alignment.It is well-known in the art being used for the method that aligned sequences is used for comparison, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses the algorithm ((1970) J Mol Biol 48:443-453) of Needleman and Wunsch to find the comparison that makes the maximization of coupling number and make minimized two complete sequence of room number.BLAST algorithm (Altschul etc. (1990) J Mol Biol215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI obtainable.Homologue can use ClustalW multiple sequence alignment algorithm (1.83 version) for example to identify easily in acquiescence pairing comparison parameter with the methods of marking of percentage ratio.The overall percentage ratio of similarity and identity also can use one of obtainable method in the MatGAT software package and determine (Campanella etc., BMC Bioinformatics.2003 Jul 10; 4:29.MatGAT: use protein or dna sequence dna and the application that produces similarity/identity matrix).Can carry out trickle edit to optimize the comparison between the conservative motif, apparent as those skilled in the art.

Sequence identity value can be used program mentioned above, uses default parameters to determine at whole conserved domain (as indicated above) or on the nucleic acid of total length or aminoacid sequence.

Homologue also comprises directly to homologue and collateral line homologue.Directly can easily find by carrying out so-called interactivity blast search to homologue and collateral line homologue.This can be undertaken by a BLAST, and a wherein said BLAST comprises that submission search sequence (for example SEQ ID NO:1 or SEQ ID NO:2) is used for the blast search at any sequence library (as public's available ncbi database).When nucleotide sequence begins, can use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when protein sequence begins.Randomly can screen BLAST result.The full length sequence of submitting The selection result and non-The selection result subsequently to is with at carry out the blast search second time (oppositely BLAST) from the sequence of biology, wherein search sequence from described biology (be under the situation of SEQ ID NO:1 or SEQ ID NO:2 wherein in search sequence, for the second time BLAST thereby will be at arabidopsis thaliana sequence).The result who compares first and second blast searches subsequently.If the high-order position of BLAST is hit and is derived from the species identical with the species of the search sequence of deriving from the first time, then identify the collateral line homologue; If high-order position is hit and is not derived from and the identical species of search sequence deutero-species, then identify directly to homologue.Be AtHAL3a (SEQ ID NO:2) directly preferably to homologue, the straight microscler formula (SEQ IDNO:40) of AtHAL3b (SEQ ID NO:10) to homologue or AtHAL3b.It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hits by probability serendipitous low more).The calculating of E-value is well-known in the art.Under the situation of extended familys, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.Except the E-value, comparative result is also kept the score by identity percentage ratio.Identity percentage ratio refers to identical Nucleotide (or amino acid) number in the length-specific scope between two nucleic acid that compared (or polypeptide) sequence.Preferably, HAL3 homologous peptide thing with have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity or similarity (function identity) as the unmodified HAL3 polypeptide of SEQ ID NO:2 representative to increase progressively preferred sequence.Preferably, HAL3 homologous peptide thing is by the sequence representative as mentioning in the Table A.

The HAL3 polypeptide that is used for the inventive method can be the derivative of SEQ ID NO:2." derivative " comprises such peptide, oligopeptides, polypeptide, it is compared with the aminoacid sequence (as at the aminoacid sequence described in the SEQ ID NO:2) of proteinic natural existence form, comprises the interpolation of the amino-acid residue of non-natural existence to the amino-acid residue of amino acid whose displacement or non-natural existence.As the protein derivatives by listed sequence representative in the Table A is other examples that are applicable in the inventive method.

The example of the nucleic acid of coding HAL3 polypeptide includes but not limited to those nucleic acid by the sequence representative of listing in the Table A.The variant of the nucleic acid of coding HAL3 polypeptide goes in the inventive method.Suitable variant comprise coding HAL3 polypeptide nucleic acid and/or can with the part of the nucleic acid of the nucleic acid/gene recombination of coding HAL3 polypeptide.Other variant comprises the splice variant and the allelic variant of the nucleic acid of coding HAL3 polypeptide.

Term as defined herein " part " refers to the section of DNA of coded polypeptide, and wherein said polypeptide comprises following feature from aminoterminal to carboxyl terminal: (i) substrate is in conjunction with spiral, the (ii) property inserted His motif, (iii) PXMNXXMW motif and (iv) substrate identification folder; And has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to increase progressively preferred sequence with conserved regions by SEQ ID NO:8 representative.

Part can for example produce one or more disappearances by the nucleic acid to coding HAL3 polypeptide and prepare.Described part can be used or they can merge with other coding property (or non-coding) sequence with isolating form, so that for example produce in conjunction with several active protein.When merging with other encoding sequence, it is bigger that the gained polypeptide that produces during translation can be compared the polypeptide that the HAL3 part predicted.Preferably, the such polypeptide of described part coding, it has the substantially the same biologic activity of HAL3 polypeptide with SEQ ID NO:2.This part generally is at least 50,100,150 or 200 length of nucleotides, preferably at least 250,300,350 or 400 length of nucleotides, more preferably at least 450,500,550,600 or 650 length of nucleotides.

Preferably, described part is the part by the nucleic acid of the sequence representative of listing in the Table A.Most preferably, this part is the part as the nucleic acid of SEQ ID NO:1 representative.

Term " fragment ", " fragment of sequence " or " part of sequence ", " part " or " its part " mean the truncated sequence of mentioned original series.Truncated sequence (nucleotide sequence or protein sequence) can change on length significantly; Minimum size be sequence with sufficient size with at least quite function that sequence is provided mentioned original series and/or active or with of the present invention or the inventive method in used nucleic acid molecule hybridize under stringent condition, and overall dimension and non-key.In some applications, overall dimension is not more than usually basically provides the activity wanted and/or function and the size that needs to original series.Suitable function means original series at least 40%, 45% or 50%, preferred at least 60%, 70%, 80% or 90% or higher function.

The another kind of variant of nucleic acid that coding is used for the HAL3 polypeptide of the inventive method be can under the stringency that reduces, preferably under stringent condition with from as previous herein institute definition nucleic acid the nucleic acid of deutero-probe hybridization, wherein hybridization sequences is encoded and is comprised the polypeptide of following feature from aminoterminal to carboxyl terminal: (i) substrate in conjunction with spiral, (ii) the property inserted His motif, (iii) the PXMNXXMW motif and (iv) substrate discern and press from both sides; And has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to increase progressively preferred sequence with conserved regions by SEQ ID NO:8 representative.

Preferably, hybridization sequences be can with as by the nucleic acid of the sequence representative of listing in the Table A (or with from wherein deutero-probe) or with the sequence of the part hybridization of any these sequences (target sequence).Most preferably, hybridization sequences can with SEQ ID NO:1 (or with from wherein deutero-probe) hybridization.Probe usually less than 700bp or 600bp length, preferably less than 500,400bp, 300bp, 200bp or 100bp length.Usually, the probe length that is used for DNA-DNA hybridization (as southern blotting technique) 100 and 50bp between change, and the hybridization zone in the probe that is used for DNA-DNA hybridization (as at pcr amplification) is less than 50 Nucleotide usually but surpass 10 length of nucleotides, and they are preferably 15,20,25,30,35,40,45 or 50bp length years old.

The HAL3 polypeptide can be encoded by splice variant.Term as used in this article " splice variant " comprise the variant of nucleotide sequence like this, in described nucleotide sequence, downcut, replace, be shifted or add selected intron and/or exon, or wherein intron is shortened or extends.This type of variant will be the variant of the basic biologic activity of retaining protein wherein, and this can realize by the functional section of retaining protein optionally.This type of splice variant can find or can manually produce at occurring in nature.The method that is used to produce this type of splice variant is well-known in the art.

Preferred splice variant is the splice variant of the nucleic acid of coding HAL3 polypeptide, and wherein said HAL3 polypeptide comprises (i) substrate in conjunction with spiral, the (ii) property inserted His motif, (iii) PXMNXXMW motif and (iv) substrate identification folder from aminoterminal to carboxyl terminal; And has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to increase progressively preferred sequence with conserved regions by SEQ ID NO:8 representative.

It is splice variant as the nucleic acid of any sequence representative of listing in the Table A that coding comprises as other preferred splice variant of the nucleic acid of the HAL3 polypeptide of above institute defined feature herein.It most preferably is splice variant as the nucleotide sequence of SEQ ID NO:1 representative.

The HAL3 polypeptide also can be by the allelic variant of nucleic acid encoding coding, and wherein said polypeptide comprises (i) substrate in conjunction with spiral, the (ii) property inserted His motif, (iii) PXMNXXMW motif and (iv) substrate identification folder from aminoterminal to carboxyl terminal; And has at least 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to increase progressively preferred sequence with conserved regions by SEQ ID NO:8 representative.

It is allelic variant as the nucleic acid of any sequence representative of listing in the Table A that coding comprises as the preferred allelic variant of the nucleic acid of the HAL3 polypeptide of above institute defined feature herein.It most preferably is allelic variant as the nucleotide sequence of SEQ ID NO:1 representative.

Orthogenesis (or gene reorganization effect) also can be used for producing the variant of the nucleic acid of coding HAL3 polypeptide.Site-directed mutagenesis can be used for producing the variant of the nucleic acid of coding HAL3 polypeptide.Several method can be used for realizing site-directed mutagenesis, the modal method (Current that is based on PCR

Protocols in Molecular Biology.Wiley edits).

The nucleic acid of coding HAL3 polypeptide can be from any natural source or artificial source.Nucleic acid can have a mind to operate and modified by the mankind with regard to composition and/or genome environment aspect according to its natural form.HAL3 nucleic acid is preferably from plant, and also preferably from dicotyledons, more preferably from Cruciferae, this nucleic acid is most preferably from Arabidopis thaliana.

The expression of the increase of the nucleic acid of coding HAL3 polypeptide, the expression that preferentially increases in plantling can be implemented by importing genetic modification (preferably in the locus of HAL3 gene).The locus of gene as defined herein means such genome area, and it comprises goal gene and upstream of coding region or downstream 10KB.

Genetic modification can be for example by the following method any (multiple) import: T-DNA activation method, TILLING and homologous recombination method (as in " definition " part, describing) or by introduce and the preferential expansion tissue of nucleic acid that increases the HAL3 polypeptide of encoding plant in express and importing.Behind the importing genetic modification, carry out optional step subsequently, promptly select the preferentially expression of increase in the expansion tissue of nucleic acid of coding HAL3 factor polypeptide, wherein the expression that increases produces the plant of the output with increase.

T-DNA activates the label generation shows the dominant phenotype because of the improvement of the gene of the close promotor that imports transgenic plant.Promotor to be imported can be the preferential arbitrary promotor of expression in the expansion tissue of plant of nucleic acid that can increase coding HAL3 polypeptide.

Also can use TILLING (genome interior orientation inductive local damage) technology in the locus of the gene of coding HAL3 polypeptide, to import genetic modification.The plant of carrying this type of mutation variants preferentially has the expression of increase of the nucleic acid of coding HAL3 polypeptide in the distensibility plant tissue.

T-DNA activation method and TILLING are the technical examples that can produce genetic modification (nucleic acid that comprises preferential increase coding HAL3 polypeptide is expressed) in the expansion tissue of plant.

Effect of the present invention also can use homologous recombination method to reproduce.The nucleic acid for the treatment of target is preferably controlled the zone of nucleic acid natural expression in plant of coding HAL3 polypeptide.Weak promoter for expression specificity in the seedling is imported into this zone, partly replace this zone or basically wholly replace it.

The preferred method that is used for importing genetic modification (described genetic modification does not need to be positioned at the locus of HAL3 gene in this case) is to import and at the preferential coding as the nucleic acid of HAL3 polypeptide defined above herein of expressing of plantling.The nucleic acid of plant to be imported can be that total length nucleic acid maybe can be as part or the hybridization sequences or the another kind of nucleic acid variant of aforementioned definitions herein.

The inventive method depends on the expression that the nucleic acid of coding HAL3 polypeptide preferentially in the plantling tissue, preferably increases in the cell spreading belt of nutrition branch.

The present invention also provides genetic constructs and carrier to promote to import and/or in seedling, preferably preferential expression is used for the nucleotide sequence of the inventive method in the expansion tissue of plantling.

Thereby, gene construct is provided, it comprises:

The nucleic acid of coding HAL3 polypeptide as hereinbefore defined;

With one or more regulating and controlling sequences that the nucleic acid of (i) effectively is connected, wherein at least a is the seedling specificity promoter.

Used construct can use the well-known recombinant DNA technology of those skilled in the art to make up in the inventive method.This gene construct can insert the carrier that can be purchased that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed.The present invention thereby the purposes in the methods of the invention of gene construct as hereinbefore defined is provided.

Plant transforms with the carrier that comprises aim sequence (nucleic acid of the HAL3 polypeptide of promptly encoding).The technician is appreciated that successfully and transforms, selects and breed the host cell that contains aim sequence and the genetic elements that must exist very much on carrier.Aim sequence effectively is connected with one or more regulating and controlling sequences (at least with promotor).

The common known suitable promotor that function is arranged in plant.They can take the form of composing type or inducible promoter.Suitable promotor can make development-specific and/or the tissue specific expression in the many cells eukaryote become possibility; Therefore can in plant, advantageously use leaf-, root-, flower-, seed-, pore-, stem tuber-or fruit-specific promoter.

The different plant promoters that can be used in the plant are such promotors, for example USP, LegB4-, DC3 promotor or from the ubiquitin promotor of celery (parsley).

For expressing in plant, as mentioned above, nucleic acid molecule must effectively be connected or comprise this promotor with suitable promotor, and this promotor is expressed gene on correct time point and in cell or tissue specificity mode.The available promotor is constitutive promoter (Benfey etc., EMBO is (1989) 2195-2202 J.8), those constitutive promoters as plant-derived virus, as 35SCAMV (Franck etc., Cell 21 (1980) 285-294), 19S CaMV (seeing also US 5352605 and WO 84/02913), 34S FMV (Sanger etc., Plant.Mol.Biol., 14,1990:433-443), celery ubiquitin promotor or as at US 4,962, the plant promoter such as Rubisco small subunit promotor or the plant promoter PRP1[Ward etc. that describe in 028, Plant.Mol.Biol.22 (1993)], SSU, PGEL1, OCS[Leisner (1988) Proc Natl Acad Sci USA85 (5): 2553-2557], lib4, usp, mas[Comai (1990) Plant Mol Biol 15 (3): 373-381], STLS1, ScBV (Schenk (1999) Plant Mol Biol 39 (6): 1221-1230), B33, SAD1 or SAD2 (flax promotor, ain etc., Crop Science, 39 (6), 1999:1696-1701) or (1984) Nucleic Acids Res.12 (20): 7831-7846 such as nos[Shaw].Other example of constitutive plant promoters is a beet V-ATP enzyme promotor (WO 01/14572).Synthetic constitutive promoter example be super promotor (WO 95/14098) and from the G-box deutero-promotor (WO 94/12015).As required, can also further use chemical inducible promoter, relatively EP-A 388186, EP-A 335528, WO 97/06268.The stable constitutive expression of protein of the present invention in plant can be favourable.Yet if express the late period before results is favourable, and the inducible expression of polypeptide of the present invention is favourable, because the metabolic operation can cause plant-growth slow.

Also can by chemical inducible promoter promote plant gene expression (summary is seen Gatz1997, Annu.Rev.Plant Physiol.Plant Mol.Biol., 48:89-108).When needs during with temporal mode expressing gene, chemical inducible promoter is a particularly suitable.The example of this type of promotor is salicylic acid inducible promotor (WO 95/19443) and dormin-inducible promoter (EP 335 528), tsiklomitsin-inducible promoter (Gatz etc. (1992) Plant J.2,397-404), hexalin-or ethanol-inducible promoter (WO 93/21334) or other chemical inducible promoter as described herein.

Other suitable promotor is to coerce or those promotors of inanimate stress conditions reaction biological, pathogen-inducible PRP1 gene promoter (Ward etc. for example, Plant.Mol.Biol.22 (1993) 361-366), (US 5 for tomato thermal induction type hsp80 promotor, 187,267), cold induction type α-Dian Fenmei promotor (WO 96/12814) of potato or wound induction type pinII promotor (EP-A-0375 091) or other promotor as described herein.

Preferred promotor especially in tissue and organ, at seed cell such as albuminous cell and grow those promotors that cause genetic expression in the cell of embryo.Suitable promotor is that (US 5 for the rapeseed protein gene promoter of rape, 608,152), broad bean (Vicia faba) USP promotor (Baeumlein etc., Mol Gen Genet, 1991,225 (3): 459-67), Arabidopis thaliana oleosin promotor (WO98/45461), (US 5 for Kidney bean (Phaseolus vulgaris) phaseolin promoter, 504,200), Brassica plants (Brassica) Bce4 promotor (WO 91/13980), beans arc5 promotor, Radix Dauci Sativae DcG3 promotor or leguminous plants (Legumin) B4 promotor (LeB4; Baeumlein etc., 1992, Plant Journal, 2 (2): 233-9) and cause the promotor of the seed-specific expression in monocotyledons such as corn, barley, wheat, rye, rice etc.Favourable seed specific promoters is sucrose-binding proteins promotor (WO 00/26388), phaseolin promoter and rapeseed protein promotor.The suitable promotor that must take in is that barley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230) and the promotor described in WO 99/16890 are (from the hordein gene of barley, the glutenin gene of rice, the paddy rice plain gene of rice, the prolamine gene of rice, the gliadine gene of wheat, the glutenin gene of wheat, the zein spirit-soluble gene of corn, the avenaceous glutenin gene, the promotor of the secalin gene of Chinese sorghum kasirin gene and rye).Other suitable promotor is Amy32b, Amy 6-6 and Aleurain[US 5,677,474], [US 5 for Bce4 (rape), 530,149], glycinin (soybean) [EP 571 741], phosphoric acid enol pyruvic acid carboxylase (soybean) [JP 06/62870], ADR12-2 (soybean) [WO 98/08962], [US 5 for isocitrate lyase (rape), 689,040] or α-Dian Fenmei (barley) [EP 781 849].Be used in that other promotor of expressing gene is the leaf specificity promoter in the plant, regulate promotor as, pea petE promotor for example as those promotors of in DE-A 19644478, describing or light.

Other suitable plant promoter is cytosol FBP enzyme promotor or potato ST-LSI promotor (Stockhaus etc., EMBO J.8,1989,2445), the joint specificity promoter of soybean Phosphoribosyl tetra-sodium amide transferase promotor (GenBank accession number U87999) or description in EP-A-0 249 676.

In preferred embodiments, the nucleotide sequence of coding HAL3 polypeptide effectively is connected with the seedling specificity promoter.The seedling specificity promoter refers to arbitrary promotor of can the driving purposes gene expressing in plant nutrition branch tissue.Preferably, the seedling specificity promoter can the variety of priority driven goal gene in the expansion tissue of nutrition branch, promptly in the cell spreading belt of nutrition branch, express.In this article " preferentially " driven quoting to mean to drive and expressing in the cell spreading belt of nutrition branch with arbitrary sequence that the seedling specificity promoter effectively is connected of expressing in seedling, express to such an extent as to get rid of basically in plant, to drive in other position, except any residual expression due to the property revealed promoter expression.The seedling specificity promoter can natural promoter or synthetic promotor.

Preferably, the seedling specificity promoter is an isolating promotor from β-expansion protein gene, as rice β expansion albumen EXBP9 promotor (WO 2004/070039), represent as SEQ ID NO:5, or have the promotor of similar intensity and/or have the promotor (being the promotor of function equivalent) of similar expression pattern to rice β expansion protein promoter to rice β expansion protein promoter.

Be understood that suitability of the present invention is not limited to encode by the nucleic acid of the HAL3 polypeptide of SEQ ID NO:1 representative, the expression of nucleic acids of coding HAL3 polypeptide when suitability of the present invention also is not limited to be driven by β expansion protein promoter simultaneously.

Randomly, one or more terminator sequences (also being regulating and controlling sequence) can be used in the construct that imports plant.Extra regulatory element can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.One skilled in the art will recognize that or can obtain this type of sequence easily.

Genetic constructs of the present invention can also comprise need be used for the replication sequence starting point of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.

Other regulating and controlling sequence (except that promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stable element.

For detecting and/or selecting as describe the successful transfer that reaches the nucleotide sequence that uses in the methods of the invention in sequence solution, applying marking gene (=reporter gene) is favourable.These marker gene can be identified the successful transfer of nucleic acid molecule by a series of different principle, for example by fluorescence, luminous or in the noticeable light wavelength scope of eye by range estimation identify, by Herbicid resistant or antibiotic resistance, by so-called nutrition mark (auxotrophy mark) or anti-trophicity mark, by enzyme assay or pass through plant hormone.The example of this type of mark that can mention is GFP (=green fluorescent protein); Luciferin/luciferase system, beta-galactosidase enzymes substrate coloured with it be X-Gal for example, to for example imidazolone, glyphosate, the Herbicid resistant of phosphinothricin or sulfourea, to for example bleomycin, Totomycin, Streptomycin sulphate, kantlex, tsiklomitsin, paraxin, penbritin, gentamicin, Geneticin (G418), the antibiotic resistance of spectinomycin or blasticidin etc., the nutrition mark as to the utilization of seminose or wood sugar or anti-trophicity mark as resistance to the 2-deoxyglucose.This list is only represented the possible mark of minority.The technician is familiar with this type of mark very much.Depend on biology and system of selection, preferred different mark.

The present invention also comprises by the obtainable plant of the inventive method.The present invention thereby provide by the obtainable plant of the inventive method, plant part or its vegetable cell, wherein plant or its part or cell are included in the nucleic acid transgenosis of the coding HAL3 polypeptide under the control of seedling specificity promoter.

The present invention also is provided for producing the method that has the transgenic plant that increase output with respect to control plant, is included in to import in the plantling and preferential nucleic acid of expressing coding HAL3 polypeptide.

Host plant advantageously can synthesize all plants of used polypeptide in the inventive method in principle for nucleic acid used in the inventive method or carrier, expression cassette or construct or carrier.

More specifically, the invention provides the method that is used to produce the transgenic plant with increase output, described method comprises:

In plantling, import and preferential nucleic acid of expressing coding HAL3 polypeptide; With

Cell cultivates plants under the condition that promotes plant-growth and growth.

Nucleic acid can directly import vegetable cell or import plant itself (comprising any other parts that import tissue, organ or plant).According to preferred feature of the present invention, nucleic acid preferably imports plant by transforming.

Alien gene to the transfer in the Plant Genome is called conversion.When transforming, be used for transforming and be used to instantaneous conversion or stable conversion from plant tissue or vegetable cell regenerate the described method of plant.In order to select plant transformed, the vegetable material that obtains in conversion is accepted selection condition in principle and is handled, to such an extent as to plant transformed can be distinguished with unconverted plant.For example, can sow with the seed that mode mentioned above obtains, after date when initial the cultivation carries out suitable selection by spraying.Another possibility is included in the seed of growing on the agar plate that uses suitable selective agent (as required after sterilization), makes the seed that only transforms can grow into plant.Other favourable method for transformation (being particularly useful for plant) is known to the skilled and describes hereinafter.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the effable genes encoding of the plant that moves with the goal gene corotation.

As mentioned, the Agrobacterium that transforms with expression vector of the present invention also can be used to transform plant in a manner known way as testing plant for example Arabidopis thaliana or crop plants, such as grain, corn, oat, rye, barley, wheat, soybean, rice, cotton, beet, rape, Sunflower Receptacle, flax (flax), hemp (hemp), potato, tobacco, tomato, Radix Dauci Sativae, sweetbell redpepper (bell pepper), rape, cassava (tapioca, cassava), arrowroot (arrow root), Flower of Aztec Marigold, clover, lettuce (lettuce) and multiple tree, nut plant and vine species, particularly butyraceous crop plants such as soybean, peanut (peanut), castor-oil plant (castor-oil Plant), Sunflower Receptacle, corn, cotton, flax, rape, coconut, oil palm, safflower or cocoa beans are for example by soaking abrasive leaf or leaf section and cultivating them subsequently in suitable culture medium in Agrobacterium solution.

Except transformant cell (the necessary subsequently complete plant of regeneration), also might transform the merismatic cell of plant and special those cells that develop into gamete that transform.In this case, the gamete of conversion is followed natural development of plants process, produces transgenic plant.Therefore, for example the Arabidopis thaliana seed is handled with Agrobacterium and obtain seed from is grown plant, and wherein a certain proportion of described plant is transformed and is genetically modified [Feldman, KA and Marks MD (1987) Mol GenGenet 208:274-289 therefore; Feldmann K (1992).: editor C Koncz, N-H Chua and J Shell, Methods in Arabidopsis Research.Word Scientific, Singapore, 274-289 page or leaf].Alternative method based on remove inflorescence repeatedly and make in the rosette in the heart the excision position and the Agrobacterium incubation of conversion, thereby the seed that transforms can obtain at later time point equally, and (Chang (1994) Plant is J.5:551-558; Katavic (1994) Mol Gen Genet, 245:363-370).Yet especially effective means is the vacuum infiltration method of improvement, as " flower is contaminated " method.Under the situation of Arabidopis thaliana vacuum infiltration method, complete plant is under reduced pressure handled [Bechthold, N (1993) with the Agrobacterium suspension.C R Acad Sci Paris Life Sci, 316:1194-1199], and under the situation of " flower dip method ", of short duration incubation [the Clough of Agrobacterium suspension that the flower tissue and the tensio-active agent of growing handled, SJ and Bent, AF (1998) The Plant J.16,735-743].Gathered in the crops a certain proportion of transgenic seed in both cases, and these seeds can be distinguished by under aforesaid selection condition, cultivating with the non-transgenic seed.In addition, the stable conversion of plastid is favourable because plastid in most of crop with the heredity of parent mode, reduce or eliminated transgenosis through the pollen flow risk.The conversion of chloroplast gene group generally by at Klaus etc., 2004[Nature Biotechnology 22 (2), 225-229] in the exemplary method realization of being showed.In brief, sequence to be transformed be cloned into together with selectable marker gene and chloroplast gene group homologous flanking sequence between.These homologous flanking sequences instruct locus specificity to be integrated in the plastom(e).Numerous different plant species having been described plastid transforms and summarizes and can come from Bock (2001) transgenosis plastid (Transgenic plastids in basicresearch and plant biotechnology) .J Mol Biol.2001 September 21 in fundamental research and Plant Biotechnology; 312 (3): 425-38 or Maliga, P (2003) plastid transformation technology commercialization progress (Progress towardscommercialization of plastid transformation technology) .TrendsBiotechnol.21,20-28.Further the biotechnology progress has been made report with the form of unmarked plastid transformant recently, described unmarked plastid transformant can produce (Klaus etc. by the instantaneous marker gene of integrating altogether, 2004, Nature Biotechnology 22 (2), 225-229).

Can the regenerate vegetable cell of genetic modification of all methods of being familiar with by the technician.Suitable method can be seen S.D.Kung and R.Wu, Potrykus or Above-mentioned publication with Willmitzer.

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze or quantitative PCR is monitored, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clonal expansion method or classical breeding technique.For example, the first-generation (or T1) transforms plant can carry out the s-generation (or T2) transformant that selfing is isozygotied with generation, and the T2 plant further breeds by classical breeding technique.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's transformant (for example being transformed) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the root stock of the conversion of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring show with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also comprises host cell, and it contains the isolating nucleic acid of coding HAL3 polypeptide.The preferred host cell of the present invention is a vegetable cell.

The present invention also extend to plant the part gathered in the crops as, but be not limited to seed, leaf, fruit, flower, stem, rhizome, stem tuber and bulb.The invention further relates to derived from, preferred directly derived from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises the purposes of nucleic acid of coding HAL3 polypeptide and the purposes of HAL3 polypeptide, is used for increasing in the methods of the invention plant biomass as hereinbefore defined.

The nucleic acid or the HAL3 polypeptide of coding HAL3 polypeptide can be used for the procedure of breeding, wherein identify the dna marker that can be connected with the HAL3 gene hereditarily.Described nucleic acid/gene or HAL3 polypeptide can be used for defining molecule marker.This DNA or protein labeling can be used for selecting to have in the inventive method the plant of increase output as hereinbefore defined subsequently in the procedure of breeding.

The allelic variant of HAL3 nucleic acid/gene also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to import allelic variation by for example using the EMS mutagenesis that plant is made mutagenic treatment sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be used to select discuss and cause increasing the step of excellent allelic variant of the sequence of output.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises and will identify the plant and the another kind of plant hybridization of excellent allelic variant.This can be used for for example producing target phenotype combination of features.

The nucleic acid of coding HAL3 polypeptide also can be as probe so that carry out genetic mapping or physical mapping to gene, and described probe reaches the mark of the proterties related with these genes as the part of described gene.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.This purposes of HAL3 nucleic acid only needs to have the nucleotide sequence of at least 15 length of nucleotides.HAL3 nucleic acid can be used as restriction fragment length polymorphism (RFLP) mark.The Southern trace of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) MolecularCloning, A Laboratory Manual) can be used the HAL3 nuclei acid probe.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the Southern trace of the genomic dna that contains one group of individuality handling through restriction endonuclease, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for calculating the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of HAL3 nucleic acid in using the previous genetic map that obtains of this colony.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter 4:37-41.Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be can be used for mapping with other population of individuals to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics 16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces has the plant that increases output as previously described.The output of this increase also can with other economic favourable proterties combination, strengthen proterties, other inanimate coerced and biological tolerance of coercing, the proterties of regulating multiple constructivity feature and/or biochemical characteristics and/or physiologic character as other output.

The detailed description of the MADS15 that raises

Now be surprised to find the expression of nucleic acid in plant of regulating coding MADS15 polypeptide and produced the plant that has the enhanced yield correlated character with respect to control plant.Describe the MADS15 polypeptide of the certain kinds that is suitable for strengthening output correlated character in the plant hereinafter in detail.

The invention provides the method that strengthens output correlated character in the plant with respect to control plant, it comprises the expression of nucleic acid in plant of regulating coding MADS15 polypeptide.

Any MADS15 polypeptide that means as defined herein of quoting of " protein that is used for the inventive method " after this.Any nucleic acid that means this MADS15 polypeptide of to encode of quoting of " nucleic acid that is used for the inventive method " after this.

Be used for regulating preferred method that (preferred increasing) coding is used for the proteinic expression of nucleic acid of the inventive method and be plant and import and express coding as the defined proteinic nucleic acid that is used for the inventive method hereinafter.

The nucleic acid of plant to be imported (and thereby be used for implementing the inventive method) is the proteinic arbitrary nucleic acid of the present described type of coding, hereinafter is called " MADS15 nucleic acid " or " MADS15 gene " again.

Term as defined herein " MADS15 polypeptide " refers to belong to as (J.Mol.E the 62nd volume, the 15-31) protein of the group of illustrated FUL sample MADS box protein matter such as Adam.FUL sample MADS box protein matter is the part of SQUAMOSA subfamily and has typical MIKCc structure: at N-terminal MADS structural domain (M), then be participate in dimerization interleave structural domain (I), the variable domains at carboxyl terminal (C) of the Keratin sulfate structural domain (K) of high conservative and responsible binding interactions protein or transcriptional activation (Trends in Plant Science 8 such as De Bodt, 475-483).

Plant MADS15 polypeptide also can obtain because of the existence of some conservative motif identifying.The existence of these conservative motifs can be used and as indicated above be used for the method that aligned sequences is used for comparison and identify.In some cases, can adjust default parameters to regulate the severity of search.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.Like this, can identify almost accurate short coupling.In case identified the MADS15 polypeptide by the existence of these motifs, those skilled in the art can easily obtain the corresponding nucleic that encoded packets contains the polypeptide of relevant motif, and use the continuous nucleotide of the sufficient length of this nucleic acid to implement any or multiple gene silencing methods mentioned above (being used for reducing or removing substantially endogenous MADS15 genetic expression).

Usually, motif 1 to 4 existence one of at least should be enough to identify that arbitrary search sequence is MADS15, yet preferably has motif 1 and 2 at least.The monocotyledons sequence of the consensus sequence that is provided to provide in the sequence table.Those skilled in the art understand fully aware of if (for example from dicotyledons) other sequence or different sequence are used for comparison, and then can there be certain variation in consensus sequence.

Be positioned at the motif 1 (SEQ ID NO:48) of MADS structural domain:

L(L/V)KKA(H/N)EIS(VI)L(C/Y)DAE(V/I/L)(A/G)(L/A/V)(I/V)(I/V)FS(T/P/A/N)KGKLYE(Y/F)(A/S)(T/S)(D/N/E)S(K/C/R/S)M(D/E)(N/I/R/K)IL(E/D)R。

Preferably, this conserved sequence motif 1 has following sequence:

LLKKAHEISVLCDAEVA(L/A/V)I(I/V)FS(T/P)KGKLYEYATDS(C/R)M(D/E)(R/K)ILER，

Most preferably, conserved sequence motif 1 has following sequence:

LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILER

Be positioned at the motif 2 (SEQ ID NO:49) of K structural domain:

KLK(A/S)(K/R)(V/I)E(A/T/S)(L/I)(Q/N)(K/R/N)(S/C/R)(Q/H)(R/K)HLMGE

Preferably, this conserved sequence motif 2 has following sequence:

KLKAK(V/I)E(A/T)(L/I)QK(S/C)(Q/H)(R/K)HLMGE

More preferably, this conserved sequence motif 2 has following sequence:

KLKAKIETIQKCHKHLMGE

Randomly, MADS15 polypeptide or its homologue can comprise motif 3 and/or motif 4 in the C-structure territory:

Motif 3 (SEQ ID NO:50):

Q(P/Q/V/A)QTS(S/F)(S/F)(S/F)(S/F)(S/C/F)(F/M)

Motif 4 (SEQ ID NO:51):

(G/A/V/L)(L/P)XWMX(S/H)

Wherein the X residue in motif 4 can be arbitrary amino acid, but preferably L, P or H; And wherein the 2nd X residue can be arbitrary amino acid, but preferably V or L.

Alternatively, the C-structural domain (begins behind the Keratin sulfate structural domain, i.e. R175 place in SEQ ID NO:44) can be feature (normally 3.93% with the glutamine occurrence rate that increases, here be 8.77% at least, maximum 26.73%), in addition, the content of L-Ala, proline(Pro) and/or Serine also can increase (being higher than 7.8%, 4.85% and 6.89% respectively).

That propose, that be used for the inventive method in addition preferred MADS15 protein comprises the aminoterminal MADS structural domain corresponding to SMART structural domain SM00432 (Pfam PF00319) at least, be subsequently with Pfam in be defined as the corresponding Keratin sulfate structural domain in K box district of PF01486, more preferably, MADS15 protein has conservative label SEQ ID NO:48 and have conservative label SEQ ID NO:49 in its K-structural domain in its MADS structural domain, most preferably, MADS15 protein has the sequence that provides in SEQ ID NO:44.

Protein that is used for the inventive method and the example of encoding this proteinic nucleic acid have been provided hereinafter among the table D of embodiment 8.

What also be used for the inventive method is the homologue of any aminoacid sequence of providing among the D at table.

Also be used for the inventive method be the derivative of any polypeptide of providing among the D at table or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue.Preferred derivative is the derivative of SEQ IDNO:44.The derivative of given polypeptide is the other example that can be suitable in the methods of the invention among the table D.The derivative that is used for the inventive method preferably have to described derivative from similar biologic activity and the functionally active of the non-modifying protein of deutero-wherein.

The present invention transforms plant by the rice nucleotide sequence of using the coded polypeptide sequence SEQ ID NO:44 that is represented by SEQ ID NO:43 and is illustrated, yet enforcement of the present invention is not limited to these sequences.The inventive method can use coding to be used for the inventive method proteinic any nucleic acid as defined herein and advantageously to carry out, and described nucleic acid comprises directly to homologue and collateral line homologue, as the arbitrary nucleotide sequence that provides in table D.The aminoacid sequence that provides among the D at table can be considered as by the MADS15 polypeptide of SEQ IDNO:44 representative directly to homologue and collateral line homologue.

Directly can easily find by carrying out so-called interactivity blast search to homologue and collateral line homologue.This is usually directed to a BLAST, and a wherein said BLAST comprises that submission search sequence (for example arbitrary sequence of listing among the use table D) is used for the blast search at arbitrary sequence library (as public's available ncbi database).When nucleotide sequence begins, generally use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when protein sequence begins.Randomly can screen BLAST result.The full length sequence of submitting The selection result and non-The selection result subsequently to is with at carry out reverse BLAST (the 2nd BLAST) from the sequence of biology, wherein search sequence is from described biology (be under the situation of SEQ ID NO:43 or SEQ ID NO:44 in search sequence wherein, the 2nd BLAST thereby will be at the rice sequence).The result who compares first and second blast searches subsequently.If from the high-order position of a BLAST hit be derived from search sequence from identical species of deutero-species wherein, oppositely BLAST produces search sequence subsequently ideally as the highest hitting, and then identifies the collateral line homologue; If the high-order position among the BLAST hit be not derived from search sequence from identical species of deutero-species wherein, and preferably when reverse BLAST, produce the search sequence that belongs to the highest row that hit, then identify directly to homologue.

It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hit because of the chance odds low more).The calculating of E-value is well-known in the art.Except the E-value, comparative result is also kept the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.Under the situation of large-scale family, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.

Table D provides the proteinic straight example to homologue and collateral line homologue by the MADS15 of SEQ ID NO 44 representatives.Other directly can use BLAST method mentioned above and identify easily to homologue and collateral line homologue.

Protein of the present invention is because of the existence of conservative MADS structural domain and/or Keratin sulfate structural domain but appraisable (showing among Fig. 5).There is the specialized database that is used to identify structural domain, as SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95,5857-5864; Letunic etc. (2002) Nucleic Acids Res 30,242-244), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and in the function of automatization sequence interpretation. (and) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002)).The one group of instrument that is used for analysing protein sequence on the computer chip can obtain (to have (Gasteiger etc. by Switzerland information biology institute on ExPASY proteomics server, ExPASy: be used for the protein science server of deep understanding and analysing protein, Nucleic Acids Res.31:3784-3788 (2003)).

Structural domain also can use routine techniques as identifying by sequence alignment.It is well-known in the art being used for the method that aligned sequences is used for comparison, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses Needleman and Wunsch algorithm ((1970) J Mol Biol 48:443-453) to find (promptly covering complete sequence) the of overall importance comparison that makes the maximization of coupling number and make minimized two complete sequence of room number.BLAST algorithm (Altschul etc. (1990) JMol Biol 215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI (NCBI) obtainable.Homologue can use for example ClustalW multiple sequence alignment algorithm (1.83 version) evaluation easily to give tacit consent to pairing comparison parameter and methods of marking (with percentage ratio).The overall percentage ratio of similarity and identity also can use one of obtainable method in the MatGAT software package and determine (Campanella etc., BMC Bioinformatics.2003 Jul 10; 4:29.MatGAT: with protein or dna sequence dna and the application that produces similarity/identity matrix).Can carry out trickle edit to optimize the comparison between the conservative motif, apparent as those skilled in the art.In addition as using full length sequence, also can use ad hoc structure territory (as MADS structural domain or Keratin sulfate structural domain or one of defined motif above) to identify substituting of homologue.Use program mentioned above, use default parameters, measured the sequence identity value that is shown as percentage ratio among the embodiment 10 hereinafter at whole nucleotide sequence or aminoacid sequence and/or at the structural domain of selecting or conservative motif.

In addition, MADS15 protein also can because of can or can not in conjunction with DNA and with other protein interaction but appraisable.Dna binding activity and protein-protein interaction can use technology well-known in the art and easily external or determine in vivo.The example that is used for the external test method of dna binding activity comprises: gel retardation assay (West etc. (1998) the Nucl Acid Res 26 (23): 5277-87), or the yeast one-hybrid analysis that uses known MADS box DNA binding domains.The example that is used for the external test method of protein-protein interaction is yeast two-hybrid analysis (Fields and Song (1989) Nature 340:245-6).Known and OsMADS15 interacting proteins comprise other MADS protein (as those MADS protein of the signal effect complex body that participates in blooming), receptor-like kinase enzyme such as Clavata1 and Clavata2, Erecta, BRI1 or RSK.Other details provides in embodiment 13.

It is total length nucleic acid that the proteinic nucleic acid that coding is used for the inventive method need not, and uses the total length nucleotide sequence because the enforcement of the inventive method does not rely on.The nucleic acid example that is suitable in implementing the inventive method is included in the nucleotide sequence that provides among the table D, but is not limited to these sequences.The nucleic acid variant also can be used to put into practice the inventive method.The example of nucleic acid variant comprise used proteinic nucleic acid in the code book inventive method part, with the code book inventive method in the variant of used proteinic nucleic acid in the allelic variant of used proteinic nucleic acid and the code book inventive method that obtains by gene reorganization effect in the splice variant, code book inventive method of used proteinic nucleic acid in the nucleic acid, code book inventive method of used proteinic nucleic acid hybridization.Term " partly, hybridization sequences, splice variant, allelic variant and gene reorganization " will be described now.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, the part of the straight nucleic acid to homologue, collateral line homologue or homologue that is included in the plant any aminoacid sequence of providing among the part that imports and be expressed in any nucleotide sequence that provides among the D or the coding schedule D.

The part coding that is used for the inventive method belongs to the polypeptide of the range of definition of nucleic acid, wherein said nucleic acid encoding be used for the inventive method and have with show D in the protein of the identical biologic activity of the aminoacid sequence that provides.Preferably, this part is the part of any nucleic acid of providing among the D at table.This part generally has at least 400 continuous nucleotide length, preferably at least 600 continuous nucleotide length, more preferably at least 700 continuous nucleotide length and at least 800 continuous nucleotide length most preferably, and wherein said continuous nucleotide is any nucleotide sequence that provides in table D.Most preferably, this part is the part of nucleic acid SEQ ID NO:43.Preferably, this part coding comprise as defined herein (any or multiple) MADS structural domain and the aminoacid sequence of Keratin sulfate structural domain.

The part of the coding proteinic nucleic acid of MADS15 as defined herein can for example prepare by this nucleic acid is produced one or more disappearances.Described part can be used or they can merge with other coding property (or non-coding) sequence with isolating form, so that for example produce several active protein of combination.When merging with other encoding sequence, it is bigger that the gained polypeptide that produces during translation can be compared the polypeptide that the MADS15 protein portion predicted.

The another kind of nucleic acid variant that is used for the inventive method is can be under the stringency that reduces, preferably under stringent condition with the coding proteinic nucleic acid hybridization of MADS15 as defined herein, or with the nucleic acid of as defined herein part hybridization.

The polypeptide that the hybridization sequences coding that is used for the inventive method has MADS structural domain and/or Keratin sulfate structural domain (seeing the comparison result of Fig. 6) and has the biologic activity identical substantially with MADS15 protein, wherein said MADS15 protein is by the arbitrary aminoacid sequence representative that provides among the table D.This hybridization sequences generally has at least 400 continuous nucleotide length, preferably at least 600 continuous nucleotide length, more preferably at least 700 continuous nucleotide length and at least 800 continuous nucleotide length most preferably, and wherein said continuous nucleotide is any nucleotide sequence that provides in table D.Preferably, hybridization sequences be arbitrary nucleic acid hybridization that can provide among the D with table or with the sequence of the part hybridization of arbitrary these sequences, a wherein said part is as hereinbefore defined.Most preferably, hybridization sequences can with as the nucleic acid of SEQ ID NO:43 representative or with its part hybridization.Preferably, hybridization sequences coding comprise any or multiple as defined herein motif or the aminoacid sequence of structural domain.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the plant import and express can with the nucleic acid of any nucleic acid hybridization of providing among the table D, or be included in the plant and import and to express such nucleic acid, its can be coded in arbitrary nucleotide sequence of providing among the table D directly to the nucleic acid hybridization of homologue, collateral line homologue or homologue.

The another kind of nucleic acid variant that is used for the inventive method is as hereinbefore defined the proteinic splice variant of MADS15 of encoding.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the splice variant that imports and be expressed in any nucleotide sequence that provides among the D in the plant, or like this splice variant of nucleic acid, arbitrary aminoacid sequence that wherein said nucleic acid encoding provides in table D directly to homologue, collateral line homologue or homologue.

Preferred splice variant be by the splice variant of the nucleic acid of SEQ ID NO:43 representative or coding SEQ ID NO:44 directly to the splice variant of the nucleic acid of homologue or collateral line homologue.Preferably, comprise any or multiple motif or structural domain as defined herein by described splice variant amino acid sequence coded.

The another kind of nucleic acid variant that is used for implementing the inventive method is the allelic variant of the proteinic nucleic acid of MADS15 as hereinbefore defined of encoding.The allelic variant that is used for the inventive method has with the MADS15 protein of SEQ ID NO:44 goes up identical biologic activity substantially.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the allelic variant that imports and be expressed in any nucleic acid that provides among the D in the plant, or be included in the plant allelic variant that imports and express nucleic acid like this, arbitrary aminoacid sequence that wherein said nucleic acid encoding provides in table D directly to homologue, collateral line homologue or homologue.

Preferably, this equipotential variant be the allelic variant of SEQ ID NO:43 or coding SEQ IDNO:44 directly to the allelic variant of the nucleic acid of homologue or collateral line homologue.Preferably, comprise any or multiple motif or structural domain as defined herein by described allelic variant amino acid sequence coded.

The another nucleic acid variant that is used for the inventive method is the nucleic acid variant that obtains by gene reorganization effect.Gene reorganization effect or orthogenesis also can be used for producing the variant of the proteinic nucleic acid of MADS15 that coding defines as mentioned.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the variant that imports and be expressed in any nucleotide sequence that provides among the D in the plant, or be included in the plant variant that imports and express nucleic acid like this, arbitrary aminoacid sequence that wherein said nucleic acid encoding provides among the D at table directly to homologue, collateral line homologue or homologue, wherein variant nucleic acid obtains by gene reorganization effect.

Preferably, the variant nucleic acid encoding that obtains by gene reorganization effect comprise any or multiple as defined herein motif or the aminoacid sequence of structural domain.

In addition, the nucleic acid variant also can be by site-directed mutagenic obtained.Several method can be used for realizing site-directed mutagenesis, the method for the modal PCR of being based on (Current Protocols inMolecular Biology.Wiley edits)

The proteinic nucleic acid of coding MADS15 can be derived from any natural source or artificial source.Nucleic acid can have a mind to operate and modified by the mankind with regard to composition and/or genome environment aspect according to its natural form.Preferably, the coding MADS15 nucleic acid from plant, also preferably from monocotyledons, more preferably from Gramineae, this nucleic acid most preferably from rice.

In this article to the proteinic any MADS15 protein of definition as mentioned of quoting thereby mean of MADS15.The proteinic any nucleic acid of the MADS15 like this that encodes is applicable to enforcement the inventive method.

The present invention also comprises by the obtainable plant of the inventive method or its part (comprising seed).This plant or its part comprise the proteinic nucleic acid transgenosis of MADS15 that coding defines as mentioned.

The present invention also provides genetic constructs and carrier to promote to import and/or express the nucleotide sequence that is used for the inventive method in plant.This gene construct can insert the carrier that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed, and wherein said carrier can obtain by commercial sources.The present invention also provides the purposes in the methods of the invention of gene construct as defined herein.

More specifically, the invention provides construct, it comprises

Coding is the proteinic nucleic acid of MADS15 of definition as mentioned;

Can drive one or more regulating and controlling sequences of the nucleotide sequence expression of (a); Randomly

Transcription termination sequence.

Plant transforms (nucleic acid of the MADS15 polypeptide of definition as mentioned of promptly encoding) with the carrier that comprises aim sequence.The technician is appreciated that successfully and transforms, selects and breed the host cell that contains aim sequence and the genetic elements that must exist very much on carrier.Aim sequence effectively is connected with one or more regulating and controlling sequences (at least with promotor).

Advantageously, the promotor of any kind can be used for driving the expression of nucleotide sequence.

Promotor can be a constitutive promoter; Alternatively, promotor can be an inducible promoter, promptly has the transcripting starting of being induced when chemical replied or increasing or the promotor of stress induced promoter or pathogen-inducible.

Additionally or alternatively, promotor can be organ specificity or tissue-specific promoter, or promotor can be the omnipresence promotor, or promotor can be to grow modulability.In addition, promotor can be organ specificity or tissue specificity or cell-specific.

Preferably, MADS15 nucleic acid or its variant effectively are connected with constitutive promoter.Preferred constitutive promoter is the omnipresence constitutive promoter of expressing basically.Promotor is also preferably from plant, more preferably obtain from monocotyledons.Most preferably use GOS2 promotor (for example from rice, SEQID NO:47 or SEQ ID NO:108).Be understood that suitability of the present invention is not limited to the MADS15 nucleic acid by SEQ IDNO:43 representative, suitability of the present invention also is not limited to MADS15 expression of nucleic acids when the GOS2 promoters driven simultaneously.Also can be used for driving other constitutive promoter of MADS15 expression of nucleic acid or example demonstration in " definition " part of functional equivalent promotor.

Randomly, one or more terminator sequences can be used in the construct that imports plant.Extra regulatory element can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.One skilled in the art will recognize that or can obtain this type of sequence easily.

Also can add intron sequences to be increased in the amount of the ripe information that accumulates in the endochylema to 5 ' non-translational region (UTR) or in encoding sequence.

Other regulating and controlling sequence (except that promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stabilization element.One skilled in the art will recognize that or can obtain this type of sequence easily.

Genetic constructs of the present invention can also comprise need be used for the replication orgin sequence of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.

For detecting as successful transfer of used nucleotide sequence in the methods of the invention and/or the transgenic plant that selection comprises these nucleic acid, applying marking gene (or reporter gene) is favourable.Thereby genetic constructs can randomly comprise selectable marker gene.

The present invention also provides the method that produces the transgenic plant with enhanced yield correlated character with respect to control plant, is included in to import and express the coding proteinic any nucleic acid of MADS15 as hereinbefore defined in the plant.

More specifically, the invention provides the method that is used to produce the transgenic plant with increase output, described method comprises:

In plant or vegetable cell, import and express MADS15 nucleic acid or its variant; With

Cell cultivates plants under the condition that promotes plant-growth and growth.

Nucleic acid can directly import vegetable cell or import plant itself (comprising any other parts that import tissue, organ or plant).According to preferred feature of the present invention, nucleic acid preferably imports plant by transforming.

The vegetable cell of genetic modification can be regenerated by all methods that the technician is familiar with.Suitable method can be at S.D.Kung and R.Wu, Potrykus or

With find in the aforementioned publication of Willmitzer.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the expressive gene of plant coding that moves with the goal gene corotation.For selecting plant transformed, the vegetable material that obtains in conversion is accepted selection condition in principle and is handled, to such an extent as to plant transformed can be distinguished with unconverted plant.For example.Can sow with the seed that mode mentioned above obtains, after date when initial the cultivation carries out suitable selection by spraying and handles.Another possibility is included in cultivates seed (as required after sterilization) on the agar plate that uses suitable selective agent, to such an extent as to only the seed of Zhuan Huaing can grow into plant.Alternatively, plant transformed is screened the existence of selective marker (those selective markers as indicated above).

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze monitoring, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clone's property method of proliferating or classical breeding technique.For example, the s-generation (or T2) transformant that the conversion plant of the first-generation (or T1) can carry out selfing and select to isozygoty, and the T2 plant can further breed by classical breeding technique subsequently.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's property transformant (for example conversion) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the conversion rhizome of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring show with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also comprises host cell, and it contains the proteinic isolating nucleic acid of coding MADS15 as hereinbefore defined.The preferred host cell of the present invention is a vegetable cell.

Host plant advantageously can synthesize whole plants of used polypeptide in the inventive method in principle for used nucleic acid or carrier, expression cassette or construct or carrier in the inventive method.

The present invention also extend to plant the part gathered in the crops as, but be not limited to seed, leaf, fruit, flower, stem, root stock, stem tuber and bulb.The invention further relates to from oneself, preferably directly from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

According to preferred feature of the present invention, the expression of being regulated is the expression that increases.

As mentioned, the preferred method that is used for regulating (preferred increasing) proteinic expression of nucleic acid of coding MADS15 is to import and express the proteinic nucleic acid of coding MADS15 plant; Yet the effect of implementing described method promptly strengthens the output correlated character also can use well-known other technology to realize.Hereinafter be to some description in these technology.

A kind of such technology is that T-DNA activates labelization.The transgenic plant that produce show the dominant phenotype because of the modification of the gene of the close promotor that imports.

Effect of the present invention also can be used TILLING technology (genome interior orientation inductive local damage) or pass through homologous recombination and reproduce.

In this article the reference of enhanced yield correlated character is meant biomass (weight) increase of one or more parts of plant, described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.

With the corn is example, and the output increase can show as following one or more indexs: the increase of the increase of the per hectare or the increase of acre plant number, every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

In specific embodiment, this type of can gather in the crops part is root, and the enforcement of the inventive method produces the root growth with respect to the appropriate control plant, has the plant of the root growth of increase.Root development is the key determinative of plant-growth and crop yield, because root is to extract nutraceutical principal passage from environment.

The root output that increases can for example itself show as following one or more indexs: a) the root biomass quantity of Zeng Jiaing (thereby can be by setting certain threshold value thick with thin root between make differentiations), b) average root diameter, the c of increase) the root total biomass and the d of increase) the root biomass seedling biomass ratio of increase.Be purpose of the present invention, be to be understood that term " root growth " is included in aspect growth whole of the different piece that constitutes root system in monocotyledons and the dicotyledons on the different growth perioies.The enhanced growth that will be appreciated that root can be because of due to the enhanced growth of one or more parts (comprising main root, lateral root, adventive root etc.) of root, and they all are in the scope of the present invention.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of control plant, these plants might show on the corresponding stage in its life cycle increase growth velocity (its life cycle during the small part).The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Plant with growth velocity of increase can possess short life cycle.The life cycle of plant can be considered as meaning from dry mature seed and grow to the needed time in stage that plant has produced the dry mature seed similar to parent material.This life cycle can be influenced by following factors, as early stage vigor, growth velocity, green degree index, flowering time and seed maturity speed.The increase of growth velocity can take place during life cycle on the one or more stages in life cycle or in whole plants basically plant.The growth velocity that increases during plant early stage in life cycle can reflect the enhanced vigor.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this can not (similar effect can obtain with flowering time early).If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

According to preferred feature of the present invention, the enforcement of the inventive method produces the plant that has the growth velocity of increase with respect to control plant.Thereby according to the present invention, provide with respect to the method that increases plant growth rate, described method comprises regulating expresses, preferably increases the expression of the defined coding proteinic nucleic acid of MADS15 in plant herein.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term as used in this article " non-coercing " condition is the envrionment conditions that allows the plant optimum growh.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method is with respect to the appropriate control plant of cultivating under comparable conditions, gives under the non-stress conditions or the output that increases of the plant of cultivating under slight drought condition.Thereby according to the present invention, be provided under the non-stress conditions or increase the method for output in the plant of under slight drought condition, cultivating, described method comprises that the nucleic acid that increases coding MADS15 polypeptide expresses in plant.

The enforcement of the inventive method produces with respect to the appropriate control plant of cultivating under comparable conditions, the plant of under the nutritive deficiency condition, especially cultivating under nitrogen shortage condition that increases output that has.Thereby, increasing the method for output in the plant that is provided under the nutritive deficiency condition, cultivating according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding MADS15 polypeptide.Nutritive deficiency can because of nutraceutical shortage or excessive due to, described nutrition for example is nitrogen, phosphoric acid salt and other P contained compound, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.

In the preferred embodiment of the invention, the increase of output and/or growth velocity occurs under non-stress conditions according to the inventive method.

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise being selected from following feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

The present invention also comprises the purposes and the proteinic purposes of these MADS15 of the proteinic nucleic acid of MADS15 described in coding this paper, is used for strengthening the output correlated character of plant.

Proteinic nucleic acid of MADS15 or MADS15 protein itself can be used for the procedure of breeding described in coding this paper, wherein identify the dna marker that can be connected with the gene of coding MADS15 hereditarily.Described nucleic acid/gene or MADS15 protein itself can be used for defining molecule marker.This DNA or protein labeling can be used for selecting to have in the inventive method the plant of enhanced yield correlated character as hereinbefore defined subsequently in the procedure of breeding.

The allelic variant of the proteic nucleic acid/gene of coding MADS15 also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to import allelic variation by for example using the EMS mutagenesis that plant is made mutagenic treatment sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be used to select discuss and cause increasing the step of excellent allelic variant of the sequence of output.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises and will identify the plant and the another kind of plant hybridization of excellent allelic variant.This can be used for for example producing target phenotype combination of features.

The proteic nucleic acid of coding MADS15 also can be as probe so that carry out genetic mapping or physical mapping to gene, and described probe reaches the mark of the proterties related with these genes as the part of described gene.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.The nucleotide sequence that this purposes of the proteic nucleic acid of coding MADS15 only needs to have at least 15 length of nucleotides.The proteic nucleic acid of coding MADS15 can be used as restriction fragment length polymorphism (RFLP) mark.The Southern trace of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) can be used the proteic nuclei acid probe of coding MADS15.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the Southern trace of the genomic dna that contains one group of individuality handling through restriction endonuclease, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of the proteic nucleic acid of calculation code MADS15 in using the previous genetic map that obtains of this colony.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter 4:37-41.Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be can be used for mapping with other population of individuals to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mam malian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces the plant with enhanced yield correlated character, as mentioned before.These proterties also can be favourable with economy the combination of other proterties, strengthen proterties, other inanimate coerced and biological tolerance of coercing, the proterties of regulating multiple structural attitude and/or biochemical characteristics and/or physiologic character as other output.

The detailed description of the MADS15 of downward modulation

Now be surprised to find the level that reduces endogenous MADS15 polypeptide and/or actively produced the plant that has the enhanced yield correlated character with respect to corresponding wild-type plant, especially increases output.The present invention thereby provide with respect to control plant and strengthen the output correlated character, be particularly useful for increasing the method for plant biomass, it comprises level and/or the activity that reduces endogenous MADS15 polypeptide.In this article quoting of " control plant " meant the corresponding wild-type plant that the activity of endogenous MADS15 polypeptide does not wherein reduce.

Advantageously, the enforcement of the inventive method produces the plant that has the biomass of the output of increase, the seed production that especially increases and/or increase with respect to corresponding wild-type plant.

The biomass (weight) that term as defined herein " output of increase " means one or more parts of plant increases, and described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.

Especially, this type of can be gathered in the crops part and comprise trophicity biomass and/or seed, and the enforcement of the inventive method produces the plant that has the output (aspect trophicity biomass and/or seed) of increase with respect to the output of control plant.

With the corn is example, the output increase can show as following one or more indexs: the increase of per hectare or acre plant number, the increase of the increase of every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

The increase of seed production also can show as the increase of seed size and/or seed volume.This can increase the quantity of material in the seed (as oil, protein and carbohydrate) or change its composition.

According to preferable feature, the enforcement of the inventive method produces has the output of increase, the biomass that especially increases and/or the plant of seed production.Thereby, being provided for increasing the method for plant biomass according to the present invention, described method comprises the activity level that preferably reduces MADS15 polypeptide or its homologue by the expression of downward modulation MADS15 gene or its homologue.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of corresponding wild-type plant, these plants might show on the corresponding stage in its life cycle the growth velocity that increases (its life cycle during the small part).The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Plant with growth velocity of increase can possess short life cycle.The life cycle of plant can be considered as meaning from dry mature seed and grow to the needed time in stage that plant has produced the dry mature seed similar to parent material.This life cycle can be influenced by following factors, as early stage vigor, growth velocity, flowering time and seed maturity speed.The increase of growth velocity can take place during life cycle on the one or more stages in life cycle or in whole plants basically plant.The growth velocity that increases during plant early stage in life cycle can reflect the enhanced vigor.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this can not (similar effect can obtain with flowering time early).If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

The enforcement of the inventive method produces the plant of the growth velocity with increase.Thereby, providing the method that increases plant growth rate with respect to control plant according to the present invention, described method comprises expression level and/or the activity of preferential reduction endogenous MADS15 gene in plant.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term as used in this article " non-coercing " condition is the envrionment conditions that allows the plant optimum growh.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method is with respect to the appropriate control plant of cultivating under comparable conditions, gives under the non-stress conditions or the output that increases of the plant of cultivating under slight drought condition.Thereby according to the present invention, be provided under the non-stress conditions or increase the method for output in the plant of under slight drought condition, cultivating, described method comprises that reducing endogenous MADS15 gene expresses in plant.

The enforcement of the inventive method produces with respect to the appropriate control plant of cultivating under comparable conditions, the plant of under the nutritive deficiency condition, especially cultivating under nitrogen shortage condition that increases output that has.Thereby, increasing the method for output in the plant that is provided under the nutritive deficiency condition, cultivating according to the present invention, described method comprises the expression of nucleic acid in plant that reduces coding MADS15 polypeptide.Nutritive deficiency can because of nutraceutical shortage or excessive due to, described nutrition for example is nitrogen, phosphoric acid salt and other P contained compound, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.

In the preferred embodiment of the invention, the increase of output and/or growth velocity occurs under non-stress conditions according to the inventive method.

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

In this article to endogenous MADS15 protein in plant level " reduction " quote the endogenous MADS15 protein level that means with respect to existing in the corresponding wild-type plant, endogenous MADS15 protein on protein concn reduction or remove endogenous MADS15 protein substantially.This reduction or basic removal can cause the MADS15 protein active that reduces in the plant or eliminate basically.

In this article " reduction " of endogenous MADS15 protein active in the plant quoted the endogenous MADS15 protein active level that means with respect to existing in the wild-type plant, the basic removal of the reduction of MADS15 protein active or endogenous MADS15 protein active.

Preferably, endogenous MADS15 protein level and/or active reduction obtain by downward modulation endogenous MADS15 expression of gene.

" endogenous " MADS1 gene of mentioning herein not only refers to the MADS15 gene that exists with its natural form (promptly not having human any intervention) as in the plant, also refers to import subsequently the isolated M ADS15 gene of plant.For example, containing the genetically modified transgenic plant of MADS15 can meet with the reduction of MADS15 transgene expression or remove substantially and/or the reduction or basic removal of endogenous MADS15 genetic expression.

This reduction of endogenous MADS15 genetic expression (or basic removal) can be used any or multiple of several known gene silencing methods and realize.As used herein, " gene silencing " of expression or ＂ downward modulation " refer to the reduction or basic removal of MADS15 genetic expression and/or MADS15 polypeptide level and/or MADS15 polypeptide active.

Being used to reduce or remove a kind of of endogenous MADS15 genetic expression substantially is the down regulation of gene expression (the reticent effect of RNA) of RNA mediation as this method.Silence acts in this case by triggering with the double-stranded RNA sequence (dsRNA) of target MADS15 dna homolog basically in the plant.This dsRNA is further processed into about about 26 Nucleotide of 20-by plant, is called short interferential RNA (siRNA).SiRNA is impregnated in the reticent mixture of RNA inducibility (RISC), and wherein said RISC cuts the mRNA transcript of MADS15 target gene, thereby reduces or eliminate basically the number of the proteinic MADS15mRNA of one-tenth MADS15 to be translated.

An example of RNA silencing methods comprises encoding sequence or its part that is in sense orientation to the plant importing.This extra gene or its part can make endogenous MADS15 gene silencing, produce to be called inhibiting altogether phenomenon.When several additional copies imported plant, the reduction meeting of MADS15 genetic expression was more obvious, because have positive correlation between the inhibiting together triggering of high transcript level.

Another example of RNA silencing methods comprises use antisense MADS15 nucleotide sequence.

This antisense nucleic acid can use expression vector to produce in the biology mode, and in the described expression vector, nucleic acid is with antisense orientation subclone (promptly the RNA that transcribes from the nucleic acid that inserts will be antisense orientation with the purpose target nucleic acid, and this will describe in following trifle).Preferably, the generation of antisense nucleic acid in plant is by the transgenosis of stable integration and take place, and wherein said transgenosis comprises promotor, antisense oligonucleotide and the terminator that is effective to constitutive expression in plant.

Be used for reducing or the preferred method of removing endogenous MADS15 genetic expression substantially is to use expression vector by the reticent effect of RNA, wherein MADS15 gene or its fragment are cloned as (partially or completely) inverted repeats that is separated by transcribed spacer (non-coding DNA).

In another embodiment, reduction that endogenous MADS15 expresses or basic removal can obtain by using ribozyme.For example, can make up the derivative of thermophilas (Tetrahymena) L-19IVS RNA, wherein nucleotide sequence complementation to be cut among the mRNA of the nucleotide sequence of reactive site and coding MADS15.

If have sudden change on the endogenous MADS15 gene and/or importing existence sudden change on the isolated M ADS15 gene of plant subsequently, then gene silencing also can be realized by inserting mutagenesis.The reduction of MADS15 protein active or basic removal can be caused by non-functional MADS15.For example, MADS15 and multiple interaction protein bound; One or more sudden changes in the MADS of MADS15 box and/or brachymemma effect thereby such MADS15 protein can be provided: it still can binding interactions protein, but can not show the normal function as transcription factor.

The method of another kind of gene silencing is that target decide nucleotide sequence, the complementary triple-helix structure of transcribing in target cell with formation prevention MADS15 gene of its regulatory region (for example MADS15 promotor and/or enhanser) with the MADS15 gene.

The method of another gene silencing is described by Hiratsu etc. (Plant J.34,733-739,2003).This method does not rely on the sequence homology with target gene, but comprise the inhibition sequence domains of using in the gene fusion thing of transcribing, and the proterties (Fujita etc. of agronomy meaning have been used for improveing, Plant Cell 17,3470-3488,2005 and Mitsuda at al., Plant Cell 17,2993-3006,2005).Generally, can influence target energetically at coding decides the gene of the protein of genetic expression (as transcription activating protein) and encodes to produce the chimeric fusions of Nucleotide between the nucleotide fragments that suppresses structural domain.When expressing described mosaic gene fusions, decided expression of gene by target and be suppressed, suppressed in the dominant mode usually.It is well-known in the art suppressing structural domain, the EAR motif that for example exists in some AP2 and zinc finger transcription factor.Be very suitable for overcoming the gene redundancy of being decided gene in the selected plant species by target based on the method that suppresses structural domain.

Mentioned above is the example that is used for the several different methods of gene silencing (being used for reducing or removing substantially endogenous MADS15 genetic expression).The inventive method depends on and reduces the expression of endogenous MADS15 gene in plant.For example, those skilled in the art will can adjust the preceding method that is used for gene silencing easily, to such an extent as to by using suitable promotor whole strain plant or in its part, realize gene silencing.

Should be understood that marrow of the present invention is the favourable and wonderful result who is found when reducing or remove endogenous MADS15 genetic expression in the plant substantially, and be not limited to be used for so reducing or removing substantially any concrete grammar of endogenous MADS15 protein active.The activity of MADS15 polypeptide also can reduce or eliminates by importing genetic modification (preferably in the locus of MADS15 gene).The locus of gene as defined herein means such genome area, and it comprises goal gene and upstream of coding region or downstream 10kb.

Genetic modification can be for example any (or multiple) by following method import: T-DNA inactivation method, TILLING, site-directed mutagenesis, orthogenesis method, homologous recombination method.After importing genetic modification, carry out the active step of the reduction of selecting the MADS15 polypeptide, wherein active reduction obtains having the plant of the output of increase.

T-DNA inactivation labeling acts is included in the genome area of goal gene or the upstream of gene coding region or downstream 10kb insert T-DNA with structure like this, decides expression of gene to such an extent as to this T-DNA suppresses target.Usually, deciding the regulating effect that the natural promoter of gene decides genetic expression to target by target is damaged.T-DNA inserts Plant Genome at random, for example passes through agroinfection, and causes near the downward modulation of the genetic expression insertion T-DNA.The transgenic plant that produce show phenotype because of the downtrod expression of gene near the T-DNA that imports.

Also can use TILLING (genome interior orientation inductive local damage) technology in the locus of MADS15 gene, to import genetic modification.

Site-directed mutagenesis and random mutagenesis can be used for producing the variant of MADS15 nucleic acid.Several method can be used for realizing site-directed mutagenesis, the method for the modal PCR of being based on (CurrentProtocols in Molecular Biology.Wiley edits).

Orthogenesis also can be used for producing the variant of MADS15 nucleic acid.This is shuttled back and forth by DNA repeatedly, suitably screening and/or select to form (Castle etc., (2004) Science 304 (5674): 1151-4 subsequently to produce MADS15 nucleic acid variant or its variant of MADS15 polypeptide that coding has (be reduce or cancellation) biologic activity of modification herein; United States Patent (USP) 5,811,238 and 6,395,547).

T-DNA activation method, TILLING, site-directed mutagenesis and orthogenesis method are the technical examples that can produce neomorph and MADS15 variant.

Effect of the present invention also can be used homologous recombination method and reproduce.Treat that the fixed nucleic acid of target can be to be used for replacing the coding inactive protein matter of native gene or to have the active proteinic allelotrope of reduction, perhaps can except that described native gene, import again, and need be by the locus of target to the MADS15 gene.

Other method, as using at the antibody of endogenous MADS15 suppressing the function of this MADS15 in plant, or the signal pathway that disturbs MADS15 to participate, will be that the technician is well-known.Alternatively, can set up the natural variant of screening procedure with identification of M ADS15 gene, wherein said variant has the MADS15 activity of reduction, or does not have the MADS15 activity.This type of natural variant also can be used for method of the present invention.

Be optimum performance, be used to reduce or the gene silent technology of removing endogenous MADS15 genetic expression substantially need use from monocotyledonous MADS15 nucleotide sequence to be converted in the monocotyledons.Preferably, the MADS15 nucleic acid from any given plant species imports in the same species.For example, will be converted into rice plant from the MADS15 nucleic acid (it should be total length MADS15 sequence or fragment) of rice.MADS15 nucleic acid does not need to import identical plant variety.

In this article to the two strands of quoting the arbitrary length that means polymerized form of " MADS15 gene " or " MADS15 nucleic acid " or the deoxyribonucleotide polymkeric substance or the ribonucleoside acid polymer of strand, or its analogue, they have the essential characteristic of natural nucleus sugar nucleotide, because they can be with mode and the nucleic acid hybridization to be similar to naturally occurring polynucleotide." MADS15 gene " or " MADS15 nucleic acid " refer to encode Nucleotide of successive basically of the sufficient length of carrying out gene silencing of gene of MADS15; Described length may be as little to 20 or Oligonucleotide more.Coding (functional) proteinic gene is not that discussed above to be used to reduce or to remove substantially the several different methods that the MADS15 native gene expresses required.

The inventive method can use the sufficient length of the Nucleotide of successive basically of MADS15 gene/nucleic acid to carry out, wherein said length can by 20 or more Oligonucleotide form, these Nucleotide can be from any part of MADS15 gene/nucleic acid, as very conservative in the MADS15 gene family or coding 5 ' end of the coding region of one of conservative motif hereinafter described.

The MADS15 gene is well-known in the art, and the MADS15 gene that is used for the inventive method is the Nucleotide of successive basically of plant MADS15 gene/nucleic acid of describing in (Plant Physiol.120,1193-1204,1999) such as Moon.

Other MADS15 gene/nucleotide sequence also can be used for the inventive method, and can be identified by those skilled in the art easily.Can be because of there be one or more evaluation of several well-known characteristic (seeing below) in the MADS15 polypeptide.When identifying the MADS15 polypeptide, those skilled in the art can use conventional techniques and easily draw respective coding nucleic acid, and use the continuous nucleotide of the sufficient length of this nucleic acid to implement any or multiple gene silencing methods mentioned above.

Term as defined herein " MADS15 polypeptide or its homologue " refers to belong to as (J.Mol.E the 62nd volume, the 15-31) protein of the group of illustrated FUL sample MADS box protein matter such as Adam.FUL sample MADS box protein matter is the part of SQUAMOSA subfamily and has typical MIKCc structure: at N-terminal MADS structural domain (M), then be participate in dimerization interleave structural domain (I), the variable domains at carboxyl terminal (C) of the Keratin sulfate structural domain (K) of high conservative and responsible binding interactions protein or transcriptional activation (Trends in Plant Science 8 such as De Bodt, 475-483).

Plant MADS15 polypeptide also can obtain because of the existence of some conservative motif identifying.The existence of these conservative motifs can be used and as indicated above be used for the method that aligned sequences is used for comparison and identify.In some cases, can adjust default parameters to regulate the severity of search.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.Like this, can identify almost accurate short coupling.In case identified the MADS15 polypeptide by the existence of these motifs, those skilled in the art can easily obtain the corresponding nucleic that encoded packets contains the polypeptide of relevant motif, and use the continuous nucleotide of the sufficient length of this nucleic acid to implement any or multiple gene silencing methods mentioned above (being used for reducing or removing substantially endogenous MADS15 genetic expression).

Usually, motif 1 to 4 existence one of at least should be enough to identify that arbitrary search sequence is MADS15, yet preferably has motif 1 and 2 at least.The monocotyledons sequence of the consensus sequence that is provided to provide in the sequence table.Those skilled in the art understand fully aware of if (for example from the dicotyledons sequence) other sequence or different sequence are used for comparison, and then can there be certain variation in consensus sequence.

Be positioned at the motif 1 (SEQ ID NO:114) of MADS structural domain:

L(L/V)KKA(H/N)EIS(VI)L(C/Y)DAE(V/I/L)(A/G)(L/A/V)(I/V)(I/V)FS(T/P/A/N)KGKLYE(Y/F)(A/S)(T/S)(D/N/E)S(K/C/R/S)M(D/E)(N/I/R/K)IL(E/D)R。

Preferably, this conserved sequence motif 1 has following sequence:

LLKKAHEISVLCDAEVA(L/A/V)I(I/V)FS(T/P)KGKLYEYATDS(C/R)M(D/E)(R/K)ILER，

Most preferably, conserved sequence motif 1 has following sequence:

LLKKAHEISVLCDAEVAAIVFSPKGKLYEYATDSRMDKILER

Be positioned at the motif 2 (SEQ ID NO:115) of K structural domain:

KLK(A/S)(K/R)(V/I)E(A/T/S)(L/I)(Q/N)(K/R/N)(S/C/R)(Q/H)(R/K)HLMGE

Preferably, this conserved sequence motif 2 has following sequence:

KLKAK(V/I)E(A/T)(L/I)QK(S/C)(Q/H)(R/K)HLMGE

More preferably, this conserved sequence motif 2 has following sequence:

KLKAKIETIQKCHKHLMGE

Randomly, MADS15 polypeptide or its homologue can comprise motif 3 and/or motif 4 in the C-structure territory:

Motif 3 (SEQ ID NO:116):

Q(P/Q/V/A)QTS(S/F)(S/F)(S/F)(S/F)(S/C/F)(F/M)

Motif 4 (SEQ ID NO:117):

(G/A/V/L)(L/P)XWMX(S/H)

Wherein the X residue in motif 4 can be arbitrary amino acid, but preferably L, P or H; And wherein the 2nd X residue can be arbitrary amino acid, but preferably V or L.

Alternatively, the C-structural domain (begins behind the Keratin sulfate structural domain, i.e. R175 place in SEQ ID NO:110) can be feature (normally 3.93% with the glutamine occurrence rate that increases, here be 8.77% at least, maximum 26.73%), in addition, the content of L-Ala, proline(Pro) and/or Serine also can increase (being higher than 7.8%, 4.85% and 6.89% respectively).

That propose, that be used for the inventive method in addition preferred MADS15 protein comprises the aminoterminal MADS structural domain corresponding to SMART structural domain SM00432 (Pfam PF00319) at least, be subsequently with Pfam in be defined as the corresponding Keratin sulfate structural domain in K box district of PF01486, more preferably, MADS15 protein has conservative label SEQ ID NO:114 and have conservative label SEQ ID NO:115 in its K-structural domain in its MADS structural domain, most preferably, MADS15 protein has the sequence that provides in SEQ ID NO:110.

Ding Yi homologue can use conventional techniques well-known in the art as identifying easily by sequence alignment as mentioned; The homologue of OsMADS15 may be named in a plurality of plant species, thereby genes matter title should not be used for identifying directly to homologue or collateral line homologue.It is well-known in the art being used for the method that aligned sequences is used for comparison, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses the algorithm ((1970) J Mol Biol 48:443-453) of Needleman and Wunsch to find the comparison that makes the maximization of coupling number and make minimized two complete sequence of room number.BLAST algorithm (Altschul etc. (1990) J Mol Biol215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI obtainable.Homologous sequence can use ClustalW multiple sequence alignment algorithm (1.83 version) for example to identify easily in acquiescence pairing comparison parameter with the methods of marking of percentage ratio.Can carry out trickle edit to optimize the best comparison (seeing below) between the conservative motif, apparent as those skilled in the art.

A plurality of structural structural domain in the MADS15 protein can use for example SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95,5857-5864 of specialized database; Letunic etc. (2002) Nucleic Acids Res 30,242-244), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318; ), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and in the function of automatization sequence interpretation. (In) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002)) identified.

In addition, MADS15 protein also can because of can in conjunction with DNA and with other protein interaction but appraisable.Dna binding activity and protein-protein interaction can use technology well-known in the art and easily external or determine in vivo.The example that is used for the external test method of dna binding activity comprises: gel retardation assay (West etc. (1998) the Nucl Acid Res 26 (23): 5277-87), or the yeast one-hybrid analysis that uses known MADS box DNA binding domains.The example that is used for the external test method of protein-protein interaction is yeast two-hybrid analysis (Fields and Song (1989) Nature 340:245-6).Known and OsMADS15 interacting proteins comprise other MADS protein (as those MADS protein of the signal effect complex body that participates in blooming), receptor-like kinase enzyme such as Clavata1 and Clavata2, Erecta, BRI1 or RSK.

Thereby in case utilize one or more feature and identification of M ADS15 polypeptide as mentioned above, those skilled in the art can easily obtain the corresponding nucleic of coding said polypeptide, and use the continuous nucleotide of the sufficient length of this nucleic acid to implement any or multiple gene silencing methods mentioned above (being used for reducing or removing substantially endogenous MADS15 genetic expression).

Be preferred in the inventive method is the Nucleotide of successive basically of the sufficient length of SEQ ID NO:109 (OsMADS15), or use the Nucleotide of successive basically of the sufficient length of nucleotide sequence like this, wherein said nucleic acid sequence encoding OsMADS15 (SEQ ID NO:109) directly to homologue or collateral line homologue.This type of that OsMADS15 is provided in following table D is directly to the example of homologue and collateral line homologue.Close relative's homologue of OsMADS15 is by the protein of SEQ ID NO:147,149,151,153,155,157,159,161,163,165,167,169,171 and 173 representatives.

Directly can easily find by carrying out so-called interactivity blast search in monocotyledons species for example to homologue.This can be undertaken by a BLAST, and a wherein said BLAST comprises search sequence (for example SEQ ID NO:109 or SEQ ID NO:110) is carried out blast search at arbitrary sequence library (as public's available ncbi database).When nucleotide sequence begins, can use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when protein sequence begins.Randomly can screen BLAST result.The full length sequence of submitting The selection result and non-The selection result subsequently to is with at carry out reverse blast search (the 2nd BLAST) from the sequence of biology, search sequence from described biology, derive (be under the situation of SEQ ID NO:109 or SEQ IDNO:110 wherein, the 2nd BLAST thereby will be) wherein at the rice sequence in search sequence.The result who compares first and second blast searches subsequently.If from the high-order position of a BLAST hit be derived from search sequence from identical species of deutero-species wherein, then identify the collateral line homologue; If high-order position hit be not derived from search sequence from identical species of deutero-species wherein, then identify directly to homologue.It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hit because of the chance odds low more).The calculating of E-value is well-known in the art.Under the situation of large-scale family, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.

The source of the Nucleotide of successive basically of MADS15 gene/nucleic acid can be arbitrary plant origin or artificial source.Be optimum performance, be used to reduce or the gene silent technology of removing endogenous MADS15 genetic expression substantially need use from monocotyledonous MADS15 sequence to be converted in the monocotyledons.Preferably, will be transformed into grass from MADS15 sequence gramineous.Also preferably, MADS15 nucleic acid (it should be total length MADS15 sequence or the fragment) conversion from rice enters rice plant.MADS15 nucleic acid does not need to import identical plant variety.Most preferably, be the Nucleotide of successive basically or Nucleotide of successive basically of the sufficient length of nucleotide sequence like this of the sufficient length of SEQ ID NO:109 (OsMADS15) from the MADS15 nucleic acid of rice, wherein said nucleic acid sequence encoding OsMADS15 (SEQ ID NO:109) directly to homologue or collateral line homologue.As mentioned, what those skilled in the art can fully understand formation is intended to carry out the above sufficient length of the Nucleotide of successive basically of arbitrary gene silencing methods that defines, and this length can be 20 or the Nucleotide of successive basically still less in some cases.

The present invention also provides genetic constructs and carrier to promote to import and/or express the nucleotide sequence that is used for the inventive method.

Thereby, provide such gene construct, to such an extent as to it comprises and can drive justice and/or antisense MADS15 nucleotide sequence and express and make endogenous MADS15 gene reticent one or more regulating and controlling sequences in plant in plant; And randomly comprise transcription termination sequence.Preferably, regulating and controlling sequence is constitutive promoter and omnipresence promotor.

The preferred construct that is used for gene silencing is the construct that comprises MADS15 gene or its segmental inverted repeats (it preferably can form hairpin structure), and wherein said inverted repeats is in constitutive promoter control down.

Thereby, the invention provides construct, it comprises:

Can form the MADS15 nucleic acid of hairpin structure;

Can drive one or more regulating and controlling sequences of the nucleotide sequence expression of (a); Randomly

Transcription termination sequence.

Used construct can use the well-known recombinant DNA technology of those skilled in the art to produce in the inventive method.This gene construct can insert the carrier that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed, and wherein said carrier can obtain by commercial sources.The present invention thereby the purposes in the methods of the invention of gene construct as hereinbefore defined is provided.

Aim sequence effectively is connected with one or more regulating and controlling sequences (at least with promotor) that can increase expression in plant.

Advantageously, the promotor of any kind can be used for driving the expression of nucleotide sequence.Promotor can be an inducible promoter, is promptly responding to the transcripting starting that has induced or increase when developmental character, chemical, environment or physical property stimulate.Additionally or alternatively, promotor can be to organize preferred property promotor or the preferred property of cell promotor, promptly can be preferentially some tissue as leaf, root, seed tissue etc. in or even startup is transcribed in specific cells promotor.Only can in some tissue or cell, start the promotor of transcribing and be called " tissue specificity " or " cell-specific " in this article respectively.

Preferably, MADS15 nucleic acid or its functional variant are connected with constitutive promoter effectively.Preferably, this promotor is the omnipresence promotor and mainly expresses in whole strain plant scope.Preferably, the constitutive promoter that nucleic acid is preferentially expressed in whole strain plant scope has the express spectra suitable with the GOS2 promotor.More preferably, this constitutive promoter has and the identical express spectra of rice GOS2 promotor, most preferably, can make nucleic acid preferentially in whole strain plant scope expression promoter be GOS2 promotor (SEQ ID NO:113 or SEQ ID NO:174) from rice.Be understood that suitability of the present invention is not limited to the MADS15 nucleic acid by SEQ ID NO:109 representative, suitability of the present invention also is not limited to express MADS15 nucleic acid when the GOS2 promoters driven simultaneously.The substituting constitutive promoter that is used for the inventive method is high mobility group protein promotor (PRO0170, the SEQ ID NO:40 among the WO2004070039).The example that also can be used for driving other constitutive promoter of MADS15 expression of nucleic acid shows in " definition " part.

Randomly, one or more terminator sequences can be used in the construct that imports plant.Extra regulatory element can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.One skilled in the art will recognize that or can obtain this type of sequence easily.

Genetic constructs of the present invention can also comprise need be used for the replication orgin sequence of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.Genetic constructs can randomly comprise selectable marker gene.

The present invention also comprises by the obtainable plant of the inventive method, comprises plant part, and wherein said plant has the output of increase with respect to control plant and has endogenous MADS15 genetic expression reduction or that remove basically.

The present invention also provides the method that produces the transgenic plant of the output with increase with respect to control plant, and wherein said transgenic plant have endogenous MADS15 genetic expression reduction or that remove basically.

More specifically, the invention provides the method for the transgenic plant that are used to produce the seed production with increase, described method comprises:

In plant, plant part or vegetable cell, import and the expressing gene construct, to such an extent as to described gene construct comprise can variety of priority driven MADS15 nucleotide sequence inverted repeats in plant, expresses one or more regulating and controlling sequences that make endogenous MADS15 gene silence in plant; With

Under the condition that promotes plant-growth and growth, cultivate described plant, plant part or vegetable cell.

Preferably, the construct of importing plant is to comprise the MADS15 gene or the construct of its segmental (partially or completely) inverted repeats (it preferably can form hairpin structure).

According to preferred feature of the present invention, this construct imports plant by transforming.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the expressive gene of plant coding that moves with the goal gene corotation.

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze or quantitative PCR is monitored, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clone's property method of proliferating or classical breeding technique.For example, the first-generation (or T1) transforms plant can carry out the s-generation (or T2) transformant that selfing is isozygotied with generation, and the T2 plant further breeds by classical breeding technique.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's property transformant (for example conversion) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the root stock of the conversion of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring demonstrate with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also extends to the part gathered in the crops of plant, as seed and from, preferred directly from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises MADS15 nucleic acid and is used for reducing or removes plant endogenous MADS15 genetic expression substantially to increase the purposes of plant seed output as hereinbefore defined.

Proteinic nucleic acid of MADS15 or MADS15 protein itself can be used for the procedure of breeding described in coding this paper, wherein identify the dna marker that can be connected with the gene of coding MADS15 hereditarily.Described nucleic acid/gene or MADS15 protein itself can be used for defining molecule marker.This DNA or protein labeling can be used for selecting to have in the inventive method the plant of enhanced yield correlated character as hereinbefore defined subsequently in the procedure of breeding.

The allelic variant of the proteic nucleic acid/gene of coding MADS15 also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to import allelic variation by for example using the EMS mutagenesis that plant is made mutagenic treatment sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be used to select discuss and cause increasing the step of excellent allelic variant of the sequence of output.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises and will identify the plant and the another kind of plant hybridization of excellent allelic variant.This can be used for for example producing target phenotype combination of features.

The proteic nucleic acid of coding MADS15 also can be as probe so that carry out genetic mapping or physical mapping to gene, and described probe reaches the mark of the proterties related with these genes as the part of described gene.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.The nucleotide sequence that this purposes of the proteic nucleic acid of coding MADS15 only needs to have at least 15 length of nucleotides.The proteic nucleic acid of coding MADS15 can be used as restriction fragment length polymorphism (RFLP) mark.The Southern trace of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) can be used the proteic nuclei acid probe of coding MADS15.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the Southern trace of the genomic dna that contains one group of individuality handling through restriction endonuclease, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of the proteic nucleic acid of calculation code MADS15 in using the previous genetic map that obtains of this colony.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter 4:37-41.Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be can be used for mapping with other population of individuals to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics 16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces the plant with enhanced yield correlated character, as mentioned before.These proterties also can be favourable with economy the combination of other proterties, strengthen proterties, other inanimate coerced and biological tolerance of coercing, the proterties of regulating multiple structural attitude and/or biochemical characteristics and/or physiologic character as other output.

The detailed description of PLT

Now be surprised to find the expression of nucleic acid that increases coding PLT transcription factor polypeptide produce with respect to control plant have the enhanced yield correlated character, the plant of the output that especially increases.

According to the present invention, the method that is used to increase plant biomass with respect to control plant is provided, comprise the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide.

The biomass (weight) that term as defined herein " output of increase " means one or more parts of plant increases, and described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.

Especially, this type of can gather in the crops part is seed, and the enforcement of the inventive method produces the plant that has the seed production of increase with respect to the seed production of control plant.

With the corn is example, the output increase can show as following one or more indexs: the increase of per hectare or acre plant number, the increase of the increase of every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

According to preferred feature of the present invention, the enforcement of the inventive method produces the plant that has the seed production of increase with respect to control plant.Thereby, providing the method that is used for increasing the plant seed production with respect to the seed production of control plant according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of corresponding wild-type plant, these plants might show on the corresponding stage in its life cycle the growth velocity that increases (its life cycle during the small part).The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Have increase growth velocity plant in addition can show prematurity.The increase of growth velocity can take place on the one or more stages in life cycle or during whole plants life cycle plant.The growth velocity that increases can reflect enhanced vigor (the seedling vigor that increases) when emerging during plant early stage in life cycle.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this is with impossible.If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

The enforcement of the inventive method produces the plant of the growth velocity with increase.Thereby, being provided for increasing the method for plant growth rate according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term as used in this article " non-coercing " condition is the envrionment conditions that allows the plant optimum growh.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method give under the non-stress conditions or the plant of under slight drought condition, cultivating with respect to the control plant enhanced yield correlated character of under suitable condition, cultivating.Thereby according to the present invention, be provided for strengthening in output correlated character under the non-stress conditions or in the plant of cultivating under slight drought condition, be particularly useful for increasing the method for output, described method comprises the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide.

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.The example that cultivation is used for the common crop plants of oil plant production comprises soybean, Sunflower Receptacle, cotton, canola oil dish, peanut or palm.The example that cultivation is used for the common crop plants of Starch Production comprises rice, wheat, barley, corn or potato.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

Term as defined herein " PLT transcription factor polypeptide " refers to arbitrary polypeptide of having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ ID NO:191 representative.

Preferably, the PLT transcription factor polypeptide of selection is the PLT transcription factor polypeptide of expressing in the root (underground part) plant basically under its natural genotypic environment.

Also preferably, PLT transcription factor polypeptide comprises a kind of motif, but preferably comprise simultaneously as SEQ ID NO:192PK (V/L) (A/E) the DFLG representative motif 1 and as the motif 2 of SEQ ID NO:209 (V/L) FX (M/V) WN (D/E) representative, wherein X can be arbitrary amino acid; Sequence (V/L) F (T/S/N) (M/V) WN (D/E) of motif 2 with SEQ ID NO:193 preferably.

Increase progressively the protein that example that preferred sequence comprises the PLT transcription factor polypeptide of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity is the sequence representative that provides in the table 1 by the embodiment part with the AP2 structural domain of SEQ ID NO:191 representative.Preferred examples is by SEQ ID NO:176 and SEQ ID NO:178 representative.

AP2 structural domain in the PLT transcription factor polypeptide can use for example SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95,5857-5864 of specialized database; Letunic etc. (2002) Nucleic Acids Res 30,242-244), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and in the function of automatization sequence interpretation. (In) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002)) identified.The AP2 structural domain of PLT transcription factor comprises two tumor-necrosis factor glycoproteins R1 and R2, and each tumor-necrosis factor glycoproteins is about 68 amino acid and separates (Figure 13) by connector area.

Can use as the AP2 structural domain of SEQ ID NO:191 representative and as indicated above be used for the method that aligned sequences is used for comparison and identify.In some cases, can adjust default parameters to regulate the severity of search procedure.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.By this way, can identify almost accurate short coupling, comprise as the motif 1 of SEQ ID NO:192 representative with as SEQ ID NO:209 representative, preferably as the motif 2 of SEQ ID NO:193 representative.

The homologue of PLT transcription factor polypeptide also can be used for implementing method of the present invention.Homologue (or homologous protein) can use conventional techniques well-known in the art as identifying easily by sequence alignment.It is well-known in the art being used for the method that aligned sequences is used for comparison, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses Needleman and Wunsch algorithm ((1970) J Mol Biol 48:443-453) to find the comparison that makes the maximization of coupling number and make minimized two complete sequence of room number.BLAST algorithm (Altschul etc. (1990) JMol Biol 215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI obtainable.It is the methods of marking of percentage ratio and identifying easily with acquiescence pairing comparison parameter and unit that homologue can use ClustalW multiple sequence alignment algorithm (1.83 version) for example.Can carry out trickle edit to optimize the comparison between the conservative motif, apparent as those skilled in the art.

Homologue also comprises directly to homologue and collateral line homologue.Directly can easily find by carrying out so-called interactivity blast search to homologue and collateral line homologue.This can be undertaken by a BLAST, and a wherein said BLAST comprises that search sequence (for example SEQ ID NO:175, SEQID NO:176, SEQ ID NO:177 or SEQ ID NO:178) carries out blast search at arbitrary sequence library (as public's available ncbi database).When nucleotide sequence begins, can use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when protein sequence begins.Randomly can screen BLAST result.The full length sequence of submitting The selection result and non-The selection result subsequently to is with at carry out reverse blast search (the 2nd BLAST) from the sequence of biology, search sequence from described biology, derive (be under the situation of SEQ ID NO:175, SEQ ID NO:176, SEQ IDNO:177 or SEQ ID NO:178 wherein, the 2nd BLAST thereby will be) wherein at arabidopsis thaliana sequence in search sequence.The result who compares first and second blast searches subsequently.If from the high-order position of a BLAST hit be derived from search sequence from identical species of deutero-species wherein, then identify the collateral line homologue; If high-order position hit be not derived from search sequence from identical species of deutero-species wherein, then identify directly to homologue.It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hit because of the chance odds low more).The calculating of E-value is well-known in the art.Under the situation of large-scale family, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.Except the E-value, comparative result is also kept the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.PLT transcription factor homologous peptide thing is to increase progressively preferred sequence and to have at least 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity or similarity (function identity) as the unmodified PLT transcription factor polypeptide of SEQ ID NO:176 or SEQ ID NO:178 representative.It is generally acknowledged that the identity percentage ratio beyond the AP2 structural domain is very low between the PLT transcription factor homologous peptide thing.Preferably, PLT transcription factor homologous peptide thing comprises such AP2 structural domain, and it has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ IDNO:191 representative.Also preferably, PLT transcription factor homologous peptide thing is as being represented by SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQID NO:186, SEQ ID NO:188, SEQ ID NO:200 and SEQ ID NO:202.

The PLT polypeptide can be the derivative of SEQ ID NO:176 or SEQ ID NO:178." derivative " comprises such peptide, oligopeptides, polypeptide, they are compared with the aminoacid sequence of the protein (as a kind of protein that shows in SEQ IDNO:176 or SEQ ID NO:178) of natural existence form, comprise the interpolation of the amino-acid residue that the amino-acid residue that exists with non-natural exists amino acid whose displacement or non-natural.The derivative of SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ IDNO:186, SEQ ID NO:188, SEQ ID NO:200 and SEQ ID NO:202 is the other example that can be suitable in the methods of the invention, and prerequisite is that they have same or analogous biologic activity.

In addition, PLT transcription factor polypeptide (at least with its natural form) generally has dna binding activity and activation structure territory.Those skilled in the art can use conventional techniques and method and determine that easily the existence in activation structure territory and DNA-are in conjunction with activity.Can use standard technique to those skilled in the art easily to be identified with PLT transcription factor polypeptide (for example in transcription complex) interacting proteins.

The example of the nucleic acid of coding PLT transcription factor polypeptide is drawn together but is not limited to those nucleic acid by any representative of SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:199 and SEQ ID NO:201.The variant of the nucleic acid of coding PLT transcription factor polypeptide goes in the inventive method, and prerequisite is that they have same or analogous biologic activity is provided.Suitable variant comprise coding PLT transcription factor polypeptide nucleic acid and/or can with the part of the nucleic acid of the nucleic acid/gene recombination of coding PLT transcription factor polypeptide.Other variant comprises the splice variant and the allelic variant of the nucleic acid of coding PLT transcription factor polypeptide.

Term as defined herein " part " refers to the section of DNA of coded polypeptide, and wherein said polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ ID NO:191 representative.This part also can comprise a kind of motif, but preferably comprises simultaneously as the motif 1 of SEQ ID NO:192 representative with as the motif 2 of SEQ ID NO:209 representative; Preferably, motif 2 has the sequence as SEQ ID NO:193 representative.

Part can for example produce one or more disappearances by the nucleic acid to coding PLT transcription factor polypeptide and prepare.Described part can merge with other coding property (or non-coding) sequence with isolating form use or they, so that for example produce in conjunction with several active protein.When merging with other encoding sequence, it is bigger that the gained polypeptide that produces during translation can be compared the polypeptide that PLT transcription factor part predicted.Preferably, the such polypeptide of described part coding, it has the substantially the same biologic activity of PLT transcription factor polypeptide with SEQ IDNO:176 and SEQ ID NO:178.

Preferably, described part is the part of the nucleic acid of any sequence representative of listing in the Table I by embodiment.Most preferably, this part is the part as the nucleic acid of SEQ ID NO:175 and SEQ ID NO:177 representative.

The another kind of variant of nucleic acid that coding is used for the PLT transcription factor polypeptide of the inventive method is can be under the stringency that reduces, preferably under stringent condition with from the nucleic acid of nucleic acid deutero-probe hybridization as hereinbefore defined, the such polypeptide of hybridization sequences coding wherein, it comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ ID NO:191 representative.Hybridization sequences also can comprise any motif, but preferably comprises simultaneously as the motif 1 of SEQ ID NO:192 representative and/or as SEQ ID NO:209 representative, preferably as the motif 2 of SEQ ID NO:193 representative.

Preferably, this hybridization sequences is such sequence, its can with the nucleic acid of any sequence representative of listing in the Table I by embodiment (or from its probe) hybridization, or with the part hybridization of arbitrary aforementioned sequence (target sequence).Most preferably, hybridization sequences can with SEQ ID NO:175 or with SEQ ID NO:177 (or with from wherein deutero-probe) hybridization.Probe usually less than 1000bp length, preferably less than 500bp length.Usually, for the probe length of DNA-DNA hybridization (as the Southern trace) be 100 and 50bp between change, and the hybridization zone is less than 50 Nucleotide usually but surpasses 10 length of nucleotides in the probe that is used for DNA-DNA hybridization (as at pcr amplification).

PLT transcription factor polypeptide can be encoded by splice variant.Term " splice variant " comprises the variant of nucleotide sequence like this as used in this article, excise, replace, replace in described nucleotide sequence or add selected intron and/or exon, or wherein intron shortens or extends.This type of variant will be the variant of the main biologic activity of retaining protein wherein, and this can realize by the functional section of retaining protein optionally.This type of splice variant can find or can manually produce at occurring in nature.The method that is used to produce this type of splice variant is well-known in the art.

Preferred splice variant is the splice variant of the nucleic acid of coding PLT transcription factor polypeptide, and wherein said PLT transcription factor polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ ID NO:191 representative.This splice variant also can comprise any motif, but preferably comprises simultaneously as the motif 1 of SEQ ID NO:192 representative and/or as SEQ ID NO:209 representative, preferably as the motif 2 of SEQ ID NO:193 representative.

It is splice variant by the nucleic acid of any sequence representative of listing in the table 1 of embodiment that coding comprises other preferred splice variant of the nucleic acid of the PLT transcription factor polypeptide of feature as hereinbefore defined.It most preferably is splice variant as the nucleotide sequence of SEQ ID NO:175 and SEQ ID NO:177 representative.

PLT transcription factor polypeptide also can be by the allelic variant coding of nucleic acid encoding, and wherein said polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the AP2 structural domain that increases progressively preferred sequence and SEQ ID NO:191 representative.This equipotential variant also can comprise any motif, but preferably comprises simultaneously as the motif 1 of SEQ ID NO:192 representative and/or as SEQ ID NO:209 representative, preferably as the motif 2 of SEQ ID NO:193 representative.

Coding comprises the splice variant that the preferred allelic variant of the nucleic acid of the PLT transcription factor polypeptide of feature as hereinbefore defined is the nucleic acid of any sequence representative of listing in the Table I by embodiment.It most preferably is allelic variant as the nucleotide sequence of SEQ ID NO:175 and SEQ ID NO:177 representative.

The increase of the expression of nucleic acid of coding PLT transcription factor polypeptide causes the corresponding mRNA level or the polypeptide level that raise, and this can be equal to the PLT transcription factor polypeptide active of rising; Or should activity also can when the horizontal no change of polypeptide even when the polypeptide level reduces, raise.By producing when having the natural characteristics that more highly active mutant form changes polypeptide than wild type peptide, this situation can occur when for example.

Orthogenesis (or gene reorganization) can be used for producing the variant of the nucleic acid of coding PLT transcription factor polypeptide.This is shuttled back and forth by DNA repeatedly, subsequently suitably screening and/or select with produce coding PLT transcription factor polypeptide or have the homologue of improvement biologic activity or its part nucleic acid variant or its part form (Castle et al., (2004) Science 304 (5674): 1151-4; United States Patent (USP) 5,811,238 and 6,395,547).

Site-directed mutagenesis can be used for producing the variant of the nucleic acid of coding PLT transcription factor polypeptide.Several method can be used for realizing site-directed mutagenesis, the method for the modal PCR of being based on (CurrentProtocols in Molecular Biology.Wiley edits).

Orthogenesis and site-directed mutagenesis are the technical examples that can produce the variant of the nucleic acid of coding PLT transcription factor polypeptide, and wherein said PLT transcription factor polypeptide has the modified activity that is used to implement the inventive method.

The nucleic acid of coding PLT transcription factor polypeptide can be from any natural source or artificial source.Nucleic acid can just be formed and/or operation and modify from its natural form is had a mind to by human in genome environment aspect.PLT transcription factor nucleic acid is preferably from plant, and preferably from dicotyledons, also preferably from Cruciferae, more preferably from Arabidopsis, this nucleic acid is most preferably from Arabidopis thaliana.

The inventive method depends on the expression that the nucleic acid of coding PLT transcription factor polypeptide increases in plant.This nucleic acid can be that total length nucleic acid maybe can be as the defined part of preamble or hybridization sequences or another kind of nucleic acid variant.

The inventive method depends on the expression that the nucleic acid of coding PLT transcription factor polypeptide increases in plant.

The present invention also provides genetic constructs and carrier to promote to import and/or express the nucleotide sequence that is used for the inventive method in plant.

Thereby, gene construct is provided, it comprises:

The nucleic acid of coding PLT transcription factor polypeptide as hereinbefore defined;

One or more regulating and controlling sequences, wherein at least a is the constitutive promoter of medium tenacity.

Used construct can use the well-known recombinant DNA technology of those skilled in the art to make up in the inventive method.This gene construct can insert the carrier that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed, and wherein said carrier can obtain by commercial sources.The present invention thereby the purposes in the methods of the invention of gene construct as hereinbefore defined is provided.

Plant transforms with the carrier (nucleic acid of the PLT transcription factor polypeptide of promptly encoding) that comprises aim sequence.The technician is appreciated that successfully and transforms, selects and breed the host cell that contains aim sequence and the genetic elements that must exist very much on carrier.Aim sequence is connected with one or more regulating and controlling sequences (at least with promotor) effectively.

The constitutive promoter that the nucleic acid of coding PLT transcription factor polypeptide or its variant effectively connect to form type promotor, preferred medium tenacity.Constitutive promoter is in growth and the major part of growing but transcriptional activity was arranged during non-whole stage and express with medium level omnipresence ground basically.Promotor intensity and/or expression pattern can be as analyzing described in " definition " part.Promotor intensity and/or expression pattern can be for example with the seedling of abundant sign preferably with reference to promotor such as Cab27 promotor (weak expression, GenBank AP004700) or the promotor intensity of the protochlorophyllide reductase enzyme promotor of inferring (strongly expressed, GenBank AL606456) and/or expression pattern relatively.Preferably, the promotor that is used for the inventive method is from plant, also preferably from monocotyledons, the most preferred GOS2 promotor (from rice) (SEQ ID NO:194 or alternatively SEQ ID NO:210) that is to use.Be understood that suitability of the present invention is not limited to the nucleic acid by SEQ ID NO:175 or SEQ ID NO:177 representative, the expression of nucleic acids of coding PLT transcription factor polypeptide when suitability of the present invention also is not limited to by the GOS2 promoters driven simultaneously.Also can be used for driving example demonstration in " definition " part of other constitutive promoter of the expression of nucleic acid of coding PLT transcription factor polypeptide.

Randomly, one or more terminator sequences (also being regulating and controlling sequence) can be used in the construct that imports plant.Extra regulatory element can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.Other regulating and controlling sequence (except that promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stabilization element.One skilled in the art will recognize that or can obtain this type of sequence easily.

Genetic constructs of the present invention can also comprise need be used for the replication orgin sequence of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.Genetic constructs can randomly comprise selectable marker gene.

The present invention also comprises by the obtainable plant of the inventive method.The present invention thereby provide by the obtainable plant of the inventive method, plant part or its vegetable cell, wherein said plant or its part or cell comprise the nucleic acid transgenosis that is coded in the PLT transcription factor polypeptide under constitutive promoter, the preferred non-viral constitutive promoter control.

The present invention also provides the method that produces the transgenic plant of the output with increase with respect to control plant, is included in to import in the plant and preferential nucleic acid of expressing coding PLT transcription factor polypeptide.

More specifically, the invention provides the method for the transgenic plant that are used to produce the output with increase, described method comprises:

In plant, import and express the nucleic acid of coding PLT transcription factor polypeptide; With

Cell cultivates plants under the condition that promotes plant-growth and growth.

Nucleic acid can directly import vegetable cell or import plant itself (comprising any other parts that import tissue, organ or plant).According to preferred feature of the present invention, nucleic acid preferably imports plant by transforming.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the expressive gene of plant coding that moves with the goal gene corotation.

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze or quantitative PCR is monitored, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clone's property method of proliferating or classical breeding technique.For example, the first-generation (or T1) transforms plant can carry out the s-generation (or T2) transformant that selfing is isozygotied with generation, and the T2 plant further breeds by classical breeding technique.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's property transformant (for example conversion) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the root stock of the conversion of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring demonstrate with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also comprises host cell, and it contains the nucleic acid of separated coding PLT transcription factor polypeptide.The preferred host cell of the present invention is a vegetable cell.

The present invention also extends to the part gathered in the crops of plant, as is not limited to seed, leaf, fruit, flower, stem, root stock, stem tuber and bulb.The invention further relates to from, preferred directly from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

The present invention also comprises the purposes of nucleic acid of coding PLT transcription factor polypeptide and the purposes of PLT transcription factor polypeptide, is used for increasing in the methods of the invention plant biomass as hereinbefore defined.

The expression that the nucleic acid of coding PLT transcription factor polypeptide increases can be implemented by importing genetic modification (preferably in the locus of PLT transcription factor gene).The locus of gene as defined herein means such genome area, and it comprises goal gene and upstream of coding region or downstream 10kb.

This genetic modification can be for example imports by alternative approach one of as indicated above, for example any (or multiple) by following method: T-DNA activation method, TILLING and homologous recombination method.Be the optional step that expression that the nucleic acid to coding PLT transcription factor polypeptide increases is selected after importing genetic modification, wherein the expression that increases produces the plant of the output with increase.

T-DNA activates the label generation shows the dominant phenotype because of the modification of the gene of the close promotor that imports transgenic plant.Promotor to be imported is the constitutive promoter that can increase the medium tenacity that the nucleic acid of coding PLT transcription factor polypeptide expresses in plant.

Also can use TILLING (genome interior orientation inductive local damage) technology in the locus of coding PLT transcription factor polypeptide, to import genetic modification.

Effect of the present invention also can use homologous recombination to reproduce.With the locus of nucleic acid to be imported (it can be the nucleic acid of coding as preamble defined PLT transcription factor polypeptide or its variant) target to the PLT gene.The nucleic acid for the treatment of target can be the improved allelotrope that is used for replacing native gene, perhaps can be except that described native gene and import.

T-DNA activation method, TILLING and homologous recombination are to produce the genetic modification technical examples of (comprising the expression of nucleic acid in plant that increases coding PLT transcription factor polypeptide).

The nucleic acid of coding PLT transcription factor polypeptide, or PLT transcription factor polypeptide can be used for the procedure of breeding, wherein identifies the dna marker that can be connected with the PLT transcription factor gene hereditarily.Described nucleic acid/gene or PLT transcription factor polypeptide can be used for defining molecule marker.This DNA or protein labeling can be used for selecting to have in the inventive method the plant of increase output as hereinbefore defined subsequently in the procedure of breeding.

The allelic variant of PLT transcription factor nucleic acid/gene also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to handle and import allelic variation by for example using the EMS mutagenesis that plant is made mutagenicity sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be used to select discuss and cause increasing the step of excellent allelic variant of the sequence of output.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises makes certified plant of wherein excellent allelic variant and another kind of plant hybridization.This may be used for for example producing target phenotype combination of features.

The nucleic acid of coding PLT transcription factor polypeptide also can be used as probe so that gene is carried out genetic mapping or physical mapping, they as the part of described gene and as with the mark of the proterties of those gene linkages.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.The nucleotide sequence that this purposes of PLT transcription factor nucleic acid only needs to have at least 15 length of nucleotides.PLT transcription factor nucleic acid can be used as restriction fragment length polymorphism (RFLP) mark.The southern blotting technique of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) can be used PLT transcription factor nuclei acid probe.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the NDA trace of the genomic dna of the restriction endonuclease processing that contains one group of individuality, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for calculating the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of PLT transcription factor nucleic acid in using the previous genetic map that obtains of this colony.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter 4:37-41.Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be can be used for mapping with other population of individuals to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics 16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces the plant of the output with increase, as mentioned before.The output of this increase also can be favourable with economy the combination of other proterties, described proterties as other output strengthen proterties, other inanimate coerced proterties with biological tolerance of coercing, the multiple constructivity feature of adjusting and/or biochemical characteristics and/or physiologic character.

Detailed description to bHLH

The expression of nucleic acid in plant that now has been surprised to find the bHLH transcription factor of regulating the coding certain kinds produced the plant that has the enhanced yield correlated character with respect to control plant.Describe in detail hereinafter and be applicable to the bHLH transcription factor that strengthens the certain kinds of output correlated character in the plant.

The invention provides the method that strengthens output correlated character in the plant with respect to control plant, comprise the expression of nucleic acid in plant of regulating coding certain kinds bHLH transcription factor.

The preferred method that is used for regulating the expression of nucleic acids of (preferred increasing) coding bHLH transcription factor be plant import and express coding as hereinafter further the bHLH of the certain kinds of definition transcribe because of nucleic acid.

The nucleic acid of plant to be imported (and thereby be used for implementing the inventive method) is any nucleic acid of the bHLH transcription factor of the present described type of coding.BHLH as defined herein transcribes because of referring to the polypeptide by any representative of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ ID NO:299 and SEQ ID NO:301.The present invention is illustrated by using rice sequence by the coded polypeptide SEQ ID NO:213 of SEQ ID NO:212 representative to transform plant.From the SEQ IDNO:215 of rice (by SEQ ID NO:214 coding) with from the SEQ ID NO:217 (by SEQ IDNO:216 coding) of rice is the collateral line homologue of the polypeptide of SEQ ID NO:213.From the SEQID NO:219 of Arabidopis thaliana (by SEQ ID NO:218 coding) with from the SEQ ID NO:225 (by SEQ ID NO:224 coding) of Arabidopis thaliana be SEQ ID NO:213 polypeptide directly to homologue.From the SEQ ID NO:221 of Arabidopis thaliana (by SEQ ID NO:220 coding) with from the SEQ IDNO:223 (by SEQ ID NO:222 coding) of Arabidopis thaliana is the variant of SEQ ID NO:218 and SEQ ID NO:219.

Directly can easily find by carrying out so-called interactivity blast search to homologue and collateral line homologue.This generally relates to a BLAST, and a wherein said BLAST comprises that search sequence (for example SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ IDNO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ IDNO:219, SEQ ID NO:224 or SEQ ID NO:225) carries out blast search at arbitrary sequence library (as public's available ncbi database).When nucleotide sequence begins, generally use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when protein sequence begins.Randomly can screen BLAST result.Subsequently with the full length sequence of The selection result and non-The selection result at carry out reverse blast search (the 2nd BLAST) from the sequence of biology, wherein search sequence from described biology (be under the situation of SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQID NO:215, SEQ ID NO:216 or SEQ ID NO:217 wherein in search sequence, the 2nd BLAST thereby will be at the rice sequence; Be under the situation of SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:224 or SEQ ID NO:225 wherein in search sequence, the 2nd BLAST thereby will be at arabidopsis thaliana sequence).The result who compares first and second blast searches subsequently.If from the high-order position of a BLAST hit be derived from search sequence from identical species of deutero-species wherein, so oppositely BLAST produces search sequence subsequently ideally as the highest hitting, and then identifies the collateral line homologue; If for the first time the high-order position among the BLAST hit be not derived from search sequence from identical species of deutero-species wherein, and preferably when reverse BLAST, produce the search sequence that belongs to the highest row that hit, then identify directly to homologue.

It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hit because of the chance odds low more).The calculating of E-value is well-known in the art.Except the E-value, comparative result is also kept the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.Preferably, scoring is greater than 50, more preferably greater than 100; And preferably, the E-value is more preferably less than e-6 less than e-5.Straight detailed description example to homologue and the evaluation of collateral line homologue is provided in this paper embodiment 31 and embodiment 30 respectively.Under the situation of large-scale family, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.

By above mentioned BLAST method obtain directly to the example of homologue be from puncture vine clover (Medicago truncatula) SEQ ID NO:227 (by SEQ ID NO:226 coding) and from the SEQ ID NO:229 (by SEQ ID NO:228 coding) of barley (Horderum vulgare).Other directly can use BLAST method mentioned above and the evaluation easily according to the method that provides to homologue and collateral line homologue in the embodiment part.

The protein of SEQ ID NO:297,299 and 301 representatives is unknown so far.Thereby the present invention also provides the nucleic acid of the unknown so far bHLH transcription factor and the bHLH transcription factor of encoding.

According to another embodiment of the present invention, thereby provide isolated nucleic acid molecule, it comprises:

Nucleic acid by any representative of SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300;

Complementary sequence by the nucleic acid of any representative of SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300;

The nucleic acid of coding bHLH transcription factor, wherein said bHLH transcription factor has (a) with the aminoacid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% of any representative of increasing progressively preferred sequence and SEQ ID NO:297, SEQ ID NO:299 and SEQ ID NO:301,99% or higher sequence identity, and (b) bHLH structural domain.

According to another embodiment of the present invention, thereby provide isolated polypeptide, it comprises:

Aminoacid sequence by any representative of SEQ ID NO:297, SEQ ID NO:299 and SEQ ID NO:301;

Aminoacid sequence, its (a) has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the aminoacid sequence of any representative of increasing progressively preferred sequence and SEQ ID NO:297, SEQ IDNO:299 and SEQ ID NO:301, and (b) has the bHLH structural domain;

At above (i) or the derivative of the arbitrary aminoacid sequence that provides (ii).

Directly all comprise the bHLH structural domain by the polypeptide of any representative of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ IDNO:227, SEQ ID NO:229, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301 or arbitrary aforementioned SEQ ID NO to homologue or collateral line homologue.

The bHLH structural domain is well-known in the art and can be identified by those skilled in the art easily.This family is by the definition of bHLH label construction territory, and wherein said bHLH label construction territory is made of about 60 amino acid, and two visibly different zones of function are arranged.Be positioned at the aminoterminal alkalescence zone of this structural domain participate in DNA in conjunction with and constitute by the amino acid about 15, have a large amount of alkaline residues.Play a role as the dimerization structural domain in the HLH zone of C-terminal and mainly comprise the hydrophobic residue that forms two both sexes spirals, described spiral is separated by sequence and adjustable length ring district.

The bHLH structural domain can use and be used for the method that aligned sequences is used for comparison and identify.In some cases, can adjust default parameters to regulate the severity of search procedure.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.By this way, can identify almost accurate short coupling.

It is well-known in the art being used for the method that aligned sequences is used for comparison, and these class methods comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses Needleman and Wunsch algorithm ((1970) J Mol Biol 48:443-453) to find the overall comparison that makes the maximization of coupling number and make minimized two sequences of room number (the covering whole sequence).BLAST algorithm (Altschul etc. (1990) J Mol Biol 215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI (NCBI) obtainable.It is the methods of marking of percentage ratio and identifying easily with acquiescence pairing comparison parameter and unit that homologue can use ClustalW multiple sequence alignment algorithm (1.83 version) for example.Can carry out trickle edit to optimize the comparison between the conservative motif, apparent as those skilled in the art.

There is the specialized database that is used to identify structural domain.BHLH structural domain in the bHLH transcription factor can use for example SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA95,5857-5864; Letunic etc. (2002) Nucleic Acids Res 30,242-244), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and in the function of automatization sequence interpretation. (In) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002)) identified.HLH structural domain structure is named as IPR001092 and IPR011598 in the InterPro database; Called after PF00010 in the Pfam database; In the SMART database called after SM00353 and in PROSITE called after PS50888.In addition, the comparison result that shows in Figure 18 has highlighted the bHLH structural domain of the bHLH transcription factor that is used for the inventive method.

Directly conservative bHLH structural domain outside show generally that to homologue or collateral line homologue huge sequence departs from by the polypeptide of any representative of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301 or arbitrary aforementioned SEQ ID NO.

Figure 19 a is the matrix that shows proteinic global similarity of bHLH type mentioned above and identity (runic).Even if identity is seemingly low relatively, however the polypeptide with the sequence identity in the scope shown in the matrix that drops on can be considered as arbitrary SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219 or SEQ ID NO:225 directly to homologue or collateral line homologue; This can interactivity BLAST method by mentioned earlier confirm.Generally, the coding nucleic acid that is used for the bHLH transcription factor of the inventive method has at least 13%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher sequence identity to increase progressively preferred sequence and bHLH transcription factor by any representative of SEQ ID NO:213, SEQID NO:215, SEQ ID NO:217, SEQ ID NO:219 or SEQ ID NO:225.

The matrix display device of describing among Figure 19 b is shown in similarity and the identity (runic) in the bHLH structural domain, is higher when wherein this worthwhile right ratio is considered full length protein.Generally, the nucleic acid of bHLH transcription factor that coding is used for the inventive method has such bHLH structural domain, and it has at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher sequence identity to increase progressively preferred sequence and bHLH structural domain by any representative polypeptide of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219 or SEQ ID NO:225.

The aminoacid pattern that is called the 5-9-13 configuration can find on three positions in the alkalescence zone of bHLH structural domain (sees Heim etc., 2003 (Mol.Biol.E the 20th volume (5): Fig. 4 735-747) and Figure 18 herein, wherein the arrow of 3 points upwards shows described configuration).By the polypeptide of any representative of SEQ IDNO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ IDNO:225, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue preferably in the bHLH structural domain, comprise K/R ER configuration, more preferably RER configuration in the general alkaline zone at this structural domain.The existence of this configuration is not a prerequisite of implementing the inventive method, thereby some variations in K/R ER configuration are acceptable.Arabidopis thaliana bHLH polypeptide is divided into 12 subfamilies (seeing Heim etc., Fig. 4 of 2003) according to structural similarity.The member of IX group constitutes the bHLH polypeptide with RER configuration.In addition, a member of VI group comprises the RER configuration.

The structural domain that directly also can comprise (except that the bHLH structural domain with randomly the K/R ER5-9-13 motif, preferred RER motif) called after PFB26111 (seeing the Pfam database) by the polypeptide of any representative of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301 or arbitrary aforementioned SEQ ID NO to homologue or collateral line homologue.This structural domain also is shown in Figure 18.

Usually, the nucleic acid of coding (definition as mentioned) bHLH transcription factor is used for the inventive method, wherein said bHLH transcription factor have the bHLH structural domain and with increase progressively preferred sequence with by SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, the PFB26111 structural domain (seeing Figure 18) of any polypeptide of any representative of SEQ ID NO:299 or SEQ ID NO:301 has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher sequence identity.

Coding needs not be total length nucleic acid by the polypeptide of any representative of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301 or the straight nucleic acid to homologue or collateral line homologue of arbitrary aforementioned SEQ ID NO, uses the total length nucleotide sequence because the enforcement of the inventive method does not rely on.The example of the nucleic acid that is suitable in implementing the inventive method includes but not limited to those nucleic acid by any representative of SEQID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ IDNO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ IDNO:228, SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300.The nucleic acid variant also can use in implementing the inventive method.The example of this type of nucleic acid variant comprises the allelic variant of nucleic acid of splice variant, coding bHLH transcription factor as defined herein of nucleic acid of part, coding bHLH transcription factor as defined herein of the nucleic acid of coding bHLH transcription factor as defined herein and the coding that obtains by the gene reorganization effect variant of the nucleic acid of bHLH transcription factor as defined herein.Term " partly, splice variant, allelic variant and gene reorganization " will be described now.

The part of the nucleic acid of coding bHLH transcription factor as defined herein can for example prepare by this nucleic acid is produced one or more disappearances.Described part can be used or they can merge with other coding property (or non-coding) sequence with isolating form, so that for example produce in conjunction with several active protein.When merging with other encoding sequence, it is bigger that the gained polypeptide that produces during translation can be compared the polypeptide that bHLH transcription factor part predicted.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the plant and import and to express the part of coding by the nucleic acid of arbitrary SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, SEQ ID NO:301 representative bHLH transcription factor, or express the arbitrary aforementioned SEQ ID NO of coding directly to the part of the nucleic acid of homologue, collateral line homologue or homologue.

The such polypeptide of part coding that is used for the inventive method, it has (as indicated above) bHLH structural domain and has substantially the same biologic activity with the straight bHLH transcription factor to homologue or collateral line homologue representative of SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, any representative of SEQ ID NO:301 or arbitrary aforementioned SEQ ID NO.This part generally has at least 150 continuous nucleotide length, preferably at least 300 continuous nucleotide length, more preferably at least 400 continuous nucleotide length and at least 500 continuous nucleotide length most preferably.Preferably, this part is the part as the nucleic acid of SEQ ID NO:212, SEQ ID NO:214, SEQID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:296, SEQ IDNO:298 and any representative of SEQ ID NO:300.Most preferably, this part is the part as the nucleic acid of SEQ ID NO:212 representative.

The another kind of nucleic acid variant that is used for the inventive method is can be under the stringency that reduces, preferably under stringent condition with the nucleic acid hybridization of coding bHLH transcription factor as defined herein, or with the nucleic acid of as defined herein part hybridization.

The such polypeptide of hybridization sequences coding that is used for the inventive method, its have (as indicated above) bHLH structural domain and with bHLH transcription factor by SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, any representative of SEQ ID NO:301 have substantially the same biologic activity or with arbitrary aforementioned SEQ ID NO directly have substantially the same biologic activity to the bHLH transcription factor of homologue or collateral line homologue representative.This hybridization sequences generally has at least 150 continuous nucleotide length, preferably at least 300 continuous nucleotide length, more preferably at least 400 continuous nucleotide length and at least 500 continuous nucleotide length most preferably.Preferably, this hybridization sequences be can with by SEQ ID NO:212, SEQ ID NO:214, SEQ IDNO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:296, SEQ IDNO:298, with arbitrary nucleic acid hybridization of SEQ ID NO:300 representative or the sequence of hybridizing with the part of arbitrary aforementioned sequence, a wherein said part defines as mentioned.Most preferably, hybridization sequences can with as the nucleic acid hybridization of SEQ ID NO:212 representative or with its part hybridization.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, its be included in the plant import and express can with coding by SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, the nucleic acid of the nucleic acid hybridization of any representative bHLH transcription factor of SEQ ID NO:301, or be included in the plant import and express can with the arbitrary aforementioned SEQ ID NO of coding directly to homologue, the nucleic acid of the nucleic acid hybridization of collateral line homologue or homologue.

The another kind of nucleic acid variant that is used for the inventive method is the splice variant of bHLH transcription factor as hereinbefore defined of encoding.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, be included in the plant and import and to express the splice variant of coding by the nucleic acid of SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ ID NO:299, any representative bHLH transcription factor of SEQ ID NO:301, or arbitrary aforementioned SEQ ID NO that encodes directly to the splice variant of the nucleic acid of homologue, collateral line homologue or homologue.

Preferred splice variant is the splice variant of coding by the nucleic acid of SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, any representative bHLH transcription factor of SEQ ID NO:301, or arbitrary aforementioned SEQ ID NO that encodes directly to the splice variant of the nucleic acid of homologue or collateral line homologue.Also preferably by the splice variant of the nucleic acid of any representative of SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300.Most preferably as the splice variant of the nucleic acid of SEQ IDNO:212 representative.

The another kind of nucleic acid variant that is used for implementing the inventive method is the allelic variant of nucleic acid of bHLH transcription factor as hereinbefore defined of encoding.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, it is included in the plant and imports and to express the allelic variant of coding by the nucleic acid of SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, any representative bHLH transcription factor of SEQ ID NO:301, or be included in import in the plant and express the arbitrary aforementioned SEQ ID NO of coding directly to the allelic variant of the nucleic acid of homologue, collateral line homologue or homologue.

Allelic variant can be the allelic variant of coding by the nucleic acid of SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, any representative bHLH transcription factor of SEQ ID NO:301, or arbitrary aforementioned SEQ ID NO that encodes directly to the allelic variant of the nucleic acid of homologue or collateral line homologue.Also preferably by the allelic variant of the nucleic acid of any representative of SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300.Most preferably as the allelic variant of the nucleic acid of SEQ IDNO:212 representative.

The another nucleic acid variant that is used for the inventive method is the nucleic acid variant that obtains by gene reorganization effect.Gene reorganization or orthogenesis also can be used for producing the variant of the nucleic acid of the bHLH transcription factor that coding defines as mentioned.

According to the present invention, be provided for strengthening the method for output correlated character in the plant, it is included in and imports in the plant and express and encode by SEQ ID NO:213, SEQ ID NO:215, SEQ IDNO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ ID NO:297, SEQ IDNO:299, the variant of the nucleic acid of the bHLH transcription factor of any representative of SEQ ID NO:301, or comprise import and express the arbitrary aforementioned SEQ ID NO of coding directly to homologue, the variant of the nucleic acid of collateral line homologue or homologue, wherein said nucleic acid obtains by gene reorganization effect.

In addition, the nucleic acid variant also can be by site-directed mutagenic obtained.Several method can be used for realizing site-directed mutagenesis, the method for the modal PCR of being based on (Current Protocols inMolecular Biology.Wiley edits).

What also be used for the inventive method is such nucleic acid, its coding by the homologue of any aminoacid sequence representative of SEQ ID NO:213, SEQID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301 or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue.

What also be used for the inventive method is such nucleic acid, its coding by the derivative of any aminoacid sequence representative of SEQ ID NO:213, SEQID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:225, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301 or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue.The derivative of SEQ IDNO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:223, SEQ ID NO:225, SEQ ID NO:227 and SEQ ID NO:229, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301 is the other example that can be suitable in the methods of the invention.

In addition, bHLH transcription factor (at least with its natural form) generally has dna binding activity and activation structure territory.Those skilled in the art can use habitual instrument and technology to determine that easily the existence in activation structure territory and DNA-are in conjunction with activity.

The nucleic acid of coding bHLH transcription factor can be from any natural source or artificial source.Nucleic acid can be according to having a mind to operate and natural form is modified by the mankind with regard to composition and/or genome environment aspect.Preferably, the nucleic acid of coding bHLH transcription factor is from plant, also preferably from monocotyledons, more preferably from Gramineae, this nucleic acid most preferably from rice.

In this article bHLH transcription factor arbitrary quoted thereby means the bHLH transcription factor of definition as mentioned.Arbitrary nucleic acid of the described bHLH transcription factor of encoding is suitable in implementing the inventive method.

The present invention also comprises by the obtainable plant of the inventive method or its part (comprising vegetable cell).This plant or its part comprise the nucleic acid transgenosis of the bHLH transcription factor that coding defines as mentioned.

The present invention also provides genetic constructs and carrier to promote to import and/or express the nucleotide sequence that is used for the inventive method in plant.

Thereby, gene construct is provided, it comprises:

Arbitrary nucleic acid of coding bHLH type transcription factor as hereinbefore defined;

With effective one or more regulating and controlling sequences that are connected of the nucleic acid of (i).

Used construct can use the well-known recombinant DNA technology of those skilled in the art to make up in the inventive method.This gene construct can insert the carrier that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed, and wherein said carrier can obtain by commercial sources.The present invention thereby the purposes in the methods of the invention of gene construct as hereinbefore defined is provided.

Plant transforms with the carrier (nucleic acid of the bHLH type transcription factor of promptly encoding) that comprises aim sequence.The technician is appreciated that successfully and transforms, selects and breed the host cell that contains aim sequence and the genetic elements that must exist very much on carrier.Aim sequence is connected with one or more regulating and controlling sequences (at least with promotor) effectively.

Advantageously, the promotor of any kind (no matter being natural or synthetic) can be used for driving the expression of nucleotide sequence.Promotor can be an inducible promoter, is promptly responding to the transcripting starting that has induced or increase when developmental character, chemical, environment or physical property stimulate.Promotor can be a tissue-specific promoter, promptly can preferentially start the promotor of transcribing in some organizes as leaf, root, seed tissue etc.

According to a preferred feature of the present invention, the nucleic acid of coding bHLH type transcription factor is connected with constitutive promoter effectively.Constitutive promoter in the major part of g and D but transcriptional activity is arranged during in fact not being whole stages and basically omnipresence ground express.This constitutive promoter is the GOS2 promotor preferably, more preferably, this constitutive promoter is a rice GOS2 promotor, also preferably by similar to SEQ ID NO:230 or SEQ ID NO:233 basically nucleotide sequence representative, this constitutive promoter is most preferably as SEQ ID NO:233 representative for this constitutive promoter.

Be understood that suitability of the present invention is not limited to encode by the nucleic acid of the bHLH transcription factor of SEQ ID NO:212 representative, this expression of nucleic acids of coding bHLH transcription factor when suitability of the present invention also is not limited to by the GOS2 promoters driven simultaneously.The example that also can be used for implementing other constitutive promoter of the inventive method shows in " definition " part.

Randomly, one or more terminator sequences (also being regulating and controlling sequence) can be used in the construct that imports plant.Extra regulatory element can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.One skilled in the art will recognize that or can obtain this type of sequence easily.

Genetic constructs of the present invention can also comprise need be used for the replication orgin sequence of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.Other regulating and controlling sequence (except that promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stabilization element.Genetic constructs can randomly comprise selectable marker gene.

The present invention also provides the method that produces the transgenic plant with enhanced yield correlated character with respect to control plant, is included in the arbitrary nucleic acid that imports and express coding bHLH type transcription factor polypeptide as hereinbefore defined in the plant.

More specifically, the invention provides the method that is used to produce the transgenic plant with enhanced yield correlated character, described method comprises:

In vegetable cell, import and express the nucleic acid of coding (as defined herein) bHLH type transcription factor; With

Cell cultivates plants under the condition that promotes plant-growth and growth.

Nucleic acid can directly import vegetable cell or import plant itself (comprising any other parts that import tissue, organ or plant).According to preferred feature of the present invention, nucleic acid preferably imports plant by transforming.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the expressive gene of plant coding that moves with the goal gene corotation.

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze or quantitative PCR is monitored, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clone's property method of proliferating or classical breeding technique.For example, the first-generation (or T1) transforms plant can carry out the s-generation (or T2) transformant that selfing is isozygotied with generation, and the T2 plant further breeds by classical breeding technique.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's property transformant (for example conversion) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the root stock of the conversion of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring demonstrate with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also comprises host cell, and it contains the nucleic acid of separated coding bHLH transcription factor as hereinbefore defined.The preferred host cell of the present invention is a vegetable cell.

The present invention also extends to the part gathered in the crops of plant, as is not limited to seed, leaf, fruit, flower, stem, root stock, stem tuber and bulb.The invention further relates to from, preferred directly from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

According to preferred feature of the present invention, the expression of being regulated is the expression that increases.

As mentioned, the preferred method that is used for regulating the expression of nucleic acid of (preferred increasing) coding bHLH transcription factor is the nucleic acid that imports and express coding bHLH transcription factor plant; Yet implement the effect of described method, promptly strengthen the output correlated character and also can use well-known other technology and realize.Hereinafter be to some description in these technology.

A kind of such technology is that T-DNA activates labelization.The transgenic plant that produce show phenotype because of the modification of the gene of the close promotor that imports.Effect of the present invention also can be used TILLING (genome interior orientation inductive local damage) technology and reproduce.Effect of the present invention also can use homologous recombination method to reproduce.

The biomass of quoting the one or more parts that mean plant (weight) to term " enhanced yield correlated character " increases in this article, and described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.

Especially, this type of can gather in the crops part is seed, and the enforcement of the inventive method produces the plant that has the seed production of increase with respect to the seed production of control plant.

With the corn is example, the output increase can show as following one or more indexs: the increase of per hectare or acre plant number, the increase of the increase of every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of corresponding wild-type plant, these plants might show on the corresponding stage in its life cycle the growth velocity that increases (its life cycle during the small part).The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Have increase growth velocity plant in addition can show prematurity.The increase of growth velocity can take place on the one or more stages in life cycle or during whole plants life cycle plant.The growth velocity that increases can reflect enhanced vigor (the seedling vigor that increases) when emerging during plant early stage in life cycle.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this is with impossible.If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term as used in this article " non-coercing " condition is the envrionment conditions that allows the plant optimum growh.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method is with respect to the appropriate control plant of cultivating under suitable condition, gives under the non-stress conditions or the output that increases of the plant of cultivating under slight drought condition.Thereby according to the present invention, the method for output in the plant that is provided for being increased under the non-stress conditions or under slight drought condition, cultivates, described method comprises the expression of nucleic acid in plant that increases coding bHLH transcription factor.

The enforcement of the inventive method is created under the nutritive deficiency condition, especially has the plant that increases output the appropriate control plant of cultivating under the nitrogen shortage condition with respect to cultivating under suitable condition.Thereby, increasing the method for output in the plant that is provided under the nutritive deficiency condition, cultivating according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding MADS15 polypeptide.Nutritive deficiency can because of nutraceutical shortage or excessive due to, as nitrogen, phosphoric acid salt and other P contained compound, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.

According to preferred feature of the present invention, such plant that the enforcement of the inventive method produces, it has the increase growth velocity with respect to control plant, cause early stage vigor, especially at development of plants early stage (general 3 weeks after sprouting under the situation of rice and corn, but these will be different according to species).Thereby, being provided for increasing the method for plant growth rate according to the present invention, described method comprises adjusting, the preferred expression of nucleic acid in plant that increases coding bHLH transcription factor.The present invention thereby the method that is used to obtain have the plant of early stage vigor with respect to control plant also is provided, described method comprises adjusting, the preferred expression of nucleic acid in plant that increases coding bHLH transcription factor.

Early stage vigor can cause also or show as the plant adaptability that increases with respect to control plant that its reason is that for example plant adapts to its environment (promptly more can tackle multiple inanimate or the biological factor of coercing) better because of the plant adaptability that increases with respect to control plant.Plant with early stage vigor also demonstrates crop and better sets up (crop fitly grows, and promptly most of plants reach each stage of growth on the substantially the same time), and shows better growth and often better output.Thereby early stage vigor can be determined by the multiple factor such as growth of seedling speed, thousand seed weight, sprouting percentage ratio, the percentage ratio of emerging, seedling height, root length and seedling biomass and numerous other factors

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.The plant that wherein early stage vigor is special proterties of wishing comprises rice, corn, wheat, Sunflower Receptacle, Chinese sorghum.

The present invention also comprises the purposes of nucleic acid of coding bHLH transcription factor and the purposes of bHLH transcription factor polypeptide, is used to strengthen the output correlated character.

The nucleic acid or the bHLH transcription factor itself of coding bHLH transcription factor polypeptide can be used for the procedure of breeding, wherein identify the dna marker that can be connected with the gene of coding bHLH transcription factor hereditarily.Described nucleic acid/gene or bHLH transcription factor itself can be used for defining molecule marker.This DNA or protein labeling can be used for selecting to have in the inventive method the plant of increase output as hereinbefore defined subsequently in the procedure of breeding.

The allelic variant of the nucleic acid/gene of coding bHLH transcription factor also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to handle and import allelic variation by for example using the EMS mutagenesis that plant is made mutagenicity sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be used to select discuss and cause increasing the step of excellent allelic variant of the sequence of output.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises makes certified plant of wherein excellent allelic variant and another kind of plant hybridization.This can be used for for example producing target phenotype combination of features.

The nucleic acid of coding bHLH transcription factor also can be used as probe so that gene is carried out genetic mapping or physical mapping, they as the gene of the part of described gene and as with the mark of the proterties of those gene linkages.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.The nucleotide sequence that this purposes of the nucleic acid of coding bHLH transcription factor only needs to have at least 15 length of nucleotides.The nucleic acid of coding bHLH transcription factor can be used as restriction fragment length polymorphism (RFLP) mark.The southern blotting technique of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, ALaboratory Manual) can be used the nuclei acid probe of coding bHLH transcription factor.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the southern blotting technique of the genomic dna that contains one group of individuality handling through the restriction enzyme enzymatic nucleic acid, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for the position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) of nucleic acid in using the previous genetic map that obtains of this colony of calculation code bHLH transcription factor.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (1986) Plant Mol.Biol.Reporter 4:37-41.Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be can be used for mapping with other population of individuals to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics 16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces the plant with enhanced yield correlated character, as mentioned before.These proterties also can be favourable with economy the combination of other proterties, described proterties as other output strengthen proterties, other inanimate coerced proterties with biological tolerance of coercing, the multiple constructivity feature of adjusting and/or biochemical characteristics and/or physiologic character.

Detailed description to SPL15

Now being surprised to find the nucleic acid that increases coding SPL15 transcription factor polypeptide expresses in plant and has produced the plant that has the enhanced yield correlated character with respect to control plant, especially has the output of increase.Thereby, the invention provides the method that increases output in the plant with respect to control plant, comprise the expression of nucleic acid in plant that increases coding SPL15 transcription factor polypeptide.

The preferred method that is used for increasing the expression of nucleic acid of coding SPL15 transcription factor polypeptide is the nucleic acid that imports and express coding SPL15 transcription factor polypeptide plant.

Term as defined herein " SPL15 transcription factor polypeptide " refers to such polypeptide, and it comprises from aminoterminal to carboxyl terminal: (i) as the motif 1 of SEQ ID NO:276 representative; And (ii) at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity is arranged with the SPL DBD that increases progressively preferred sequence and SEQ ID NO:277 representative; And (iii) as the motif 2 of SEQ ID NO:278 representative.

The most conservative amino acid is XLXFGXXXYFX in the motif 1, and the most conservative amino acid are DSXXALSLLSX (wherein X are for the different amino acid whose particular subset in each position, as shown in SEQ ID NO:276 and the SEQ ID NO:277) in the motif 2.The arbitrary locational one or more conservative propertys of permission in motif 1 and motif 2 change, and/or arbitrary locational one, two or three non-conservations changes.

In addition, SPL15 transcription factor polypeptide can comprise following any one or two: (a) be in the sequence that is rich in G/S before the SPLDBD; (b) at the W of this polypeptide C-terminal (S/T) L tripeptides.

The example of SPL15 transcription factor polypeptide as hereinbefore defined is as representing in SEQ ID NO:235, and wherein said SPL15 transcription factor polypeptide comprises from aminoterminal to carboxyl terminal: (i) as the motif 1 of SEQID NO:276 representative; And (ii) at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity is arranged with the SPL DBD that increases progressively preferred sequence and SEQ ID NO:277 representative; (iii) as the motif 2 of SEQ ID NO:278 representative; And comprise extraly: (a) be in the SPL DBD sequence that is rich in G/S before; (b) at the W of this polypeptide C-terminal (S/T) L tripeptides.Other this type of example is by SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:283, SEQ ID NO:285, any representative of SEQ ID NO:287 or arbitrary aforementioned SEQ ID NO directly represent to homologue or collateral line homologue.The present invention transforms plant by the arabidopsis thaliana sequence by SEQ ID NO:234 representative with coded polypeptide SEQ ID NO:235 and is illustrated.SEQ ID NO:237 (by SEQ ID NO:236 coding) from Arabidopis thaliana is the collateral line homologue of polypeptide SEQ ID NO:235.SEQ ID NO:239 (is encoded by SEQ ID NO:238, from Aquilegia formosa x Aquilegia pubescens), SEQ ID NO:241 (is encoded by SEQID NO:240, from upland cotton), SEQ ID NO:243 (is encoded by SEQ ID NO:242, from lead a cow (Ipomoea nil)), SEQ ID NO:245 (is encoded by SEQ ID NO:244, from lettuce), SEQ ID NO:247 (is encoded by SEQ ID NO:246, from apple (Malusdomestica)), SEQ ID NO:249 (is encoded by SEQ ID NO:248, from the puncture vine clover), SEQ ID NO:251 (is encoded by SEQ ID NO:250, from Nicotiana bentamiana), SEQ ID NO:253 (is encoded by SEQ ID NO:252, from rice), SEQ ID NO:255 (is encoded by SEQ ID NO:254, from rice), SEQ ID NO:257 (is encoded by SEQ ID NO:256, from potato), SEQ ID NO:259 (is encoded by SEQ ID NO:258, from grape), SEQID NO:261 (is encoded by SEQ ID NO:260, from Zea mays), SEQ ID NO:263 is (by SEQID NO:262, Zea mays), SEQ ID NO:283 is (by SEQ ID NO:282, overgrown with weeds blue or green (Brassicarapa)), SEQ ID NO:285 is (by SEQ ID NO:284, soybean) and SEQ ID NO:287 (by SEQ ID NO:286, Populus tremuloides (Populus tremuloides)) be polypeptide SEQ ID NO:235 directly to homologue.

Directly can easily find by carrying out so-called interactivity blast search to homologue and collateral line homologue.This generally relates to by a BLAST and being undertaken, and a wherein said BLAST comprises that search sequence (for example SEQ ID NO:234 or SEQ ID NO:235) carries out blast search at any sequence library (as public's available ncbi database).When nucleotide sequence begins, can use BLASTN or TBLASTX (using the standard default value), and, can use BLASTP or TBLASTN (use standard default value) when when peptide sequence begins.Randomly can screen BLAST result.Subsequently with the full length sequence of The selection result and non-The selection result at carry out reverse blast search (the 2nd BLAST) from the sequence of biology, wherein search sequence is from described biology (be under the situation of SEQ ID NO:234 or SEQ ID NO:235 in search sequence wherein, the 2nd BLAST thereby will be at arabidopsis thaliana sequence).The result who compares first and second blast searches subsequently.If from the high-order position of a BLAST hit be derived from search sequence from identical species of deutero-species wherein, oppositely BLAST produces search sequence subsequently ideally as the highest hitting, and then identifies the collateral line homologue; If high-order position hit be not derived from search sequence from identical species of deutero-species wherein, and preferably when reverse BLAST, produce the search sequence that belongs to the highest row that hit, then identify directly to homologue.It is that with low E-value those hit that high-order position is hit.The E-value is low more, mark remarkable more (or in other words, this hit because of the chance odds low more).The calculating of E-value is well-known in the art.Except the E-value, comparative result is also kept the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.Straight detailed description example to homologue and the evaluation of collateral line homologue is provided in embodiment 41.Under the situation of large-scale family, can use ClustalW, use subsequently in abutting connection with the tree method to show the cluster of genes involved and so that identify directly to homologue and collateral line homologue so that help.In Figure 22, the collateral line homologue of SPL15 transcription factor polypeptide with directly be in the same place to the homologue cluster.

By SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ IDNO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ IDNO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:283, SEQ IDNO:285, the SPL15DBD that directly all comprises to increase progressively preferred sequence and SEQ ID NO:277 representative to homologue or collateral line homologue of the polypeptide of any representative of SEQ ID NO:287 or arbitrary aforementioned SEQ ID NO has at least 65%, 70%, 75%, 80%, 85%, 90%, the SPL DBD of 95% or 98% sequence identity.

SPL DBD is well-known in the art and can be identified by those skilled in the art easily.SPL DBD can use and be used for the method evaluation that aligned sequences compares.Be used for the method that aligned sequences compares and comprise GAP, BESTFIT, BLAST, FASTA and TFASTA.GAP uses Needleman and Wunsch algorithm ((1970) J Mol Biol 48:443-453) to find the comparison that makes the maximization of coupling number and make minimized two complete sequence of room number.BLAST algorithm (Altschul etc. (1990) J Mol Biol 215:403-10) sequence of calculation identity percentage ratio and execution are to the statistical study of similarity between two sequences.Being used to carry out software that BLAST analyzes and being the public, to pass through NCBI obtainable.Homologue can use for example obtainable ClustalW multiple sequence alignment algorithm (1.83 version) on the GenomeNet server of university student's thing information science center, capital of a country (Kyoto University Bioinformatics Center), identifies easily with acquiescence pairing comparison parameter with in the methods of marking of percentage ratio.Can carry out trickle edit to optimize the comparison between the conservative motif, apparent as those skilled in the art.Comparison result shown in Figure 23 and 24 highlights the SPL DBD in the SPL15 transcription factor polypeptide.Preferably, the SPL15 transcription factor polypeptide that is used for the inventive method comprises the SPL DBD that has at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity with the SPL15 DBD that increases progressively preferred sequence and SEQ ID NO:277 representative.

In some cases, can adjust default parameters to regulate the severity of search procedure.For example use BLAST, can improve the statistical significance threshold value (being called " expectation " value) that is used to report at the coupling of database sequence to show the lower coupling of severity.By this way, can identify almost accurate short coupling.Can identify that by this way wherein said motif all is contained in being used for the SPL15 transcription factor polypeptide of the inventive method (Figure 24) as the motif 1 of SEQ ID NO:276 representative with as the motif 2 of SEQID NO:278 representative.The arbitrary locational one or more conservative propertys of permission in motif 1 and motif 2 change, and/or arbitrary locational one, two or three non-conservations changes.W (S/T) the L tripeptides (Figure 24) that can identify equally at this polypeptide C-terminal.

Also there is the specialized database that is used to identify structural domain.SPL DBD in SPL15 transcription factor polypeptide can use for example SMART (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95,5857-5864; Letunic etc. (2002) Nucleic Acids Res 30,242-244; EMBL by Heidelberg, Germany has), InterPro (Mulder etc., (2003) Nucl.Acids.Res.31,315-318; European bioinformation institute (EBI) by Britain has), Prosite (Bucher and Bairoch (1994) are used for the broad sense collection of illustrative plates grammer of biomolecular sequence motif and in the function of automatization sequence interpretation. (In) ISMB-94; Second molecular biology intelligence system international conference collected works .Altman R., Brutlag D., Karp P., Lathrop R., Searls D. edits, 53-61 page or leaf, AAAIPress, Menlo Park; Hulo etc., Nucl.Acids.Res.32:D134-D137, (2004)), ExPASy protein group server that provides to scientific circles as a service (the Switzerland information biology institute (SIB) by Switzerland has) or Pfam (Bateman etc., Nucleic Acids Research 30 (1): 276-280 (2002) is had by the Sanger institute of Britain) identified.SPL DBD is called after IPR004333 in the InterPro database, in the Pfam database called after PF03110 and in the PROSITE database called after PS51141.

In addition, also can identify exist (Figure 24) that is in the SPL DBD sequence that is rich in G/S before easily.For determining whether the polypeptide structure territory is rich in specific amino acid, the essential amino acid composition (%) can be used from the software program of ExPASy server, ProtParam instrument ((2003) ExPASy such as Gasteiger E: the protein science server .Nucleic Acids Res 31:3784-3788 that is used for deep understanding and analysing protein) calculated especially.The composition of target protein can be formed (%) relatively with the average amino acid in the Swiss-Prot protein sequence database subsequently.In the Swiss-Prot protein sequence database, average Gly (G) content and average Ser (E) content all are 6.9% (adding up to 13.8%).As an example, be in the sequence that is rich in G/S before the SPL DBD of SEQ ID NO:235 and contain 14.5% G and 25.5% S (adding up to 40%).As defined herein, the sequence that is rich in G/S has such merging G and S content (with regard to %), and its average amino acid of protein that is higher than in the Swiss-Prot protein sequence database is formed merging G and the S content that exists in (with regard to %).G and S all belong to minimum amino acid classification.

Coding is by SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ IDNO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ IDNO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ IDNO:283, SEQ ID NO:285, the straight nucleic acid to homologue or collateral line homologue of any representative polypeptide of SEQ ID NO:287 or arbitrary aforementioned SEQ ID NO needs not to be total length nucleic acid, uses the total length nucleotide sequence because the enforcement of the inventive method does not rely on.The example of the nucleic acid that is suitable in implementing the inventive method includes but not limited to the NO:234 by SEQ ID, SEQ ID NO:236, SEQ IDNO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ IDNO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ IDNO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ IDNO:262, SEQ ID NO:282, those nucleic acid of any representative of SEQ ID NO:284 and SEQ ID NO:286.The nucleic acid variant also can use in implementing method of the present invention.The example of this type of variant comprises part, splice variant, the allelic variant of nucleic acid naturally occurring or that obtain by DNA operation.

Part can for example produce one or more disappearances by the nucleic acid to coding SPL15 transcription factor polypeptide as hereinbefore defined and prepare.Described part can be used or they can merge with other coding property (or non-coding) sequence with isolating form, so that for example produce in conjunction with several active protein.When merging with other encoding sequence, it is bigger that the gained polypeptide that produces during translation can be compared the polypeptide that SPL15 transcription factor part predicted.Be used for part coding (as indicated above) SPL15 transcription factor polypeptide of the inventive method, wherein said polypeptide has and arbitrary SEQ IDNO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ IDNO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ IDNO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:283, SEQ IDNO:285, the straight substantially the same biologic activity of SPL15 transcription factor polypeptide of SEQ ID NO:287 representative or arbitrary aforementioned SEQ ID NO to homologue or collateral line homologue representative.The example of part can comprise such Nucleotide, its coding with the SPL DBD that increases progressively preferred sequence and SEQ ID NO:277 representative have at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity as the motif 1 of SEQ ID NO:276 representative with as the motif 2 of SEQ IDNO:278 representative.Part can comprise extraly that coding is rich in the sequence of G/S or the Nucleotide of W (S/T) L tripeptides (being coded in the sequence that is rich in G/S and the Nucleotide of the aminoacid sequence between W (S/T) the L tripeptides but need not to be).Described part generally is at least 250 length of nucleotides, preferably at least 500 length of nucleotides, more preferably at least 750 length of nucleotides and at least 1000 length of nucleotides most preferably.Preferably, described part is the part as the nucleic acid of any representative of SEQ ID NO:234, SEQ IDNO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ IDNO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ IDNO:260, SEQ ID NO:262, SEQ ID NO:282, SEQ ID NO:284 and SEQ IDNO:286.Most preferably, this part is the part as the nucleic acid of SEQ IDNO:234 representative.

The another kind of nucleic acid variant that is used for the inventive method is can be under the stringency that reduces, preferably under stringent condition with the nucleic acid hybridization of coding SPL15 transcription factor polypeptide as defined herein, or with the nucleic acid of as defined herein part hybridization.

Be used for the such polypeptide of hybridization sequences coding of the inventive method, it comprises from aminoterminal to carboxyl terminal: (i) as the motif 1 of SEQ ID NO:276 representative; And (ii) at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% sequence identity is arranged with the SPL DBD that increases progressively preferred sequence and SEQ ID NO:277 representative; (iii) as SEQ ID NO:278 representative motif 2, and with SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ IDNO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ IDNO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ IDNO:283, SEQ ID NO:285, the straight SPL15 transcription factor polypeptide to homologue or collateral line homologue representative of any representative of SEQ ID NO:287 or arbitrary aforementioned SEQID NO has substantially the same biologic activity.Described hybridization sequences generally is at least 250 length of nucleotides, preferably at least 500 length of nucleotides, more preferably at least 750 length of nucleotides and at least 1000 length of nucleotides most preferably.Preferably, described hybridization sequences be can with by SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:282, arbitrary nucleic acid hybridization of SEQ ID NO:284 and SEQ ID NO:286 representative or the sequence of hybridizing with the part of arbitrary aforementioned sequence, wherein said part defines as mentioned.Most preferably, described hybridization sequences can be hybridized with SEQ IDNO:234 or with its part.

The another kind of nucleic acid variant that is used for the inventive method is the splice variant of SPL15 transcription factor polypeptide as hereinbefore defined of encoding.

Preferred splice variant is that coding is by SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ IDNO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ IDNO:263, SEQ ID NO:283, SEQ ID NO:285, or the splice variant of the nucleic acid of the SPL15 transcription factor polypeptide of any representative of SEQ ID NO:287, or encode arbitrary aforementioned SEQ IDNO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ IDNO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ IDNO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:282, SEQ IDNO:284 and SEQ ID NO:286 directly to the splice variant of the nucleic acid of homologue or collateral line homologue.Most preferably as the splice variant of the nucleic acid of SEQ ID NO:234 representative.

The another kind of nucleic acid variant that is used to implement the inventive method is the allelic variant of nucleic acid of SPL15 transcription factor polypeptide as hereinbefore defined of encoding.

Allelic variant can be that coding is by SEQ ID NO:235, SEQ ID NO:237, SEQ IDNO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ IDNO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ IDNO:263, SEQ ID NO:283, SEQ ID NO:285, or the allelic variant of the nucleic acid of the SPL15 transcription factor polypeptide of any representative of SEQ ID NO:287, or arbitrary aforementioned SEQ IDNO that encodes directly to the allelic variant of the nucleic acid of homologue or collateral line homologue.Also preferably by SEQ IDNO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ IDNO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ IDNO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ IDNO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:282, the allelic variant of the nucleic acid of any representative of SEQ IDNO:284 and SEQ ID NO:286.Most preferably as the allelic variant of the nucleic acid of SEQ ID NO:234 representative.

The another nucleic acid variant that is used for the inventive method is the nucleic acid variant that obtains by gene reorganization effect.Gene reorganization or orthogenesis also can be used for producing the variant of the nucleic acid of the SPL15 transcription factor polypeptide that coding defines as mentioned.

In addition, the nucleic acid variant also can be by site-directed mutagenic obtained.Several method can be used for realizing site-directed mutagenesis, the modal method (Current Protocols inMolecular Biology.Wiley (editor)) that is based on PCR.

What also be used for the inventive method is such nucleic acid, and it is encoded by SEQ ID NO:235, SEQID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ IDNO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ IDNO:261, SEQ ID NO:263, SEQ ID NO:283, SEQ ID NO:285, SEQ IDNO:287 representative the homologue of any aminoacid sequence or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue.

What also be used for the inventive method is such nucleic acid, and it is encoded by SEQ ID NO:235, SEQID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ IDNO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ IDNO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ IDNO:261SEQ ID NO:263, SEQ ID NO:283, SEQ ID NO:285, SEQ IDNO:287 representative the derivative of any aminoacid sequence or arbitrary aforementioned SEQ ID NO directly to homologue or collateral line homologue." derivative " comprises such peptide, oligopeptides, polypeptide, they are compared with the aminoacid sequence of the natural existence form of protein (as providing in SEQ ID NO:235), comprise the interpolation of the amino-acid residue that the amino-acid residue that exists with non-natural exists amino acid whose displacement or non-natural.

In addition, SPL15 transcription factor polypeptide (at least with its natural form) generally has dna binding activity and activation structure territory.Those skilled in the art can use habitual instrument and technology and determine that easily the existence in activation structure territory and DNA-are in conjunction with activity.

The nucleic acid of coding SPL15 transcription factor polypeptide can be from any natural source or artificial source.Nucleic acid can have a mind to operate and modified by the mankind with regard to composition and/or genome environment aspect according to its natural form.The nucleic acid of coding SPL15 transcription factor polypeptide is preferably from plant, and also preferably from dicotyledons, more preferably from Cruciferae, this nucleic acid is most preferably from Arabidopis thaliana.

Arbitrary quoting to SPL15 transcription factor polypeptide means the SPL15 transcription factor polypeptide of definition as mentioned in this article.Any nucleic acid of SPL15 transcription factor polypeptide like this of encoding is applicable to the method for the present invention of implementing.

The present invention also provides genetic constructs and carrier to promote to import and/or express the nucleotide sequence that is used for the inventive method in plant.

Thereby, gene construct is provided, it comprises:

The nucleic acid of coding SPL15 transcription factor polypeptide as hereinbefore defined;

With effective one or more regulating and controlling sequences that are connected of the nucleic acid of (i).

Used construct can use the well-known recombinant DNA technology of those skilled in the art to make up in the inventive method.This gene construct can insert the carrier that is suitable for being converted in the plant and is suitable for expressing goal gene in cell transformed, and wherein said carrier can obtain by commercial sources.The present invention thereby the purposes in the methods of the invention of gene construct as hereinbefore defined is provided.

Plant transforms with the carrier (nucleic acid of the SPL15 transcription factor polypeptide of promptly encoding) that comprises aim sequence.The technician is appreciated that successfully and transforms, selects and breed the host cell that contains aim sequence and the genetic elements that must exist very much on carrier.Aim sequence is connected with one or more regulating and controlling sequences (at least with promotor) effectively.

Advantageously, the promotor of any kind (no matter being natural or synthetic) can be used for driving the expression of nucleotide sequence.Promotor can be an inducible promoter, is promptly responding to the transcripting starting that has induced or increase when developmental character, chemical, environment or physical property stimulate.Promotor can be a tissue-specific promoter, promptly can preferentially start the promotor of transcribing in some organizes as leaf, root, seed tissue etc.

According to the present invention, the nucleic acid of coding SPL15 transcription factor polypeptide effectively is connected with constitutive promoter.This constitutive promoter is HMGB (the high speed swimming B of family) promotor preferably, more preferably, this constitutive promoter is a rice HMGB promotor, also preferably by the nucleotide sequence representative similar basically to SEQID NO:279, this constitutive promoter is most preferably as SEQID NO:279 or SEQ ID NO:294 representative for this constitutive promoter.

Be understood that suitability of the present invention is not limited to encode by the nucleic acid of the SPL15 transcription factor polypeptide of SEQ ID NO:234 representative, this expression of nucleic acids of coding SPL15 transcription factor polypeptide when suitability of the present invention also is not limited to by the HMGB promoters driven simultaneously.The example that also can be used for implementing other constitutive promoter of the inventive method shows in " definition " part.

The additional adjustment element that is used to increase nucleic acid or gene or gene product expression can comprise transcriptional enhancer and translational enhancer.One skilled in the art will recognize that the terminator sequence and the enhancer sequence that can be suitable in the embodiment of this invention.Other regulating and controlling sequence (except that promotor, enhanser, silencer, intron sequences, 3 ' UTR and/or 5 ' UTR district) can be protein and/or RNA stabilization element.One skilled in the art will recognize that or can obtain this type of sequence easily.Randomly, one or more terminator sequences (also being regulating and controlling sequence) can be used in the construct that imports plant.

Also can add intron sequences to be increased in the quantity of the ripe information that accumulates in the endochylema to the encoding sequence of 5 ' non-translational region (UTR) or part coding property sequence.

Genetic constructs of the present invention can also comprise need be used for the replication orgin sequence of keeping and/or duplicating in particular cell types.An example is when needs are maintained additive type genetic elements (for example plasmid or clay molecule) with genetic constructs in bacterial cell.Preferred replication orgin includes, but are not limited to f1-ori and colE1.

This genetic constructs can randomly comprise selectable marker gene.

The present invention also provides the method that produces the transgenic plant of the output with increase with respect to control plant, and it is included in the nucleic acid that imports and express coding SPL15 transcription factor polypeptide as hereinbefore defined in the plant.

More specifically, the invention provides the method that produces the transgenic plant of the output with increase with respect to control plant, described method comprises:

In vegetable cell, import and express the nucleic acid of coding SPL15 transcription factor polypeptide; With

Cell cultivates plants under the condition that promotes plant-growth and growth.

Nucleic acid can directly import vegetable cell or import plant itself (comprising any other parts that import tissue, organ or plant).According to preferred feature of the present invention, nucleic acid preferably imports plant by transforming.

Usually after conversion, vegetable cell or cell colony are selected the existence of one or more marks, wherein said mark becomes whole strain plant with the material regeneration that transforms subsequently by the expressive gene of plant coding that moves with the goal gene corotation.

After DNA shifted and regenerates, existence, copy number and/or genome structure that the conversion plant of supposition can for example use Southern to analyze goal gene were estimated.Alternative or extraly, the expression level that newly imports DNA can use Northern and/or Western to analyze or quantitative PCR is monitored, and all technology are that those skilled in the art are well-known.

The conversion plant that produces can be bred by several different methods, as passing through clone's property method of proliferating or classical breeding technique.For example, the first-generation (or T1) transforms plant can carry out the s-generation (or T2) transformant that selfing is isozygotied with generation, and the T2 plant further breeds by classical breeding technique.

The inverting biological that produces can be taked various ways.For example, they can be the mosaics of transformant and non-transformed cell; Clone's property transformant (for example conversion) to contain whole cells of expression cassette; The transplant of transforming tissue and unconverted tissue (for example in plant) with the root stock of the conversion of unconverted grafting of tender branch.

The present invention extends to any vegetable cell or the plant by described arbitrary method generation herein clearly, and extends to whole plant parts and propagulum thereof.The present invention extends further to and comprises the former generation conversion that produces by arbitrary preceding method or the offspring of transfectional cell, tissue, organ or whole strain plant, unique requirement be the offspring demonstrate with by identical yielding characteristics and/or the phenotypic characteristic of those offsprings that parental generation produced in the inventive method.

The present invention also comprises host cell, and it contains the isolating nucleic acid of coding SPL15 transcription factor polypeptide as hereinbefore defined.The preferred host cell of the present invention is a vegetable cell.

The present invention also extends to the part gathered in the crops of plant, as is not limited to seed, leaf, fruit, flower, stem, root stock, stem tuber and bulb.The invention further relates to from, preferred directly from the product in the part gathered in the crops of this kind of plant, as dried particles or powder, oil, fat and lipid acid, starch or protein.

As mentioned, the preferred method that is used for increasing the expression of nucleic acid of coding SPL15 transcription factor polypeptide is the nucleic acid that imports and express coding SPL15 transcription factor polypeptide plant; Yet the effect of implementing described method promptly increases output also can use well-known other technology to realize.Hereinafter be to some description in these technology.

A kind of such technology is that T-DNA activates labelization.The transgenic plant that produce show phenotype because of the modification of the gene of the close promotor that imports.Effect of the present invention also can be used TILLING (genome interior orientation inductive local damage) technology and reproduce.Effect of the present invention also can use homologous recombination method to reproduce.

The biomass (weight) that " output of increase " as defined herein means one or more parts of plant increases, and described part can comprise (can gather in the crops) part and/or underground (can gather in the crops) part on the ground.

Especially, this type of can be gathered in the crops part and comprise trophicity biomass and/or seed, and the enforcement of the inventive method has produced the plant (in trophicity biomass and/or the seed) that has the output of increase with respect to the output of control plant.

With the corn is example, the output increase can show as following one or more indexs: the increase of per hectare or acre plant number, the increase of the increase of every strain plant spike number, the increase of line number, every row grain number, grain weight, thousand seed weight, mealie length/diameter, the full rate of seed (wherein the full rate of seed is that the full seed number is total and multiply by 100 divided by seed) and other.With the rice is example, and itself can show as the increase of following one or more indexs the output increase: the increase of per hectare or acre plant number, every strain plant panicle number, every panicle spikelet number, every panicle flower (Xiao Hua) number (it is expressed as the ratio of full seed number to former panicle number), the full rate of seed (wherein the full rate of seed be the full seed number divided by the seed sum and multiply by 100), the increase of thousand seed weight and other.

Because transgenic plant of the present invention have the output of increase, thereby with respect to the growth velocity of corresponding wild-type plant, these plants might show on the corresponding stage in its life cycle the growth velocity that increases (its life cycle during the small part).The growth velocity that increases can be specific for one or more parts (comprising seed) of plant, or can spread all over whole strain plant basically.Have increase growth velocity plant in addition can show prematurity.The increase of growth velocity can change the harvest cycle of plant, allows the later sowing of plant and/or than early harvest, otherwise this is with impossible.If growth velocity increases fully, can allow further to sow the seed (for example sow and gather in the crops rice plant, sow and gather in the crops other rice plant subsequently, all rice plant is all in a conventional growth period) of identical plant species.Similarly, if growth velocity sufficiently increases, can allow further to sow the seed (for example sowing and harvesting corn plant are for example sowed and optional results soybean, potato or any other suitable plant subsequently) of different plant species.The results additional times also is possible in the situation of some crop plants from identical rhizome.The harvest cycle that changes plant can cause the increase of every acre year biomass yield (number of times (as in a year) that can grow and gather in the crops because of any specified plant increases).The increase of growth velocity also can allow cultivating transgenic plant in the geographic area widely than its wild type counterparts, because the region limits of cultivating crop is often determined by the plantation time (season early) or in the adverse environment condition of results period (season in evening).If shorten harvest cycle, then can avoid this class unfavourable condition.Growth velocity can determine that this type of parameter can be by obtain multiple parameter from growth curve: T-Mid (plant reaches the time that its 50% overall dimension is spent) and T-90 (plant reaches the time that its 50% overall dimension is spent), or the like.

According to preferred feature of the present invention, the enforcement of the inventive method produces planting of the growth velocity that has increase with respect to control plant.Thereby, being provided for increasing the method for plant growth rate according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding SPL15 transcription factor polypeptide.

Compare with control plant, no matter plant is under the non-stress conditions still is that plant is exposed to multiple coercing down, and the increase of output and/or growth velocity all takes place.Plant is generally replied being exposed to coerce to make by growing slowlyer.Under the condition of serious stress of soil condition, plant even can stop growing fully.On the other hand, slightly coerce and be defined as plant in this article any of its exposure coerced, the wherein said ability that does not cause plant to stop growing fully and recover growth of coercing.Compare with the control plant under the non-stress conditions, slightly coerce and in meaning of the present invention, cause being coerced the plant-growth reduction less than 40%, 35% or 30%, preferably, be more preferably less than 14%, 13%, 12%, 11% or 10% or lower less than 25%, 20% or 15%.Because the progress on the agricultural practice (irrigation, fertilising, pesticide treatments) does not often run into condition of serious stress of soil in the raise crop plant.Therefore, by the agriculture often undesirable characteristic that goes up of the impaired growth of slight stress-inducing.Slightly coerce is that the common biological and/or inanimate (environment) that plant exposes is coerced.Inanimate coerce can because of arid or waterlogging, anaerobism are coerced, due to salt stress, chemical toxicity, oxidative stress and heat, cold or the freezing temperature.It can be to coerce (especially because arid), salt stress, oxidative stress or ion by water to coerce the osmotic stress that causes that inanimate is coerced.It generally is that those that caused by pathogenic agent such as bacterium, virus, fungi and insect are coerced that biology is coerced.

Especially, method of the present invention can implemented the plant that has the output of increase with respect to control plant to produce under the non-stress conditions or under slight drought condition.As report in (Planta (2003) 218:1-14) such as Wang, inanimate is coerced a series of morphological change, physiology variation, biological chemistry variation and the molecule that cause influencing unfriendly plant-growth and productivity and is changed.Known arid, salinity, extreme temperature and oxidative stress are also can damaging and primary cellular defect by induced growth by similar mechanism of connecting each other.Rabbani etc. (Plant Physiol (2003) 133:1755-1767) have described " cross-talk " that drought stress and high salinity are coerced a very high degree.For example, arid and/or salinification mainly show as osmotic stress, cause the destruction of cell homeostasis and ion distribution.Often follow the oxidative stress of high temperature or low temperature, salinity or drought stress can cause functional protein and structural protein sex change.Therefore, these various environment-stress usually activate similar cell signal approach and cell response, as producing stress protein matter, raising antioxidant, accumulation compatible solute and growth-inhibiting.Term " non-coercing " condition is the envrionment conditions that allows the plant optimum growh as used in this article.Those skilled in the art know that normal edaphic condition and weather condition for given place.

The enforcement of the inventive method is with respect to the appropriate control plant of cultivating under suitable condition, gives under the non-stress conditions or the output that increases of the plant of cultivating under slight drought condition.Thereby according to the present invention, the method for output in the plant that is provided for being increased under the non-stress conditions or under slight drought condition, cultivates, described method comprises the expression of nucleic acid in plant that increases coding MADS15 polypeptide.

The enforcement of the inventive method is created under the nutritive deficiency condition, especially has the plant that increases output the appropriate control plant of cultivating under the nitrogen shortage condition with respect to cultivating under suitable condition.Thereby, increasing the method for output in the plant that is provided under the nutritive deficiency condition, cultivating according to the present invention, described method comprises the expression of nucleic acid in plant that increases coding MADS15 polypeptide.Nutritive deficiency can because of nutraceutical shortage or excessive due to, as nitrogen, phosphoric acid salt and other P contained compound, potassium, calcium, cadmium, magnesium, manganese, iron and boron etc.

The inventive method advantageously is applicable to any plant.

The plant that is used in particular in the inventive method comprises the whole plants that belong to vegitabilia's superfamily, and especially monocotyledons and dicotyledons comprise feeding or feed beans, ornamental plant, food crop, tree or shrub.According to the preferred embodiment of the invention, plant is a crop plants.The example of crop plants comprises soybean, Sunflower Receptacle, canola oil dish, clover, rape, cotton, tomato, potato and tobacco.Also preferably, plant is a monocotyledons.Monocotyledonous example comprises sugarcane.More preferably, plant is a cereal.The example of cereal comprises rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

The present invention also comprises by the obtainable plant of the inventive method.The present invention thus plant is provided, by the inventive method obtainable part and cell from this type of plant, wherein said plant or part or cell comprise the nucleic acid transgenosis of the SPL15 transcription factor polypeptide that coding defines as mentioned.

The present invention comprises that also the nucleic acid of coding SPL15 transcription factor polypeptide increases the purposes of output in the plant comparing with the output in the control plant.

A kind of purposes like this relates to the output that increases plant, and wherein said output as hereinbefore defined.Output can especially comprise one or more following indexs: the every paniculiform seed sum of number, the seed production that increases, increase, the full seed number of increase, the thousand seed weight (TKW) of increase and the harvest index that increases spent of the over-ground part biomass of increase, increase.

The nucleic acid of coding SPL15 transcription factor polypeptide can be used for the procedure of breeding, wherein identifies the dna marker that can be connected with the gene of coding SPL15 transcription factor polypeptide hereditarily.The nucleic acid of coding SPL15 transcription factor polypeptide can be used for defining molecule marker.This mark can be used for selecting having the plant of the seed production of increase subsequently in the procedure of breeding.The nucleic acid of coding SPL15 transcription factor polypeptide can for example be as SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ IDNO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ IDNO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ IDNO:282, the nucleic acid of any representative of SEQ ID NO:284 and SEQ ID NO:286.

The allelic variant of the nucleic acid of coding SPL15 transcription factor polypeptide also can be used for the auxiliary procedure of breeding of mark.This procedure of breeding needs to handle and import allelic variation by for example using the EMS mutagenesis that plant is made mutagenicity sometimes; Alternatively, this program can be from one group of allelic variant of the non-artificial what is called that causes " nature " origin property.Carry out the evaluation of allelic variant subsequently, for example by the PCR method.After this be the step of the excellent allelic variant of the sequence that is used to select to be discussed and cause increasing seed production.Generally the growth performance that contains the plant of the different allelic variants that sequence is discussed to some extent by monitoring is implemented to select, and wherein said allelic variant for example is SEQ ID NO:234, SEQ IDNO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ IDNO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ IDNO:260, SEQ ID NO:262, SEQ ID NO:282, any different allelic variants of SEQ ID NO:284 and SEQ IDNO:286.Can be in the greenhouse or field monitoring growth performance.Other optional step comprises makes certified plant of wherein excellent allelic variant and another kind of plant hybridization.This can be used for for example producing target phenotype combination of features.

The nucleic acid of coding SPL15 transcription factor polypeptide also can be used as probe so that gene is carried out genetic mapping or physical mapping, described probe as the part of described gene and as with the mark of the proterties of those gene linkages.This type of information can be used for plant breeding, so that exploitation has the strain system that wants phenotype.The nucleotide sequence that this purposes of the nucleic acid of coding SPL15 transcription factor polypeptide only needs to have at least 15 length of nucleotides.The nucleic acid of coding SPL15 transcription factor polypeptide can be used as restriction fragment length polymorphism (RFLP) mark.The southern blotting technique of the plant genome DNA of restrictive diges-tion (Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning, ALaboratory Manual) can be used the nuclei acid probe of coding SPL15 transcription factor polypeptide.What produce carries out genetic analysis to make up genetic map in conjunction with graphic can use a computer subsequently program such as MapMaker (Lander etc. (1987) Genomics 1:174-181).In addition, this nucleic acid can be used for surveying the southern blotting technique of the genomic dna that contains one group of individuality handling through restriction endonuclease, and wherein said one group of individual representative has the parental generation and the offspring of definite genetic cross.The separation of dna polymorphism is marked and is used for the nucleic acid of calculation code SPL15 transcription factor polypeptide formerly uses position (Botstein etc. (1980) Am.J.Hum.Genet.32:314-331) in the genetic map that this colony obtains.

Generation and its purposes in genetic mapping of plant gene deutero-probe have been described in Bernatzky and Tanksley (GENETICS 112 (4): 887-898,1986).Numerous publications have been described the genetic mapping that uses methodology mentioned above or its modification method that specific cDNA is cloned.For example, to hand over group, the group that backcrosses, panmictic population, contiguous isozygotying mutually be that (NIL) and other population of individuals can be used for mapping to F2.This type of methodology is that those skilled in the art are well-known.

It (is the arrangement of sequence on physical map that described nucleic acid probe also can be used for physical mapping; See that Hoheisel etc. exists: Non-mammalian Genomic Analyasis:A Practical Guide, Academic press 1996, the 319-346 pages or leaves and the reference of wherein quoting).

In another embodiment, nucleic acid probe can directly use in fluorescence in situ hybridization (FISH) graphing method (Trask (1991) Trends Genet.7:149-154).(several kb are to a hundreds of kb although large-scale clone is used in current FISH graphing method support; See (1995) Genome Res.5:13-20 such as Laan), however the improvement of sensitivity can allow to use shorter probe to carry out the FISH mapping.

The multiple method based on nucleic acid amplification that is used for genetic mapping and physical mapping can be used described nucleic acid and implement.Example comprises the polymorphism (CAPS of allele specific oligonucleotide TRAP (Kazazian (1989) J.Lab.Clin.Med 11:95-96), pcr amplified fragment; Sheffield etc. (1993) Genomics 16:325-332), allele-specific connects (Landegren etc. (1988) Science241:1077-1080), Nucleotide extension (Sokolov (1990) Nucleic Acid Res.18:3671), radiation hybridization mapping (Walter etc. (1997) Nat.Genet.7:22-28) and Happy graphing method (Dear and Cook (1989) Nucleic Acid Res.17:6795-6807).For these methods, use a kind of sequence of nucleic acid to design and be created in amplified reaction or the primer that in primer extension reaction, uses right.This type of primer design is that those skilled in the art are well-known.In the method for using the PCR-based genetic mapping, possibility must be identified the dna sequence dna difference between mapping intersection parental generation in corresponding to the zone of current nucleotide sequence.Yet this is optional usually for graphing method.

The inventive method produces has the plant that increases output as previously described.These proterties also can make up with other favourable economically proterties, as other output increase proterties, other inanimate are coerced and biological tolerance of coercing the proterties of regulating multiple constructivity feature and/or biochemical characteristics and/or physiologic character.

Accompanying drawing is described

The present invention is described referring now to following accompanying drawing, wherein:

Fig. 1 represents the schematic structure of AtHAL3a polypeptide, and it comprises (i) substrate in conjunction with spiral (adding single underscore), (ii) insert His motif (adding double underline), (iii) PXMNXXMW motif (adding dotted line) and (iv) substrate identification folder (adding wave underline) from aminoterminal to carboxyl terminal.Proteinic N-terminal of HAL3 and C-terminal are not very conservative.

Fig. 2 has shown binary vector pEXP::HAL3, and it is used for the expression that the Arabidopis thaliana HAL3 nucleic acid under the control of β expansion protein promoter increases rice.

Fig. 3 show from Arabidopis thaliana AtHAL3b (At_NP_973994), dichromatism chinese sorghum (Sb), Arabidopis thaliana AtHAL3a (Ath0218), upland cotton (Cg), barley (Hv), rice (Os), grape (Vitisvinifera) (Vv), the multiple ratio of the multiple HAL3 sequence of soybean (Gm), potato (St), Zea mays (Zm) and loose (Pg) is right.

Fig. 4 describes the example of the sequence be used for implementing the inventive method in detail: SEQ ID NO:1 and SEQID NO:2 represent nucleotide sequence and the protein sequence of AtHAl3a.SEQ ID NO:3 and SEQID NO:4 are the sequences that is used for separating the forward primer and the reverse primer of AtHAL3 gene.SEQ IDNO:5 is the sequence that is used for the β expansion protein promoter of the inventive method.That SEQ ID NO:9 to SEQ ID NO:42 representative is used for the inventive method or be used to separate the example of the DNA/ protein sequence of the total length of corresponding full length sequence or part.In some cases, sequence collects from est sequence, has inferior sequencing quality.Therefore, can expect some replacement nucleic acids.

Fig. 5 (A) shows the proteinic structural domain structure of MADS15, and (B) representative has the sequence of the SEQ ID NO:44 of MADS structural domain (boldface letter) and Keratin sulfate structural domain (italics).Between inserting structure between MADS structural domain and Keratin sulfate structural domain, and the C-structural domain is positioned at the carboxyl terminal of Keratin sulfate structural domain.

Fig. 6 shows the proteinic multiple comparison result of multiple MADS15.Identical amino acid represented in asterisk, and colon is represented the high conservative displacement, and point is represented the lower displacement of conservative property.

Fig. 7 shows the binary vector that is used for increasing the coding rice MADS15 proteinic expression of nucleic acid of rice under the control of GOS2 promotor.

Fig. 8 describes the example of the sequence be used for implementing the inventive method in detail.

Fig. 9 is schematically showing of total length OsMADS15 polypeptide.Common structural domain (M, MADS have been shown; I interleaves structural domain; K, Keratin sulfate K box district; C, variable carboxyl petiolarea), show participation DNA combination, dimerization and participation protein zone with following line more than the synoptic diagram with interaction protein multimerization at this.

Figure 10 shows the hair clip construct of use under constitutive promoter (GOS2) control, is used for the binary vector pGOS2::MADS15hp of the reticent effect of rice MADS15RNA.

Figure 11 describes in detail and to be used for the example series implementing the inventive method or be used for separating this type of sequence.Sequence collects from public EST, has inferior sequencing quality.Therefore, can anticipate the minority replacement nucleic acid.When these nucleic acid sequence encoding total lengths MADS15 polypeptide, initiator codon (ATG) and terminator codon have defined nucleotide sequence.Yet 5 ' and 3 ' UTR also can be used to implement the inventive method.

Figure 12 shows according to the schematic classification of the structural domain that exists to AP2/ERF transcription factor polypeptide: have the AP2 subfamily of two AP2 tumor-necrosis factor glycoproteinss and the ERF subfamily of an AP2 tumor-necrosis factor glycoproteins is only arranged.The ERF subfamily further is subdivided into transcription factor, and they comprise B3 structural domain and AP2 tumor-necrosis factor glycoproteins (being called RAV), or do not comprise the AP2 tumor-necrosis factor glycoproteins.PLT transcription factor polypeptide belongs to the AP2 subfamily with two AP2 tumor-necrosis factor glycoproteinss.

Figure 13 represents schematically illustrating of PLT transcription factor polypeptide structure.The AP2 structural domain comprises two the AP2 tumor-necrosis factor glycoproteinss (adding collimation mark goes out) that separated by connector area.Add collimation mark and go out (A/E) approximate location of DFLG and motif 2 (V/L) FX (M/V) WN (D/E) of nuclear localization signal (NLS), motif 1PK (V/L).

Other AP2 structural domain transcription factor polypeptide of Figure 14 and (from following table 4) compares, the comparison result of (from table 4) PLT transcription factor peptide sequence.Add collimation mark and go out nuclear localization signal (NLS), motif 1PK (V/L) (A/E) DFLG and motif 2 (V/L) FX (M/V) WN (D/E).Identical residue insertion of brackets mark, the conservative property residue adds gray background.

Table 4: with the AP2 structural domain transcription factor polypeptide of the PLT transcription factor polypeptide comparison that is used for implementing the inventive method

Title	The NCBI accession number	The source
			Arath_ANT	NM_119937	Arabidopis thaliana
Arath_BBM	NM_121749.1	Arabidopis thaliana
			Brana_BBM1	AF317904	Colea
Brana_BBM2	AF317905	Colea
			Medtr_AP2BBM	AY899909	The puncture vine clover
The Nicta_ANT sample	AY461432	Tobacco (Nicotiana tabacum)
			Orysa_AP2	XM_473084	Rice
Pinth_ANTL1	AB101585	Black pine (Pinus thunbergii)

Zeama_AP2

AY109146.1

Zea mays

Figure 15 compares with 40 seeds from the transgenic plant of the PLT transcription factor expression of polypeptides with increase, from the photo of 40 seeds of control plant (left side).

Figure 16 shows binary vector pGOS2::PLT, and it is used for increasing the rice PLT transcription factor expression of nucleic acids of rice under the control of GOS2 promotor.

Figure 17 describes the example of the sequence (SEQ ID NO:175 to SEQ IDNO:188) that is used to implement the inventive method in detail.Give the partial sequence (SEQ IDNO:189 and SEQ ID NO:190) that is used to separate corresponding full length sequence.

Figure 18 shows the comparison result of bHLH transcription factor as defined herein.(AlignX program MD) is compared for InforMax, Bethesda from Vector NTI software package in the sequence use.Multiple ratio is carried out extending 0.01 with room opening point penalty 10 and room.When needing, implement trickle edit so that locate some conserved regions better.The bHLH structural domain shows that in solid frame the PFB26111 structural domain shows in frame of broken lines.Also by three arrow indication 5-9-13 configurations (K/R ER) that make progress.Single downward arrow indication is used to discern the glutaminic acid residue of E-box.

Figure 19 shows from the similarity between the bHLH transcription factor of a plurality of species and the matrix of identity. identity percentage ratio shows with boldface letter.Figure 19 a is to be the matrix on the bHLH structural domain only at the matrix on the full length sequence and Figure 19 b.In embodiment 34, provide more details.

Figure 20 shows binary vector pGOS2::bHLH, and it is used for increasing the expression of nucleic acids of the coding rice bHLH transcription factor of rice under the control of GOS2 promotor.

Figure 21 describes the example of the sequence be used to implement the inventive method in detail.

Figure 22 is to use (on the GenomeNet server of university student's thing information science center, capital of a country) CLUSTAL W (1.83) and default value, and (Blosum 62 is as weight matrix, room opening point penalty 10; Point penalty 0.05 is extended in the room) the directly adjacent method tree after homologue is done the multiple sequence comparison of whole Arabidopis thaliana SPL transcription factor polypeptide and SPL15 transcription factor polypeptide is exported.Arabidopis thaliana SPL15 transcription factor polypeptide and other SPL15 transcription factor polypeptide directly to homologue and collateral line homologue cluster, shown in curly bracket.

Figure 23 show SPL15 transcription factor polypeptide directly to the comparison result of the DNA-binding domains (DBD) of homologue and collateral line homologue.(AlignX program MD) is compared for InforMax, Bethesda from Vector NTI software package in the sequence use.Multiple ratio is carried out extending 0.01 with room opening point penalty 10 and room.Add collimation mark and go out to participate in conservative Cys of zine ion bonded and His residue.To two minutes nuclear localization signal (NLS) underline.

Figure 24 is that SPL15 transcription factor polypeptide is directly to the comparison result of homologue and collateral line homologue.(AlignX program MD) is compared for InforMax, Bethesda from Vector NTI software package in the sequence use.Multiple ratio is carried out extending 0.01 with room opening point penalty 10 and room.When needing, implement trickle edit so that locate some conserved regions better.Adding collimation mark from aminoterminal to carboxyl terminal goes out three principal character structural domains and is defined as motif 1, SPLDNA binding domains and motif 2.In addition, be in the sequence that is rich in G/S before the SPL DBD with the Xs mark and add collimation mark and go out tripeptides at the W at this polypeptide C-terminal place (S/T) L.

Figure 25 has shown binary vector pHMGB::SPL15, and it is used for increasing the Arabidopis thaliana expression of nucleic acids of the coding SPL15 transcription factor polypeptide of rice under the control of HMGB promotor.

Figure 26 has described the example of the sequence that is used for implementing the inventive method in detail.

Embodiment

The present invention is described referring now to as an illustration the following example only.The following example is not intended to the thoroughly definition or the restriction scope of the invention.

DNA operation: unless stated otherwise, recombinant DNA technology is according to (Sambrook (2001) Molecular Cloning:a laboratory manual, third edition Cold Spring HarborLaboratory Press, CSH, New York) in or Ausubel etc. (1994), CurrentProtocols in Molecular Biology, the standard method of describing in Current Protocols the 1st and 2 volumes is carried out.The standard material and the method that are used for plant molecular work are described at the Plant Molecular Biology Labfax (1993) of the R.D.D.Croy that is published by BIOS ScientificPublications Ltd (UK) and Blackwell Scientific Publications (UK).

Embodiment part A: HAL3

Embodiment 1: identify with the inventive method in the relevant sequence of used nucleotide sequence

Use the database sequence research tool, as basic local comparison instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify (full-length cDNA, EST or genome) sequence relevant in those sequences of in the Entrez Nucleotide database of NCBI (NCBI), being safeguarded with used nucleotide sequence in the inventive method.This program is used for relatively and by the significance,statistical that calculates coupling finding the zone that has local similar between sequence by nucleotide sequence or peptide sequence and sequence library.For example, nucleic acid encoded polypeptide of the present invention is used the TBLASTN algorithm, adopt default setting and filtration to offset to ignore the low-complexity sequence.The result who analyzes relatively shows by pairing property, and according to probability scoring (E-value) ordering, wherein is somebody's turn to do the specific comparison result of scoring reflection because of the accidental probability (the E-value is low more, and the significance of hitting is high more) that takes place.Except the E-value, more also keep the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.In some cases, can adjust default parameters to regulate the severity of search procedure.For example can increase the E-value to show the less coupling of severity.By this way, can identify short almost mating accurately.

Table A provides the nucleotide sequence tabulation relevant with used nucleotide sequence in the inventive method.

Table A: the example of HAL3 polypeptide:

Plant origin	Nucleic acid SEQ ID NO:	Protein s EQ ID NO:
			Arabidopis thaliana	9	10
Rice	11	12
			Common wheat	13	14
Zea mays	15	16
			European spruce (Picea abies)	17	18
Colea	19	20
			Wild cabbage (Brassica oleracea)	21	22
Tobacco	23	24
			Potato	25	26

Plant origin	Nucleic acid SEQ ID NO:	Protein s EQ ID NO:
			Soybean	27	28
Grape	29	30
			Barley	31	32
Upland cotton	33	34
			Dichromatism chinese sorghum	35	36
Tomato	37	38
			Arabidopis thaliana	39	40
Pine	41	42

In some cases, correlated series is by institute such as the tentative compilation of Joint Genome Institute (TIGR) and open to the public.Eukaryotic gene directly can be used for this type of correlated series to homologue (EGO) database, and this can utilize purpose nucleic acid or peptide sequence to carry out by using the BLAST algorithm by keyword search.

Embodiment 2: used nucleotide sequence in clone's the inventive method

Nucleotide sequence used in the inventive method is by PCR, and the Arabidopis thaliana seedling cDNA library of using customization is (in MV Sport 6.0; Invitrogen, Paisley UK) increases as template.Use Hifi Taq archaeal dna polymerase, under standard conditions, utilize the 200ng template in 50 μ lPCR mixtures to carry out PCR.Used primer is prm00957 (SEQ ID NO:3; Justice is arranged, and initiator codon is a boldface letter:

5’aaaaagcaggctcacaatggagaatgggaaaagagac 3’)

And prm00958 (SEQ ID NO:4; Antisense, complementation:

5’agaaagctgggttggttttaactagttccaccg 3’)，

Wherein said primer comprises the AttB site that is used for the Gateway reorganization.The amplification PCR fragment also uses standard method to give purifying.Implement the first step of Gateway method subsequently, i.e. BP reaction, " entering the clone " pHAL3 that reorganization is named according to Gateway with generation in PCR fragment and the pDONR201 plasmid generation body during this period.Plasmid pDONR201 conduct

The part of technology is bought from Invitrogen.

Embodiment 3: expression vector establishment

Entering clone pHAL3 uses with pEXP (a kind of purpose carrier that is used for the rice conversion) in the LR reaction subsequently.This carrier contains as functional element on the T-DNA border: plant selectable marker; Selection markers expression cassette and intention with cloned purpose nucleotide sequence in the described clone of entering the Gateway box of recombinating in the LR body taken place.Be used for the upstream that the specific expressed rice β expansion protein promoter (SEQ ID NO:5) of seedling is positioned at this Gateway box.

After the LR reconstitution steps, the expression vector pEXP::HAL3 (Fig. 2) that produces is converted into agrobacterium strains LBA4044 and subsequent transformation to rice plant.

Embodiment 4: crop transforms

The Agrobacterium that contains the conversion of expression vector is used for transforming rice plant independently.Ripe dry seed shelling with the Japanese Cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, in 2,%Hg,Cl2 30 minutes subsequently, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD600) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant was being cultivated 3 days on the SH inducing culture that contains 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K2SO4 and 100 μ m Syringylethanones in the dark altogether. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 5: evaluation method

Prepare 5.1 estimate

Produce about 30 T0 rice transformant independently.Transformant was transferred to the greenhouse from incubator for tissue culture and was used for growth and results T1 seed former generation.Stay 7 incidents, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under light.

4 T1 incidents T2 from generation to generation in according to as be used for T1 identical evaluation method from generation to generation and do further assessment, but that each incident adopts is more individual.Make plant pass through the digital imagery case for several times until the ripening stage from sowing time.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048x1536 pixel, 1,600 ten thousand colors).

5.2 statistical analysis: t check and F check

Use double factor ANOVA (variance analysis) to be used for the overall evaluation of plant phenotype feature as statistical model.All measuring parameters with whole plants of whole incidents of gene transformation of the present invention are implemented F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action (being called overall gene action again) of checking gene.The threshold value that is used for the significance of true overall gene action is arranged on 5% probability level for F check.Significance F test value indicates gene action, means that the existence of gene not only or position just cause the difference on the phenotype.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " handled in the mode identical with transgenic plant, but isolating plant has therefrom taken place in transgenosis.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

Embodiment 6: evaluation result

With sophisticated main panicle gather in the crops, count, pack, add bar code label and subsequently in loft drier 37 ° of dryings 3 days.Subsequently with panicle threshing and collection and count whole seeds.Use blowing device to separate full grain and empty grain.Discarding empty grain also counts remainder once more.Full grain is weighed on analytical balance.The full grain number that the full seed number passes through behind the counting separating step is determined.The whole full grain that seed ultimate production (seed gross weight) is gathered in the crops from plant by weighing is measured.The capsomere number that every strain plant seed sum is gathered in the crops from plant by counting is measured.Harvest index (HI) is defined as seed ultimate production and over-ground part area (mm in the present invention ²) between ratio, multiply by coefficient 106.As the every paniculiform number of always spending that defines among the present invention is seed sum and the ripe ratio of leading between the paniculiform number.As the full rate of the seed that defines among the present invention is the ratio (be expressed as %) of full seed number to seed (or Xiao Hua) sum.

As shown in Table B, compare with control plant, seed production, full seed number and harvest index increase in transgenic plant, and these transgenic plant are preferentially expressed the nucleic acid of coding HAL3 polypeptide in seedling.

Show B and show that T1 compares with control plant from generation to generation, what calculate from the transgenic event incident is mean yield increase (seed gross weight), the increase of full seed number and the harvest index increase of unit with percentage ratio.

Table B

Parameter % increases the p-value

Seed gross weight 14 0.0072

Full seed several 15 0.0052

Harvest index 10 0.0093

Embodiment 7: early stage vigor evaluation result

Early stage vigor is determined from the sum that is different from the pixel of background in the plant part of ground by counting.This value changes into physical surface value (square millimeter is represented) to the averaging of picture of taking from different perspectives and by correction on identical time point.Result hereinafter described is at the plant that sprouts 3 weeks of back.

See the early stage vigor of plant (as being determined by the over-ground part area) in six incidents of T1 generation 7 incidents, compare with control plant, the over-ground part area of transgenosis seedling is whole to increase by 27%.T2 from generation to generation in further 4 incidents in these T1 incidents of assessment, and compare with control plant, these four incidents cause that all the over-ground part area of transgenosis seedling increases, and compare with control plant, the over-ground part area of transgenosis seedling is whole to increase by 33%.Confirm that also described result is that statistics is significant, the p-value of checking from F-is lower than 0.0001 (T2 from generation to generation), shows that being seen effect may be because due to the position of transgenosis rather than Yin Jiyin or the effect of strain system, see Table C.

Table C

Parameter % increases the p-value

Early stage vigor 33 0.0000

Embodiment part B: the MADS15 of rise

Embodiment 8: identify the sequence relevant with SEQ ID NO:44 with SEQ ID NO:43

Use the database sequence research tool, as basic local comparison instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify in those sequences of in the Entrez Nucleotide database of NCBI (NCBI), being safeguarded relevant (full-length cDNA, EST or the genome) sequence of SEQ ID NO:43 and/or with the relevant protein sequence of SEQ ID NO:44.This program is used for relatively and by the significance,statistical that calculates coupling finding the zone that has local similar between sequence by nucleotide sequence or peptide sequence and sequence library.To using the TBLASTN algorithm, adopt default setting and filtration to offset to ignore the low-complexity sequence by SEQ ID NO:43 encoded polypeptides.The result who analyzes relatively shows by pairing property, and according to probability scoring (E-value) ordering, wherein is somebody's turn to do the specific comparison result of scoring reflection because of the accidental probability (the E-value is low more, and the significance of hitting is high more) that takes place.Except the E-value, more also keep the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.In some cases, can adjust default parameters to regulate the severity of search procedure.

Except can public's available core acid sequence that NCBI obtains, also searching for the patentability sequence library according to same procedure as indicated above.

Table D provides and tabulates as the protein sequence of the nucleotide sequence of SEQ ID NO:43 representative and SEQ ID NO:44 representative relevant nucleotide sequence and protein sequence.

Table D: with the nucleotide sequence that is used for the inventive method (SEQ ID NO:43) relevant nucleotide sequence and corresponding derivation polypeptide.

The title biological SEQ ID NO that originates: database retrieval number

Nucl/ protein

The title biological SEQ ID NO that originates: database retrieval number

Nucl/ protein

VRN-H1 barley 52/53 AAW82995

M5 barley 54/55 CAB97352

TaMADS#11 common wheat 56/57 BAA33457

TaVRT-1 common wheat 58/59 AAP33790

AP1 one grained wheat 60/61 AAO72630

(Triticum monococcum)

VRN-A1 common wheat 62/63 AAW73222

MADS1 rye grass (Lolium perenne) 64/65 AAO45873

MADS1 lolium temulentum (Lolium temulentum) 66/67 AAD10625

M15 Zea mays 68/69 CAD23408

M4 Zea mays 70/71 CAD23417

RMADS211 rice 72/73 AAS59822

MADS14 rice 74/75 AAF19047

Mads2 sinocalamus latiflorus 76/77 AAR32119

(Dendrocalamus latiflorus)

Mads1 sinocalamus latiflorus 78/79 AAR32118

Mads3 Zea mays 80/81 AAG43200

SbMADS2 dichromatism chinese sorghum 82/83 AAB50181

ZAP1 Zea mays 84/85 AAB00081

MADS2 lolium temulentum 86/87 AAD10626

MADS2 rye grass 88/89 AAO45874

The title biological SEQ ID NO that originates: database retrieval number

Nucl/ protein

M8 barley 90/91 CAB97354

FDRMADS3 rice 92/93 AAL09473

MADS15 rice 94/95 AAL09473

TvFL2 spidewort 96/97 AAP83415

(Tradescantia virginiana)

TvFL1 spidewort 98/99 AAP83414

TvFL3 spidewort 10,0/1 01 AAP83416

SQUA1 oil palm (Elaeis guineensis) 102/103 AAQ03221

AlFL allium 104/105 AAP83362

DOMADS2 Dendrobium grex 106/107 AAF13261

Embodiment 9: the comparison of related polypeptide sequence

From the AlignX of Vector NTI (Invitrogen) popular Clustal algorithm (Thompson etc. (1997) Nucleic Acids Res 25:4876-4882 based on the comparison of carrying out property; Chenna etc. (2003), Nucleic Acids Res 31:3497-3500).Can use in abutting connection with the clustering algorithm constructing system and set.Default value is a room opening point penalty 10, and the weight matrix that point penalty 0.1 and selection are extended in the room is Blosum 62 (if comparison polypeptide).

At Fig. 6. the multiple sequence comparison result of the polypeptide of being correlated with in the used polypeptide is used in middle demonstration in identifying enforcement the inventive method.

Embodiment 10: be used to implement the calculating of overall identity percentage ratio between the peptide sequence of the inventive method

Use one of the obtainable method in this area MatGAT (matrix overall comparison instrument) software (BMC Bioinformatics.20034:29.MatGAT: use protein sequence or dna sequence dna to produce the application .Campanella JJ of similarity/identity matrix, Bitincka L, Smalley J; Software by Ledion Bitincka all) be identified for implementing overall similarity percentage ratio and identity percentage ratio between the full-length polypeptide sequence of the inventive method.MatGAT software is that dna sequence dna or protein sequence produce similarity/identity matrix, need not the pre-comparison of data.It is a series of by to comparison that this program uses Myers and Miller overall comparison algorithm (point penalty 2 is extended in room opening point penalty 12 and room) to carry out, and for example uses Blosum 62 (for polypeptide) to calculate similarity and identity and subsequently the result is placed distance matrix.Sequence similarity shows that in marginal lower part sequence identity shows in the marginal upper part in diagonal angle.

Used parameter is relatively:

Matrix: Blosum62 keeps the score

First room: 12

Extend the room: 2

Overall similarity on peptide sequence (exclusive segment peptide sequence) total length and identity software analysis result show in table E.Identity percentage ratio is providing on the diagonal lines and similarity percentage ratio provides under diagonal lines.

Compare with SEQ ID NO:44, the identity percentage ratio that is used to implement between the peptide sequence of the inventive method can hang down to 40% amino acid identity.

Table E: the overall similarity on the peptide sequence total length and the MatGAT result of identity.

	1	2	3	4	5	6	7	8	9	10	11	12	13
														1.SEQID44		70.8	70.0	70.4	70.4	71.5	71.5	70.1	71.6	71.9	72.3	71.5	73.0
2.SEQID53	77.2		99.2	96.3	96.3	95.5	95.1	87.0	86.2	80.6	80.2	81.0	82.9
														3.SEQID55	76.4	99.2		95.5	95.5	94.7	94.3	86.2	85.4	79.8	79.4	80.2	82.1
4.SEQID57	76.0	96.7	95.9		98.8	98.0	97.5	87.0	86.6	81.8	81.4	80.6	82.5
														5.SEQID59	76.4	97.1	96.3	99.2		98.0	98.0	87.0	86.6	81.8	81.4	80.6	82.5
6.SEQID61	77.2	96.7	95.9	98.4	98.8		99.6	87.9	87.4	82.6	82.6	81.0	82.9
														7.SEQID63	77.2	96.3	95.5	98.0	98.8	99.6		87.4	87.0	82.2	82.2	80.6	82.5
8.SEQID65	76.4	92.2	91.4	92.2	92.7	93.5	93.1		97.6	81.7	80.9	82.3	83.8
														9.SEQID67	76.8	92.7	91.8	92.2	92.7	93.5	93.1	98.8		81.3	81.7	82.3	83.8
10.SEQID69	79.4	87.3	86.5	89.0	89.0	89.4	89.0	88.2	88.2		93.5	83.8	85.8
														11.SEQID71	79.4	87.3	86.5	89.0	89.0	89.4	89.0	86.9	87.3	97.1		83.8	85.8
12.SEQID73	80.1	88.1	87.4	86.6	87.0	87.4	87.0	88.5	88.1	91.3	90.9		96.4
														13.SEQID75	79.8	89.8	89.0	88.6	89.0	89.4	89.0	90.7	90.2	93.5	93.1	96.4
14.SEQID77	79.8	91.8	91.0	91.4	91.8	92.6	92.2	90.6	90.2	91.8	91.4	93.3	95.1
														15.SEQID79	80.1	92.2	91.4	91.8	92.2	93.0	92.6	91.0	90.6	92.2	91.8	93.7	95.5
16.SEQID81	86.3	79.3	78.5	77.8	78.1	78.9	78.9	77.0	77.0	81.9	80.7	80.7	80.7
														17.SEQID85	85.7	76.9	76.2	76.6	76.9	77.7	77.7	75.5	75.1	80.2	79.1	79.1	79.1
18.SEQID87	89.1	78.2	77.4	79.3	79.7	79.7	80.1	78.2	77.8	82.8	80.8	81.6	80.8
														19.SEQID89	89.5	78.2	77.4	78.9	78.9	79.3	79.3	78.5	78.2	83.1	81.2	82.0	81.2
20.SEQID91	85.9	75.4	74.6	75.7	75.7	76.4	76.4	75.4	74.6	79.3	78.6	79.0	79.0
														21.SEQID93	91.8	73.0	72.3	73.8	73.8	74.5	74.5	73.0	73.0	76.0	76.8	78.3	76.4
22.SEQID95	99.6	76.8	76.0	76.0	76.0	76.8	76.8	76.0	75.7	78.7	79.0	79.8	79.4
														23.SEQID103	75.3	82.8	82.0	81.6	82.0	82.4	82.4	84.4	84.4	82.4	82.4	85.4	84.0
24.SEQID107	70.4	77.3	76.5	76.5	74.5	77.3	76.1	74.9	74.9	76.5	75.7	75.1	77.3

	14	15	16	17	18	19	20	21	22	23	24
												1.SEQID44	72.7	73.0	82.1	82.2	87.4	87.7	82.5	90.3	99.6	64.7	56.7
2.SEQID53	84.6	85.0	70.7	68.9	71.3	71.3	68.8	65.2	70.4	70.1	61.0
												3.SEQID55	83.7	84.1	70.0	68.1	70.5	70.5	68.1	64.4	69.7	69.3	60.2
4.SEQID57	85.8	86.2	70.4	69.6	72.4	72.4	69.6	65.5	70.0	69.7	62.0
												5.SEQID59	85.8	86.2	70.7	69.6	72.4	72.4	69.6	65.5	70.0	70.1	61.5
6.SEQID61	87.0	87.4	71.1	70.0	72.4	72.4	70.3	67.2	71.2	70.9	62.5
												7.SEQID63	86.6	87.0	71.1	70.0	72.8	72.4	70.3	67.2	71.2	70.9	61.8
8.SEQID65	85.7	86.1	70.5	69.3	71.4	71.8	69.3	66.8	69.8	71.8	60.7
												9.SEQID67	85.7	86.1	71.2	70.1	72.1	72.5	68.6	66.0	70.5	71.8	60.7
10.SEQID69	84.9	85.3	71.5	71.4	73.9	74.3	69.9	67.8	71.9	71.8	62.5
												11.SEQID71	84.5	84.9	71.5	71.4	73.6	73.9	69.6	69.3	71.9	72.2	61.0
12.SEQID73	89.3	89.7	72.0	71.1	72.4	72.8	69.9	68.0	71.2	71.9	60.2
												13.SEQID75	91.5	91.9	73.0	71.8	73.9	74.3	70.7	68.3	72.7	72.0	60.6
14.SEQID77		99.6	71.9	71.1	73.6	73.6	69.9	67.8	72.7	73.3	61.8
												15.SEQID79	99.6		72.2	71.4	73.9	73.9	70.3	68.2	72.7	73.7	62.2
16.SEQID81	79.3	79.6		93.4	81.9	82.3	79.0	76.8	81.8	64.3	56.8
												17.SEQID85	77.7	78.0	96.3		82.5	82.9	81.3	77.0	81.9	63.6	57.2
18.SEQID87	80.5	80.8	87.4	86.1		99.6	88.4	82.5	87.0	64.6	58.0
												19.SEQID89	80.5	80.8	87.8	86.4	99.6		88.8	82.9	87.4	64.6	58.0
20.SEQID91	76.8	77.2	86.2	88.0	90.2	90.6		77.5	82.1	62.7	55.8
												21.SEQID93	76.0	76.4	83.3	82.1	85.4	85.8	81.5		89.9	61.0	54.1
22.SEQID95	79.8	79.8	85.9	85.3	88.8	89.1	85.5	91.4		64.3	56.3
												23.SEQID103	84.8	85.2	75.2	73.6	75.9	75.9	73.6	72.3	74.9		70.1
24.SEQID107	78.5	78.9	71.5	71.4	72.8	72.8	70.3	67.4	70.0	82.4

Embodiment 11: identify at the structural domain that is used to implement comprise in the peptide sequence of the inventive method

(InterPro) database is based on the integrated interface of common used tag database of the search of text and sequence to the integrated resource in protein families, structural domain and site for Integrated Resouce of ProteinFamilies, Domain and Site.The InterPro database has made up these databases, and described database uses different methods to learn biological information with in various degree relevant fully profiling protein matter to obtain protein tag.The cooperation database comprises SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs.Interpro resides at the European bioinformation institute of Britain.

The result of the peptide sequence of InterPro scanning as SEQ ID NO:44 representative is in showing F.

Table F: as the InterPro scanning result of the peptide sequence of SEQ ID NO:44 representative

Database	Searching number	The retrieval title
			PRINTS	PR00404	MADSDOMAIN
PFAM	PF00319	SRF-TF
			SMART	SM00432	MADS
PROFILE	PS50066	MADS_BOX_2
			SUPERFAMILY	SSF55455	The SRF sample
PFAM	PF01486	The K-box
			SUPERFAMILY	SSF46589	The tRNA-brachium conjunctivum
PANTHER	PTHR11945	The MADS box protein
			PANTHER	PTHR11945：SF19	The MADS box protein

Embodiment 12: be used to implement the topology prediction (Subcellular Localization is striden film ...) of the peptide sequence of the inventive method

The Subcellular Localization of TargetP 1.1 prediction eukaryotic proteins.The existence of the prediction that is based on any aminoterminal presequence is distributed in the position: chloroplast transit peptides (cTP), Mitochondrially targeted peptide (mTP) or Secretory Pathway signal peptide (SP).Unactual as keeping the score of final fundamentals of forecasting is probability, and they may not be integrated.Yet having the highest position of keeping the score is most possible according to TargetP, and the relation between keeping the score (reliability category) can be an index of the certainty of prediction.Reliability category (RC) is 1-5, the wherein the strongest prediction of 1 expression.TargetP safeguards on the server of Technical University Of Denmark.

Contain the sequence of aminoterminal presequence for prediction, also can predict the potential cleavage site.

Numerous parameters have been selected, as biology group (non-plant or plant), critical setting (do not have, critical predefine setting or critical user specify settings) with to the calculating of cleavage site prediction (be or deny).

The result who analyzes as the TargetP 1.1 of the peptide sequence of SEQ ID NO:44 representative shows in table G.Select " plant " biological group, undefined threshold value and need the prediction length of transit peptides.Subcellular Localization as the peptide sequence of SEQ ID NO:44 representative can be tenuigenin or nuclear, does not predict transit peptides.

Table G: the TargetP 1.1 as the peptide sequence of SEQ ID NO:44 representative analyzes

Length (AA)	267
		Chloroplast transit peptides	0.088
The mitochondrial transport peptide	0.492
		The Secretory Pathway signal peptide	0.046
Other ubcellular target	0.655
		The position of prediction	Chloroplast(id)
Reliability category	5
		The transit peptides length of prediction	/

Numerous other algorithms can be used for carrying out this alanysis, comprising:

Resident ChloroP 1.1 on Technical University Of Denmark (Technical University of Denmar) server;

At (the Institute forMolecular Bioscience of molecular biosciences institute of Brisbane ,Australia University of Queensland, University of Queensland, Brisbane, resident protein Prowler Subcellular Localization predictor (Protein ProwlerSubcellular Localisation Predictor) is the 1.2nd edition on server Australia);

At Canadian Alberta province Edmonton city University of Alberta (University of Alberta, Edmonton, Alberta, resident PENCE Proteme AnalystPA-GOSUB 2.5 on server Canada);

Resident TMHMM on Technical University Of Denmark's server.

Embodiment 13: with the relevant analytical method of peptide sequence that is used to implement the inventive method

Lim etc. (PMB 44,513-527,2000) have described the two kinds of analytical methods that are used to measure protein-protein interaction that go for MADS15.In the yeast two-hybrid analytical method, the clipped form of MADS1 (comprise complete MADS box, I-structural domain and K-structural domain, but lack the carboxyl terminal part of C-structural domain) is as the interaction of bait with test and MADS15.In external drop-down analytical method, produced GST and merge the MADS1 protein of GST and they are fixed on the glutathione S epharose 4B.The GST/GST-MADS1 of resin-bonded mixes with MADS15 protein or its fragment of 35S-mark.After washing, analyze with interaction protein wash-out and by SDS-PAGE.It will be understood by those skilled in the art that MADS15 protein can be used as bait or also can be fixed on the gsh resin in double cross screening.When using according to the inventive method in addition, MADS15 protein will cause the root output (being measured as the ratio of root biomass to the increase of seedling biomass) that increases in the rice.

Embodiment 14: the clone is as the nucleotide sequence of SEQ ID NO:43 representative

Rice MADS15 gene is by PCR, and (Invitrogen, Paisley UK) increase as template to use rice seedling cDNA library.With extracting behind the RNA of seedling reverse transcription, cDNA is cloned into pCMV Sport 6.0.The insertion sequence mean size in storehouse is that 1.5kb and clone's initial number is 1.59 * 107cfu magnitude.In the original titre of the first round of 6 * 1011cfu/ml amplification back mensuration is 9.6 * 105cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ lPCR mixtures.With primer prm06892 (SEQ ID NO:45; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaaca atgggcgggggaaggt-3 ') and prm06893 (SEQ ID NO:46 justice is arranged, and initiator codon is a boldface letter, and the AttB1 site is an italic:; Antisense, complementation, the AttB2 site is an italics: 5 '-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3 ') be used for pcr amplification, wherein said primer comprises the AttB site that is used for the Gateway reorganization.In standard conditions, use the HifiTaq archaeal dna polymerase to carry out PCR.Also use the PCR fragment of standard method amplification and the about 915bp of purifying.Implement the first step of Gateway method subsequently, i.e. BP reaction is recombinated in PCR fragment and the pDONR201 plasmid generation body during this period to produce " the entering the clone " according to the Gateway name, pMADS15.Plasmid pDONR201 conduct The part of technology is bought from Invitrogen.

Embodiment 15: use as SEQ ID NO:43 representative nucleotide sequence construction of expression vector

Entering clone pMADS15 uses with pGOS2 (a kind of purpose carrier that is used for the rice conversion) in the LR reaction subsequently.This carrier contains as functional element on the T-DNA border: plant selectable marker; The selection markers expression cassette is with intention and cloned the Gateway box of recombinating in the described purpose nucleotide sequence generation LR body that enters in the clone.The rice GOS2 promotor (SEQ ID NO:108, alternatively, SEQ ID NO:47 is same useful) that is used for constitutive expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, the expression vector pGOS2::MADS15 (Fig. 7) of generation is converted among the agrobacterium strains LBA4044 according to method well-known in the art.

Embodiment 16: Plant Transformation

Rice transforms

The Agrobacterium that contains expression vector is used for transforming rice plant.Ripe dry seed shelling with the Japanese Cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, in 2,%Hg,Cl2 30 minutes subsequently, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD600) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978 Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant was being cultivated 3 days on the SH inducing culture that contains 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K2SO4 and 100 μ m Syringylethanones in the dark altogether. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 17: the phenotype evaluation method

Prepare 17.1 estimate

Produce about 35 T0 rice transformant independently.Transformant was transferred to the greenhouse from incubator for tissue culture and was used for growth and results T1 seed former generation.Stay 6 incidents, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under light.

4 T1 incidents T2 from generation to generation in according to as be used for T1 identical evaluation method from generation to generation and do further assessment, but that each incident adopts is more individual.Make plant pass through the digital imagery case for several times until the ripening stage from sowing time.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

17.2 statistical study: F-check

Use double factor ANOVA (variance analysis) to be used for the overall evaluation of plant phenotype feature as statistical model.All measuring parameters with whole plants of whole incidents of gene transformation of the present invention are implemented F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action (being called overall gene action again) of checking gene.The threshold value that is used for the significance of true overall gene action is arranged on 5% probability level for F check.Significance F test value indicates gene action, means that the existence of gene not only or position just cause the difference on the phenotype.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " handled in the mode identical with transgenic plant, but isolating plant has therefrom taken place in transgenosis.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

17.3 the parameter of measuring

From sowing time until the ripening stage, make plant pass through the digital imagery case for several times.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

Plant shoot divides area (or leaf biomass) to measure by counting is different from the pixel of background on the digital picture of dividing from plant shoot sum.This value changes into square physical surface value of mm expression to the averaging of picture of taking from different perspectives on identical time point and by correction.The over-ground part plant area that experiment confirm is measured by this way is relevant with the biomass of ground plant part.The over-ground part area is to have reached area measured on the time point of its maximum leaf biomass plant.Early stage vigor is plant (seedling) over-ground part area of sprouting 3 weeks of back.The increase of root biomass is expressed as the increase of root total biomass (be measured as during plant life viewed maximum biomass); Or be expressed as the increase of root/seedling index (being measured as when the active growth of root and seedling the ratio between interim biomass and the seedling biomass).

Embodiment 18: the phenotype evaluation result of transgenic plant

As mentioned above to plant analysis the time, the inventor finds to compare with lacking the genetically modified plant of MADS15, has more coca output with MADS15 gene construct plant transformed, and it is expressed as root/seedling index.Be 9.4% (p-value 0.0207) among the described T1 of being increased in and be 25.7% (p-value 0.0002) in T2.The p-value shows that described increase is a significance.

Embodiment portion C: the MADS15 of downward modulation

The evaluation of MADS15 correlated series and sign are also referring to embodiment 8 to 13.

Embodiment 19: gene clone

Rice MADS15 gene is by PCR, and (Invitrogen, Paisley UK) increase as template to use rice seedling cDNA library.With extracting behind the RNA of seedling reverse transcription, cDNA is cloned into pCMV Sport 6.0.The insertion sequence mean size in storehouse is that 1.5kb and clone's initial number is 1.59 * 107cfu magnitude.In the original titre of the first round of 6 * 1011cfu/ml amplification back mensuration is 9.6 * 105cfu/ml.After plasmid extracts, the 200ng template is used for 50 μ lPCR mixtures.With primer prm06892 (SEQ ID NO:111; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaacaatggg cgggggaaggt-3 ') and prm06893 (SEQ ID NO:112 justice is arranged, and initiator codon is a boldface letter, and the AttB1 site is an italic:; Antisense, complementation, the AttB2 site is an italics: 5 '-ggggaccactttgtacaagaaagctgggtttggccgacgacgacgac-3 ') be used for pcr amplification, wherein said primer comprises the AttB site that is used for the Gateway reorganization.In standard conditions, use the HifiTaq archaeal dna polymerase to carry out PCR.Also use the PCR fragment of standard method amplification and the about 915bp of purifying.Implement the first step of Gateway method subsequently, i.e. BP reaction is recombinated in hair clip construct and the pDONR201 plasmid generation body during this period to produce " the entering the clone " according to the Gateway name, pMADS15hp.Plasmid pDONR201 conduct

The part of technology is bought from Invitrogen.

Embodiment 20: vector construction

Entering clone pMADS15hp uses with p01519 (a kind of purpose carrier that is used for the inverted repeats construct) in the LR reaction subsequently.This carrier contains as functional element on the T-DNA border: plant selectable marker; To such an extent as to selection markers expression cassette and intention are used for the Gateway box that reorganization is integrated as inverted repeats from the aim sequence that enters the clone in the LR body.The rice GOS2 promotor (SEQ ID NO:174, alternatively, SEQ ID NO:113 is same useful) that is used for constitutive expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, the expression vector with inverted repeats (Figure 10) of generation is converted into agrobacterium strains LBA4044 and subsequent transformation to rice plant.

Embodiment 21: Plant Transformation

Rice transforms

The Agrobacterium that contains expression vector is used for transforming rice plant.Ripe dry seed shelling with the Japanese Cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, in 2,%Hg,Cl2 30 minutes subsequently, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD600) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978 Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant was being cultivated 3 days on the SH inducing culture that contains 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K2SO4 and 100 μ m Syringylethanones in the dark altogether. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 22: to the evaluation method with the MADS15 plant that inverted repeats is transformed under the control of rice GOS2 promotor

Produce about 15-20 independent T0 rice transformant.With former generation transformant be transferred to the greenhouse from incubator for tissue culture and be used for growth and results T1 seed.Stay 8 incidents of inverted repeats construct, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, choose and contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.The T1 plant of selecting is transferred to the greenhouse.Each plant is accepted unique bar coded sticker clearly phenotype somatotype data are connected with corresponding plant.Be provided with down in following environment on the soil of T1 plant in 10cm diameter flowerpot of selecting and cultivate: photoperiod=11.5 hour, day optical density(OD)=30,000lux or higher, daylight temperature=28 ℃ or higher, nocturnal temperature=22 ℃, relative humidity=60-70%.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Make plant pass through the digital imagery case for several times until the ripening stage from sowing time.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

Plant shoot divides area (or leaf biomass) to measure by counting is different from the pixel of background on the digital picture of dividing from plant shoot sum.This value averages and changes into by correction the physical surface value of being represented by square mm to the picture of taking from different perspectives on identical time point.The over-ground part plant area that experiment confirm is measured by this way is relevant with the biomass of ground plant part.Areamax has reached local part area on the time point of its maximum leaf biomass plant.

With sophisticated main panicle gather in the crops, count, pack, add bar code label and subsequently in loft drier 37 ° of dryings 3 days.Subsequently with the panicle threshing and collect whole seeds.Use blowing device to separate full grain and empty grain.After separation, use commercially available counting machine subsequently to two seed batch countings.Discard empty grain.Full grain is weighed on analytical balance and the cross-sectional area of seed uses the digital imagery method to measure.This method produces following seed correlation parameter set:

Each is paniculiform, and to spend number be the parameter of estimating the average number of each panicle Xiao Hua on the plant, this parameter from the seed sum divided by first panicle number.Be regarded as first panicle and count with the whole panicles of the highest panicle eclipsed when the highest panicle and arranged vertical with manual mode.The full grain number that the full seed number passes through behind the counting separating step is determined.The whole full grain that seed ultimate production (seed gross weight) is gathered in the crops from plant by weighing is measured.Every strain plant seed sum is measured by the capsomere number gathered in the crops from plant of counting and corresponding to the Xiao Hua number of every strain plant.Use image analysis software, these parameters in the automatization mode from digital picture and pass through statistical analysis.Each kind of customization apparatus measures subparameter (comprising width, length, area, weight) that use is made of two major portions (weighing device and imaging device and the software that is used for image analysis that is attached thereto).

Use is made gauged double factor ANOVA (variance analysis) is used for the plant phenotype feature as statistical model total appraisal to non-equilibrium design.Whole measuring parameters with whole plants of whole incidents of described gene transformation are implemented the F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action of checking gene (being called " effect of the gene overall situation " again).If it is significant that the value of F check shows these data, then can draw the conclusion that has " gene " effect, mean that not only described effect is just caused in the existence or the position of gene.For the F check, the threshold value that is used for the significance of true overall gene action is arranged on 5% probability level.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " are handled in the mode identical with transgenic plant, but transgenosis has therefrom taken place by isolating plant.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

Embodiment 23: the output correlation parameter of measuring inverted repeats construct transformant:

As mentioned above to seed analysis the time, the inventor finds and lacks the genetically modified plant of MADS15 and compare, has higher seed production with hair clip MADS15 gene construct plant transformed, it is expressed as full seed number, seed gross weight, seed sum and each paniculiform number of spending, and with there being adopted MADS15 gene construct plant transformed to show reverse effect.The p-value shows that described increase is significant.

The result that plant obtained of T1 in the generation gathered the average that on behalf of whole test strains, this result be in table H:

Table H:

% difference p-value

% difference p-value

Full seed number+122 0,0000

Seed gross weight+112 0,0000

Seed sum+27 0,0000

Each paniculiform+25 0,0000

Spend number

Embodiment part D:PLT

Embodiment 24: identify with the inventive method in the relevant sequence of used nucleotide sequence

Use the database sequence research tool, as basic local comparison instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify (full-length cDNA, EST or genome) sequence relevant in those sequences of in the Entrez Nucleotide database of NCBI (NCBI), being safeguarded with used nucleotide sequence in the inventive method.This program is used for relatively and by the significance,statistical that calculates coupling finding the zone that has local similar between sequence by nucleotide sequence or peptide sequence and sequence library.For example, nucleic acid encoded polypeptide of the present invention is used the TBLASTN algorithm, adopt default setting and filtration to offset to ignore the low-complexity sequence.The result who analyzes relatively shows by pairing property, and according to probability scoring (E-value) ordering, wherein is somebody's turn to do the specific comparison result of scoring reflection because of the accidental probability (the E-value is low more, and the significance of hitting is high more) that takes place.Except the E-value, more also keep the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.In some cases, can adjust default parameters to regulate the severity of search procedure.For example can increase the E-value to show the less coupling of severity.By this way, can identify short almost mating accurately.

Following Table I provides the nucleotide sequence tabulation relevant with used nucleotide sequence in the inventive method.

Table I: with the relevant nucleotide sequence of used nucleotide sequence in the inventive method

Title

Nucleic acid SEQ

Polypeptide

The NCBI searching number

The source is biological

	ID NO	SEQ ID NO
						Arath_PLT1	175	176	Total length	NM_112975(At3g20840)	Arabidopis thaliana
Arath_PLT2	177	178	Total length	NM_103997(At1g51190)	Arabidopis thaliana
						Glyma_P LT
	179	180	Total length	BU964973.1 CA783156.1BM309051.1BM309377.1	Soybean
						Glyma_PLT2
	181	182	Total length	BU926204.1BU547204.1CA783156.1BU927164.1	Soybean
						Medtr_PLT	183	184	Total length	AC144930.20	The puncture vine clover
Orysa_PLT	185	186	Total length	NM_190301	Rice
						Zeama_PLT	187	188	Total length	CS155772.1	Zea mays
Lotco_PLT
		189	190	Part	AP007400	Root or stem of Littleleaf Indianmulberry (Lotuscorniculatus)
Poptr_PLT I							199	200	Total length	scaff_III.1595	Populus tremuloides
	Poptr_PLT II
		201	202	Total length	scaff_I.328	Populus tremuloides
	Vitvi_PLT						203	204	Part	AM469514	Grape
Brana_P
		205	206	Part	CN730825	Colea

LT
						Phaco_PLT	207	208	Part	CA902624.1|	Scarlet runner bean (Phaseolus coccineus)

In some cases, correlated series is by institute such as the tentative compilation of Joint Genome Institute (TIGR) and open to the public.Eukaryotic gene directly can be used for this type of correlated series to homologue (EGO) database, and this can utilize purpose nucleic acid or peptide sequence to carry out by using the BLAST algorithm by keyword search.

Embodiment 25: the clone is used for the nucleotide sequence of the inventive method

Nucleotide sequence used in the inventive method is by PCR, and the Arabidopis thaliana cDNA library of using customization is (in MV Sport 6.0; Invitrogen, Paisley UK) increases as template, and wherein said cDNA library begins and produces from extracting from the RNA of different tissues.Under standard conditions, use Hifi Taq archaeal dna polymerase, utilize the 200ng template in 50 μ lPCR mixtures to carry out PCR.The primer of SEQ ID NO:175 (PLT1) of being used for increasing is prm08180 (SEQ IDNO:195; 5 ' GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGATCAATCCACACGGTG 3 ') and prm08181 (SEQ ID NO:196 justice is arranged, and initiator codon is a runic, and the AttB1 site is an italics:; Antisense, complementation, the AttB2 site is an italics: 5 ' GGGGACCACTTTGTACAAGAAAGCTGGGTTCCTTGTTTACTCATTCCACA 3 '), wherein said primer comprises the AttB site that is used for the Gateway reorganization.The primer of SEQ ID NO:177 (PLT2) of being used for increasing is prm08182 (SEQ ID NO:197; 5 ' GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGAATTCTAACAACTGGCTC 3 ') and prm08183 (SEQ ID NO:198 justice is arranged, and initiator codon is a boldface letter, and the AttB1 site is an italics:; Antisense, complementation, the AttB2 site is an italics: 5 ' GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCTTTTATTCATTCCACA 3 '),

The amplification PCR fragment also uses standard method to give purifying.The first step of implementing the Gateway method subsequently is the BP reaction, reorganization is pPLT1 and is pPLT2 for SEQ ID NO:177 for SEQ ID NO:175 producing " the entering the clone " according to the Gateway name in PCR fragment and the pDONR201 plasmid generation body during this period.Plasmid pDONR201 conduct

The component part of technology is bought from Invitrogen.

Embodiment 26: expression vector establishment

Entering clone pPLT1 and pPLT2 uses with the purpose carrier that is used for the rice conversion in the LR reaction subsequently.First carrier contains as functional element on the T-DNA border: plant selectable marker; The selection markers expression cassette is with intention and cloned the Gateway box of recombinating in the described purpose nucleotide sequence generation LR body that enters in the clone.The rice constitutive promoter is the upstream that GOS2 promotor (SEQ ID NO:210, alternatively, SEQ ID NO:194 is same useful) is positioned at this Gateway box.

Second carrier contains the same functionality element on the T-DNA border: plant selectable marker; The selection markers expression cassette is with intention and cloned the Gateway box of recombinating in the described purpose nucleotide sequence generation LR body that enters in the clone.Be used for rice promoters that meristematic tissue expresses and be MT promotor (SEQ ID NO:211) and (PRO0126) be positioned at the upstream of this Gateway box.

After the LR reconstitution steps, be that pGOS2::PLT1, pGOS2::PLT2, pMT::PLT1 and pMT::PLT2 (Figure 16 shows the construct of band GOS2 promotor) are converted into agrobacterium strains LBA4044 and subsequent transformation independently to rice plant with the expression vector that produces.The rice plant's growth that allow to transform is also tested to parameter hereinafter described subsequently.

Embodiment 27: crop transforms

The Agrobacterium that contains the conversion of expression vector is used for transforming rice plant independently.Ripe dry seed shelling with the Japanese Cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, in 2,%Hg,Cl2 30 minutes subsequently, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD600) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

For a construct, produce about 35 independent T0 rice transformant.With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978 Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant was being cultivated 3 days on the SH inducing culture that contains 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K2SO4 and 100 μ m Syringylethanones in the dark altogether. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 28: the phenotype evaluation method

Prepare 28.1 estimate

Evaluation under the normal growth condition

Transformant was transferred to the greenhouse from incubator for tissue culture and was used for growth and results T1 seed former generation.Stay 6 incidents, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under light.

Evaluation under the nitrogen operability that reduces

Rice plant cultivates in potted plant soil under the normal condition except that nutritive medium.Flowerpot is from migrating to ripening period with containing the specific nutrition liquid pouring that nitrogen (N) concentration reduces, usually 7-8 time or number of times still less.(plant maturation, seed results) are identical with the plant of not cultivating under inanimate is coerced during all the other of cultivating.

28.2 statistical study: F-check

Use double factor ANOVA (variance analysis) to be used for the overall evaluation of plant phenotype feature as statistical model.All measuring parameters with whole plants of whole incidents of gene transformation of the present invention are implemented F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action (being called overall gene action again) of checking gene.The threshold value that is used for the significance of true overall gene action is arranged on 5% probability level for F check.Significance F test value indicates gene action, means that the existence of gene not only or position just cause the difference on the phenotype.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " handled in the mode identical with transgenic plant, but isolating plant has therefrom taken place in transgenosis.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

28.3 the parameter of measuring

The biomass correlation parameter is measured

From sowing time until the ripening stage, make plant pass through the digital imagery case for several times.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

Plant shoot divides area (or leaf biomass, maximum area) to measure by counting is different from the pixel of background on the digital picture of dividing from plant shoot sum.This value changes into square physical surface value of mm expression to the averaging of picture of taking from different perspectives on identical time point and by correction.The over-ground part plant area that experiment confirm is measured by this way is relevant with the biomass of ground plant part.The over-ground part area is to have reached area measured on the time point of its maximum leaf biomass plant.Early stage vigor is plant (seedling) over-ground part area of sprouting 3 weeks of back.The increase of root biomass is expressed as the increase of root total biomass (be measured as during plant life viewed maximum biomass); Or be expressed as the increase of root/seedling index (being measured as when the active growth of root and seedling the ratio between interim quality and the seedling quality).

The parameter measurement that seed is relevant

With sophisticated main panicle gather in the crops, count, pack, add bar code label and subsequently in loft drier 37 ° of dryings 3 days.Subsequently with panicle threshing and collection and count whole seeds.Use blowing device to separate full grain and empty grain.Discarding empty grain also counts remainder once more.Full grain is weighed on analytical balance.The full grain number that the full seed number passes through behind the counting separating step is determined.The whole full grain that the seed ultimate production is gathered in the crops from plant by weighing is measured.The capsomere number that every strain plant seed sum is gathered in the crops from plant by counting is measured.Full seed number and the extrapolated thousand seed weight of gross weight (TKW) thereof from counting.Harvest index (HI) is defined as seed ultimate production and over-ground part area (mm in the present invention ²) between ratio, multiply by coefficient 106.As the every paniculiform number of always spending that defines among the present invention is seed sum and the ripe ratio of leading between the paniculiform number.As the full rate of the seed that defines among the present invention is the ratio (be expressed as %) of full seed number to seed (or Xiao Hua) sum.

Embodiment 29: the phenotype evaluation result of transgenic plant

29.1 under constitutive promoter control, express the phenotype evaluation result of the transgenic plant of under the normal growth condition, cultivating of PLT1 or PLT2 nucleotide sequence

To normal growth conditions following the TKW measuring result of the PLT1 that cultivates and the T1 seed of PLT2 transgenosis rice plant in table J with absolute value (average incident) with as the percentage ratio demonstration of comparing with wild-type plant.Compare with wild type seeds, observe contain both one of the obvious increase of transgenic seed aspect TKW of construct.

Table J: to normal growth conditions following the results of TKW measurement of T1 seed of the PLT1 that cultivates and PLT2 transgenic plant.

	TKW(g)	% increases
			The PLT1 transgenic plant	0.0290	16％
The PLT2 transgenic plant	0.0288	15％
			WT	0.0250

Each kind of customization apparatus measures subparameter (comprising width, length, area, weight) that use is made of two major portions (weighing device and imaging device and bonded be used for the software of image analysis) with it.

The result that the seed area of the PLT1 and the T1 seed of PLT2 transgenosis rice plant and seed length are measured in table K with absolute value (average incident) with as the percentage ratio demonstration of comparing with wild-type plant.Compare with wild type seeds, observe the obvious increase of transgenic seed aspect seed area and seed length that contains arbitrary construct.

Table K: to normal growth conditions following the seed areas of T1 seed of the PLT1 that cultivates and PLT2 transgenic plant and the result of seed length measurement

	Seed area (mm 2)	% increases	Seed length (mm)	% increases
					The PLT1 transgenic plant	27.60	12％	9.4	13％
The PLT2 transgenic plant	27.35	11％	9.4	13％
					WT	24.56		8.3

The seed width is not subjected to the T1 seed influence (data not shown) of PLT and PLT2 transgenic plant significantly.

29.2 under constitutive promoter control, express the phenotype evaluation result of the transgenic plant of under nitrogen operability reduction condition, cultivating of PLT1 or PLT2 nucleotide sequence

To nitrogen operability reduction condition following the result that measures of the TKW of the PLT1 that cultivates and the T1 seed of PLT2 transgenosis rice plant in table L with absolute value (average incident) with as the percentage ratio demonstration of comparing with wild-type plant.Compare with wild type seeds, observe the obvious increase of transgenic seed aspect TKW that contains arbitrary construct.

Table L: to the result of the TKW measurement of the T1 seeds of PLT1 that cultivates of the following institute of nitrogen operability reduction condition and PLT2 transgenic plant.

	TKW(g)	% increases
			The PLT1 transgenic plant	0.0295	12％
The PLT2 transgenic plant	0.0278	8％
			WT	0.0256

29.3 under the control of meristematic tissue specificity promoter, express the phenotype evaluation result of the transgenic plant of under the normal growth condition, cultivating of PLT2 nucleotide sequence

The result that the TKW of the T1 seed of the following PLT2 transgenosis rice plant that cultivates of normal growth conditions is measured in table M with absolute value (average incident) with show that as the percentage ratio of comparing with wild-type plant wherein said PLT2 transgenosis rice plant is expressed in the PLT2 nucleotide sequence of meristematic tissue specificity promoter under controlling.Compare with wild type seeds, observe the obvious increase of transgenic seed aspect TKW that contains construct.

Table M: to the result of the TKW measurement of the T1 seeds of the following PLT2 transgenic plant of cultivating of normal growth conditions, wherein said PLT2 transgenosis rice plant is expressed in the PLT2 nucleotide sequence of meristematic tissue specificity promoter under controlling.

	TKW(g)	% increases
			The PLT2 transgenic plant	0.0263	3％
WT	0.0254

Embodiment part E:bHLH

Embodiment 30: the collateral line homologue of identifying the bHLH of SEQ ID NO:213 in the rice

Use SEQID 2, utilize the collateral line homologue in the BLASTP algorithm search rice genome, wherein said BLASTP algorithm is used at the similarity searching of given protein sequence database execution protein search sequence.The Protein Data Bank of being inquired about is corresponding to comprising 59,712 sequences; Rice protein groups-MIPS rice the database (MOsDB) of the MIPS institute of 27,051,637 total letters.The result according to as from the highest keep the score and minimum E-value the highest determined similarity sort.Identify with SEQID NO:2 is other and have at least 50 keep the score and be lower than the E-value of e-05 to hitting of homologous bHLH protein sequence.Pairing comparison result between the collateral line homologue that hereinafter is presented at search sequence and from the beginning identifies.

Inquiry: SEQID 2

Minimum

Sum

Maximum probability

Produce the high right sequence of section of keeping the score: the P that keeps the score (N) N

9629.m00093| protein spiral-ring-helical dna-binding domains ... 1132 1.3e-115 1

9629.m00090| protein spiral-ring-helical dna-binding domains ... 305 5.7e-28 1

9630.m01262| protein spiral-ring-helical dna-binding domains ... 189 1.1e-15 1

9629.m07159| protein spiral-ring-helical dna-binding domains ... 112 3.4e-05 1

9629.m00093| proteinHelix-loop-helix DNA-binding domains, predictive

Length=225

Keep the score=1132 (403.5 hit) expected value=1.3e-115, P=1.3e-115

Identity=224/224 (100%), positive=224/224 (100%)

Inquiry: 1 MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLC LKLS 60

MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS

Sbjct： 1 MKSRKNSTTSTKAAGSCHTSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLS 60

Inquiry: 61 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKK RKQQ 120

SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ

Sbjct： 61 SLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ 120

Inquiry: 121 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEE EGAE 180

AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE

Sbjct： 121 AAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAE 180

Inquiry: 181 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224

VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY

Sbjct： 181 VVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLLLQY 224

9629.m00090| proteinHelix-loop-helix DNA-binding domains, predictive

Length=363

Keep the score=305 (112.4 hit) expected value=5.7e-28, P=5.7e-28

Identity=87/230 (37%), positive=126/230 (54%)

Inquiry: 19 TSSSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHH YSTS 78

TSSSG G +++++ ERK++E+ RR MKGLC+KL+SLIP + H S

Sbjct： 18 TSSSGSGASS----TAAAAAAERKEMERRRRQDMKGLCVKLASLIP-----KEHCSMSKM 68

Inquiry: 79 SSSSPPSSTKEAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ----------------AA 122

++S TQL L++AAAYIK+LK R+DEL ++ AA

Sbjct： 69 QAASR--------TQLGSLDEAAAYIKKLKERVDELHHKRSMMSITSSRCRSGGGGGPAA 120

Inquiry: 123 ALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIRE------------ERERAVR LHEV 170

A STS GGGG ++ VV V +++E R V+ H+V

Sbjct： 121 AAGQSTSGGGGGEEEEEDMTRTTAAAAVVEVRQHVQEGSLISLDVVLICSAARPVKFHDV 180

Inquiry: 171 IGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNL 220

IVLEEEGA++++A+FS+ +YT++S+A SRIG++ASR+S RLR L

Sbjct： 181 ITVLEEEGADIISANFSLAAHNFYYTIYSRAFSSRIGIEASRISERLRAL 230

9630.m01262| proteinHelix-loop-helix DNA-binding domains, predictive

Length=211

Keep the score=189 (71.6 hit) expected value=1.1e-15, P=1.1e-15

Identity=66/214 (30%), positive=102/214 (47%)

Inquiry: 21 SSGGGGGGGNCYSSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHH-HHY STSS 79

S+GGGGGGG K +RK E+ RR M L L SL+ +A P + +S S+

Sbjct： 7 SAGGGGGGG--------KPDRKTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSN 58

Inquiry: 80 SSSPPSSTK------EAVTQLDHLEQAAAYIKQLKGRIDELKKRKQQ---AAALTT STSN 130

S + A T+ D L AA YI+Q + R+D L+++K++ +S+S+

Sbjct： 59 SKYHQRKLRILGGAAAATTRPDRLGVAAEYIRQTQERVDMLREKKRELTGGGGGGSSSSS 118

Inquiry: 131 GGGGGM---PVVEVRCQDGTLDVVVVSEAIREERERAVRLHEVIGVLEEEGAEVVN ASFS 187

G G P VEV+ L ++ + A + H + +E+ G +V NA FS

Sbjct： 119 GAGAATAAAPEVEVQHLGSGLHAILFTGAPPTD---GASFHRAVRAVEDAGGQVQNAHFS 175

Inquiry: 188 VVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLL 221

V G K YT+H+ G++ RV RL+ +

Sbjct： 176 VAGAKAVYTIHAMIGDGYGGIE-RVVQRLKEAI 207

Embodiment 31: identify SEQ ID NO:213 in the Arabidopis thaliana bHLH directly to homologue

Use SEQID NO:2, utilize in the BLASTP algorithm search arabidopsis gene group directly to homologue, wherein said BLASTP algorithm is used for carrying out at given protein sequence database the similarity searching of protein search sequence.The Protein Data Bank of being inquired about is corresponding to comprising 26,735 sequences; Arabis protein group-MIPS Arabidopis thaliana the database (MAtDB) of the MIPS institute of 11,317,104 total letters.The result according to as from the highest keep the score and minimum E-value the highest determined similarity sort.Identify with SEQID NO:2 and directly have at least 50 keep the score and be lower than the E-value of e-05 to hitting of homologous bHLH protein sequence.The straight pairing comparison result between homologue that hereinafter is presented at search sequence and from the beginning identifies.

Inquiry: SEQID 2

Database :/home/data/blast/orgs_sets/arabi

26,735 sequences; 11,317,104 total letters

Minimum

Sum

High probability

Produce the right sequence of high keeping the score property section: the P that keeps the score (N) N

At1g10585 agnoprotein matter 180 4.5e-15 1

Protein 149 8.7e-12 1 of At4g20970 hypothesis

Protein 118 2.0e-06 1 that At4g25410 infers

BHLH transcription factor (bHLH036) 105 1.5e-05 1 that At5g51780 infers

Protein 110 1.5e-05 1 that At5g51790 infers

At1g10585Agnoprotein matter

Length=122

Keep the score=180 (68.4 hit) expected value=4.5e-15, P=4.5e-15

Identity=38/119 (31%), positive=72/119 (60%)

Inquiry: 106 QLKGRIDELKKRKQQAAALTTSTSNGGGGGMPVVEVRCQDGTLDVVVVSEAIREER ERAV 165

QLK ++ LK++K+ G +P + +R +D T+++ ++ + ++R V

Sbjct： 3 QLKENVNYLKEKKRTLLQGELGNLYEGSFLLPKLSIRSRDSTIEMNLIMD-LNMKR---V 58

Inquiry: 166 RLHEVIGVLEEEGAEVVNASFSVVGDKIFYTLHSQALCSRIGLDASRVSHRLRNLL LQY 224

LHE++ + EEEGA+V++A+ + D+ YT+ +QA+ SRIG+D SR+ R+R ++ Y

Sbjct： 59 MLHELVSIFEEEGAQVMSANLQNLNDRTTYTIIAQAIISRIGIDPSRIEERVRKIIYGY 117

At4g20970The protein of supposing

Length=167

Keep the score=149 (57.5 hit) expected value=8.7e-12, P=8.7e-12

Identity=49/171 (28%), positive=86/171 (50%)

Inquiry: 33 SSSSSKMERKDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTK EAVT 92

+ S ++RK VEKNRR+ MK L +L SL+P HH ST + P EA

Sbjct： 8 TGQSRSVDRKTVEKNRRMQMKSLYSELISLLP--------HHSSTEPLTLP-DQLDEAAN 58

Inquiry: 93 QLDHLEQAAAYIKQLKGRI---DELKKRKQQ-AAALTTSTSNGGGGGMPVVEVRCQ DGTL 148

+ L+ ++ K + L+K ++++++S +P +E++ + G++

Sbjct： 59 YIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPRKLPKIEIQ-ETGSI 117

Inquiry: 149 DVVVVSEAIREERERAVRLHEVIGVLEEE-GAEVVNASFSVVGDKIFYTLH 198

+ + ++ E E+I VL EE GAE+ +A +S+V D +F+TLH

Sbjct： 118 FHIFLVTSL----EHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164

Embodiment 32: identify the bHLH from the puncture vine clover

Problem to be solved is whether search sequence (from the SEQ ID NO:227 of puncture vine clover) is the bHLH polypeptide of basis used definition herein.The arabis protein group database of SEQ ID NO:227 and MIPS institute is compared.

Use BLASTP 2.0MP-WashU algorithm to implement relatively, wherein said algorithm is carried out the similarity searching (reference: Gish, W. (1996-2002)) of protein search sequence at given protein sequence database.Used parameter is the E value in comparison: 10; Critical keeping the score (S2): 56.

The Protein Data Bank of being inquired about is corresponding to the MIPS institute arabidopsis thaliana protein group that comprises 26,735 sequences (26,735 sequences of database :/home/data/blast/orgs_sets/arabi; 11,317,104 total letters).The result according to as from the highest keep the score and minimum E-value the highest determined similarity sort.First the hitting (underlining in comparison result) of being identified shows that corresponding to SEQ IDNO:225 (At4g20970) this sequence is the bHLH polypeptide according to used definition herein.Hereinafter show from the pairing comparison result between the bHLH of search sequence of clover (Medicago) and from the beginning evaluation.

BLASTP2.0MP-WashU[2002 September 22] [decunix4.0-ev56-I32LPF642002-09-09T17:45:09]

Inquiry=SEQ ID NO:227

(140 letter)

Database :/home/data/blast/orgs_sets/arabi

26,735 sequences; 11,317,104 total letters.

Search ... .10....20....30....40....50....60....70....80....90....1 00% finishes

Minimum

Sum

High probability

Produce the right sequence s of high keeping the score property section: the P that keeps the score (N) N

Protein 248 2.8e-22 1 of At4g20970 hypothesis

BHLH transcription factor (bHLH036) 103 6.5e-07 1 that At5g51780 infers

At1g10585 agnoprotein matter 100 1.4e-06 1

BHLH transcription factor (bHLH118) 99 7.3e-06 1 that At4g25400 infers

At4g20970The protein of supposing

Length=167

Keep the score=248 (92.4 hit) expected value=2.8e-22, P=2.8e-22

Identity=52/123 (42%), positive=82/123 (66%)

Inquiry: 7 EAISVPDQLKEATNYIKKLQINLEKMKEKKNFLLG---IQRPN------VNLNRNQ KMGL 57

E +++PDQL EA NYIKKLQ+N+EK +E+K L+ +++ N V+ + + +

Sbjct： 45 EPLTLPDQLDEAANYIKKLQVNVEKKRERKRNLVATTTLEKLNSVGSSSVSSSVDVSVPR 104

Inquiry: 58 KSPKIKIQQIGLVLEVVLITGLESQFLFSETFRVLHEE-GVDIVNASYKVNEDSVF HSIH 116

K PKI+IQ+ G + + L+T LE +F+F E RVL EE G +I +A Y + +D+VFH++H

Sbjct： 105 KLPKIEIQETGSIFHIFLVTSLEHKFMFCEIIRVLTEELGAEITHAGYSIVDDAVFHTLH 164

Inquiry: 117 CQV 119

C+V

Sbjct： 165 CKV 167

Embodiment 33: identify with the inventive method in the relevant sequence of used nucleotide sequence

Use bHLH sequence (Nucleotide (full-length cDNA, EST or genomic) or protein (total length or part of polypeptide)) to utilize the database sequence research tool, as basic local comparison instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify other bHLH Nucleotide or protein sequence in those sequences of in the Entrez Nucleotide database of NCBI (NCBI), safeguarding.This program is used for relatively and by the significance,statistical that calculates coupling finding the zone that has local similar between sequence by nucleotide sequence or peptide sequence and sequence library.For example, use is (irredundant: (database: whole GenBank+EMBL+DDBJ+PDB sequences (but not having EST, STS, GSS, environmental samples or phase 0,1 or 2HTGS sequence) at nr by the conduct of SEQ ID NO:213 encoded polypeptides, 3,819,973 sequences; 16,928,533,343 total letters) the TBLASTN algorithm is used in the inquiry of database sequence, adopts default setting and filter to ignore the low-complexity sequence.The result who analyzes (hereinafter showing) shown by paired comparisons, and according to probability scoring (E-value) ordering, wherein should the specific comparison result of scoring reflection because of the probability that exists accidentally (the E-value is low more, hit significance big more).Except the E-value, more also keep the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.

On database, identify keeping the score and being equal to or less than the hitting of E-value of e-05 of the proteinic generation at least 50 of BHLH.

E keeps the score

Produce the sequence of significance comparison result: (bit) value

Gi|55765745|ref|NM_183508.2| rice (Japanese cultivar-gro 440 1e-121

Gi|42821746|dbj|AK109432.2| rice (Japanese cultivar-g... 440 1e-121

Gi|55769744|ref|XM_549862.1| rice (Japanese cultivar-gro 247 1e-105

Gi|58530787|dbj|AP008207.1| rice (Japanese cultivar-g... 263 2e-68

Gi|17385651|dbj|AP002845.3| rice (Japanese cultivar-g... 263 2e-68

Gi|32980356|dbj|AK070332.1| rice (Japanese cultivar-g... 251 9e-65

Gi|34894135|ref|NM_183504.1| rice (Japanese cultivar-gro 137 3e-30

Gi|15293050|gb|AY050959.1| Arabidopis thaliana agnoprotein matter 80.1 6e-16

The gi|79340923|ref|NM_100934.2| Arabidopis thaliana is transcribed ... 87.8 2e-15

Gi|58531195|dbj|AP008214.1| rice (Japanese cultivar-g... 51.6 4e-13

Gi|42408246|dbj|AP004557.3| rice (Japanese cultivar-g... 51.6 4e-13

Gi|42408168|dbj|AP004376.3| rice (Japanese cultivar-g... 51.6 4e-13

Gi|22328837|ref|NM_118215.2| Arabidopis thaliana DNA combination ... 78.2 1e-12

Gi|7268888|emb|AL161554.2|ATCHRIV54 Arabidopis thaliana DNA chr 77.8 2e-12

Gi|5262774|emb|AL080282.1|ATT13K14 Arabidopis thaliana DNA c... 77.8 2e-12

Gi|50906596|ref|XM_464787.1| rice (Japanese cultivar-gro 77.0 3e-12

Embodiment 34: determine overall similarity and identity between the bHLH transcription factor

But overall similarity percentage ratio between the bHLH polypeptide and identity percentage ratio use this area MatGAT software (BMC Bioinformatics.20034:29.MatGAT: use protein sequence or dna sequence dna to produce the application .Campanella JJ of similarity/identity matrix, BitinckaL, Smalley J) determined.MatGAT software produces the similarity/identity matrix at dna sequence dna or protein sequence, need not the pre-comparison of data.This program is carried out a series of pairing comparisons, calculates similarity and identity and subsequently the result is placed distance matrix.

Used parameter is relatively:

Matrix: Blosum62 keeps the score

First room: 12

Extensibility room: 2

The result shows in Figure 19 a.

Embodiment 35: determine overall similarity and identity between the bHLH structural domain in the bHLH polypeptide

BHLH structural domain use SMART software (Schultz etc. (1998) Proc.Natl.Acad.Sci.USA 95, 5857-5864Letunic etc. (2006) Nucleic Acids Res 34, D257-D260) mapping.Smart software can pass through EMBL institute, and (European Molecular Bioglogy Laboratory (EMBL) obtains.

Hereinafter provide the sequence of used bHLH structural domain.

Hv_BHLH

KESEKERRKRMKALCEKLASLIPREHCCSTTDTMTQLGSLDVGASYIKKLKERVDE

OS_NP_908393_BHLH

KEMERRRRQDMKGLCVKLASLIPKEHCSMSKMQAASRTQLGSLDEAAAYIKKLKERVDE

OS_NP_908397.1_BHLH

KDVEKNRRLHMKGLCLKLSSLIPAAAPRRHHHHYSTSSSSSPPSSTKEAVTQLDHLEQAAAYIKQL

KGRIDE

Os_XP_464787.1_BHLH

KTTERIRREQMNKLYSHLDSLVRSAPPTVNSIPSHSNSNSKYHQRKLRILGGAAAATTRPDRLGVA

AEYIRQTQERVDM

AT1G10585_bHLH

nlrekdrrmrmkhlfsilsshvsptrklpvphlidqatsymiqlkenvny

At4g20970_bHLH

KTVEKNRRMQMKSLYSELISLLPHHSSTEPLTLPDQLDEAANYIKKLQVNVEK

Overall similarity percentage ratio and identity percentage ratio use software and the parameter described in embodiment 34 to determine between the bHLH of bHLH polypeptide structural domain.

The result shows in Figure 19 b.

Embodiment 36: used nucleotide sequence in clone's the inventive method

Nucleotide sequence used in the inventive method is by PCR, and (Invitrogen, Paisley UK) increase as template to use the rice seedling cDNA library that customizes.Under standard conditions, use Hifi TaqDNA polysaccharase, utilize the 200ng template in 50 μ lPCR mixtures to carry out PCR.Used primer is prm06808 (SEQ ID NO:231; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaacaatgaagagcaggaagaacagc 3 ') and prm06809 (SEQ ID NO:232 justice is arranged, and initiator codon is a boldface letter, and the AttB1 site is an italics:; Antisense, complementation, the AttB2 site is an italics: 5 ' ggggaccactttgtacaagaaagctgggtgcagagtgaaagagtggtgtg 3 '), wherein said primer comprises the AttB site that is used for the Gateway reorganization.The amplification PCR fragment also uses standard method to give purifying.Implement the first step of Gateway method subsequently, i.e. BP reaction is recombinated in PCR fragment and the pDONR201 plasmid generation body during this period to produce " the entering the clone " according to the Gateway name, pbHLH.Plasmid pDONR201 conduct

The component part of technology is bought from Invitrogen.

Embodiment 37: expression vector establishment

Entering clone p076 uses with pGOS2 (a kind of purpose carrier that is used for the rice conversion) in the LR reaction subsequently.This carrier contains as functional element on the T-DNA border: plant selectable marker; Selection markers expression cassette and intention with cloned purpose nucleotide sequence in the described clone of entering the Gateway box of recombinating in the LR body taken place.The rice GOS2 promotor (SEQ ID NO:233, alternatively, SEQ ID NO:230 is same useful) (internal reference PRO129) that is used for constitutive expression is positioned at the upstream of this Gateway box.

After the LR reconstitution steps, the expression vector pGOS2::bHLH (Figure 20) that produces is converted into agrobacterium strains LBA4044 and subsequent transformation to rice plant.

Embodiment 38: Plant Transformation

Rice transforms

The Agrobacterium that contains expression vector is used for transforming rice plant independently.Ripe dry seed shelling with the Japanese cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, subsequently at 2%HgCl ₂In 30 minutes, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD ₆₀₀) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

For a construct, produce about 30 independent T0 rice transformant.With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges 1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978 Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant is containing 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K in the dark ₂SO ₄With cultivated altogether 3 days on the SH inducing culture of 100 μ m Syringylethanones. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 39: evaluation method

Prepare 39.1 estimate

Produce about 30 T0 rice transformant independently.Transformant was transferred to the greenhouse from incubator for tissue culture and was used for growth and results T1 seed former generation.Stay 7 incidents, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under light.

4 T1 incidents T2 from generation to generation in according to as be used for T1 identical evaluation method from generation to generation and do further assessment, but that each incident adopts is more individual.Make plant pass through the digital imagery case for several times until the ripening stage from sowing time.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

39.2 statistical study: t check and F-check

Use double factor ANOVA (variance analysis) to be used for the overall evaluation of plant phenotype feature as statistical model.All measuring parameters with whole plants of whole incidents of gene transformation of the present invention are implemented F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action (being called overall gene action again) of checking gene.The threshold value that is used for the significance of true overall gene action is arranged on 5% probability level for F check.Significance F test value indicates gene action, means that the existence of gene not only or position just cause the difference on the phenotype.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " handled in the mode identical with transgenic plant, but isolating plant has therefrom taken place in transgenosis.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

Embodiment 40: evaluation result

Early stage vigor is determined from the sum of all pixels that is different from background in the plant part of ground by counting.This value changes into the physical surface value of being represented by square mm to the averaging of picture of taking from different perspectives and by correction on identical time point.Result hereinafter described is at the plant that sprouts 3 weeks of back.

See the early stage vigor of plant (as being determined by the over-ground part area) in 6 incidents in T1 generation 7 incidents, compare with control plant simultaneously, the over-ground part area of transgenosis seedling is overall to increase by 22%.T2 from generation to generation in further 4 incidents in these T1 incidents of assessment, and compare with control plant, all these four incidents cause that all the over-ground part area of transgenosis seedling increases, and compare with control plant, and the over-ground part area of transgenosis seedling is overall to increase by 13%.Confirm that also described result is that statistics is significant, the p-value of checking from F-is 0.0007 (T2 from generation to generation), shows that being seen effect may be because due to the position of transgenosis rather than Yin Jiyin or the effect of strain system.

Embodiment part F:SPL15

Embodiment 41: identify with the inventive method in the relevant sequence of used nucleotide sequence

Use the database sequence research tool, as basic local comparison instrument (BLAST) (Altschul etc. (1990) J.Mol.Biol.215:403-410; With (1997) Nucleic Acids Res.25:3389-3402 such as Altschul) identify (full-length cDNA, EST or genome) sequence relevant in those sequences of in the Entrez of NCBI Nucleotide database, being safeguarded with used nucleotide sequence in the inventive method.This program is used for relatively and by the significance,statistical that calculates coupling finding the zone that has local similar between sequence by nucleotide sequence or peptide sequence and sequence library.Nucleic acid encoded polypeptide of the present invention is used the TBLASTN algorithm, adopt default setting and filtration to offset to ignore the low-complexity sequence.The result who analyzes relatively shows by pairing property, and according to probability scoring (E-value) ordering, wherein is somebody's turn to do the specific comparison result of scoring reflection because of the accidental probability (the E-value is low more, and the significance of hitting is high more) that takes place.Except the E-value, more also keep the score by identity percentage ratio.Identity percentage ratio refer to two compare identical Nucleotide (or amino acid) number in the length-specific scope between nucleic acid (or polypeptide) sequence.In some cases, can adjust default parameters to regulate the severity of search procedure.

Following table N provides the nucleotide sequence tabulation relevant with used nucleotide sequence in the inventive method.

Table N: with the nucleotide sequence that is used for the inventive method (SEQ ID NO:234) relevant nucleotide sequence and corresponding derivation polypeptide

Title	Nucleic acid SEQID NO	Polypeptide SEQID NO	Sequence length	The NCBI searching number	The source is biological
						Arath_SPL15(At3g57920)	234	235	Total length	NM_115654.1	Arabidopis thaliana
Arath_SPL9 (At2g42200)	236	237	Total length	AY150378	Arabidopis thaliana
						Aqufo_SPL	238	239	Total length	The folded sequence that connects of DR915312 DR949057.1	Aquilegia formosa x Aquilegia pubescens
Goshi_SPL	240	241	Total length	DT566400	Upland cotton
						Iponi_SPL
	242	243	Total length	The folded sequence that connects of BJ576204.1 BJ556115 BJ567301	(Ipomoeanil) leads a cow
						Lacsa_SPL	244	245	Total length	DY966949DW119178	Lettuce

Maldo_SPL	246	247	Total length	The folded sequence that connects of CN891102.1CO868185.1 CV523507	Apple
						Medtr_SPL
	248	249	Total length	Montage is from AC170989.2	The puncture vine clover
						Nicbe_SPL
	250	251	Total length	The folded sequence that connects of CK284078.1CK294165	Nicotianabentamiana
						Orysa_SPL	252	253	Total length	XM_483285	Rice
Orysa_SPL II
		254	255	Total length	Montage is from AC108762	Rice
Soltu_SPL							256	257	Total length	The folded sequence that connects of CK246692.1 CK254420.1	Potato
	Vitvi_SPL
		258	259	Total length	The folded sequence that connects of CV098277 CV092812.1	Grape
	Zeama_SPL
		260	261	Total length	The folded sequence that connects of EB160653 DY235599 DV029129	Zea mays
	Zeama_SPLII						262	263	Total length	The folded sequence that connects of AJ011619 DV033513.1 DY532686.1	Zea mays
Sorpr_SPL		264	265	Part	BF422188	Sorghum propinquium

Allce_SPL	266	267	Part	CF444518.1	Onion (Alliumcepa)
						Antma_SPL	268	269	Part	AMA011623	Common Snapdragon
Brana_SPL	270	271	Part	CX189447	Colea
						Sacof_SPL	272	273	Part	The folded sequence that connects of CA113070 CA254724	Sugarcane (Sacchar um officinarum)
Fesar_SPL	274	275	Part	DT706587.1	Festuca arundinacea
						Brara_SPL
	282	283	Total length	AC189445.1	Brassicarapa
						Glyma_SPL	284	285	Total length	CX708501.1BG651519.1	Soybean
Poptr_SPL
		286	287	Total length	scaff_XVI.416	Populus tremuloides
Citcl_SPL							288	289	Part	DY293795	Citrus clementina
	Betvu_SPL
		290	291	Part	BQ594361.1	Beet (Betavulgaris)
	Hevbr_SPL						292	293	Part	EC604947	Rubber tree (Hevea brasiliensis)

Embodiment 42: determine overall similarity and identity between the SPL15 transcription factor, and the SPL DBD of SPL15 transcription factor.

Overall similarity percentage ratio between the SPL15 transcription factor and identity percentage ratio use one of the obtainable method in this area MatGAT (matrix overall comparison instrument) software (BMC Bioinformatics.2003 4:29.MatGAT: use protein sequence or dna sequence dna to produce the application .Campanella JJ of similarity/identity matrix, Bitincka L, Smalley J; Software is owned by Ledion Bitincka) determined.MatGAT software produces the similarity/identity matrix at dna sequence dna or protein sequence, need not the pre-comparison of data.Use Myers and Miller overall comparison algorithm (point penalty 2 is extended in room opening point penalty 12 and room), this program is carried out a series of pairings comparison, for example uses Blosum62 (for polypeptide) to calculate similarity and identity and subsequently the result is placed distance matrix.Sequence similarity shows that in marginal lower part sequence identity shows in the marginal upper part in diagonal angle.The sequence of SEQ ID NO:235 is shown as in matrix No. 5.

Used parameter is relatively:

Matrix: Blosum62 keeps the score

First room: 12

Extensibility room: 2

In table O, show at the overall similarity of SPL15 transcription factor polypeptide total length and the software analysis result of identity.Identity percentage ratio is providing on the diagonal lines and similarity percentage ratio provides under diagonal lines.At SPL15 transcription factor collateral line homologue and straight identity percentage ranges between homologue is 30-70%, reflects described collateral line homologue and directly conservative to the relative low sequence identity outside SPL DBD between homologue.

Table O: be used for the overall similarity on SPL15 transcription factor polypeptide total length and the MatGAT result of identity.

Demonstration is at the overall similarity of the SPL DBD of SPL15 transcription factor polypeptide and the software analysis result of identity in table P.Identity percentage ratio is providing on the diagonal lines and similarity percentage ratio provides under diagonal lines.At SPL15 transcription factor collateral line homologue and straight identity percentage ranges between the SPLDBD of homologue is 70-100%.

Table P: at the overall similarity of the SPL DBD of SPL15 transcription factor polypeptide and the MatGAT result of identity.

Embodiment 43: used nucleotide sequence in clone's the inventive method

DNA operation: unless stated otherwise, recombinant DNA technology is according to (Sambrook (2001) Molecular Cloning:a laboratory manual, third edition Cold Spring HarborLaboratory Press, CSH, New York) in or Ausubel etc. (1994), CurrentProtocols in Molecular Biology, the standard method of describing in Current Protocols the 1st and 2 volumes is carried out.The standard material and the method that are used for plant molecular work are described at the Plant Molecular Biology Labfax (1993) of the R.D.D.Croy that is published by BIOS ScientificPublications Ltd (UK) and Blackwell Scientific Publications (UK).

Nucleotide sequence used in the inventive method is by PCR, and the Arabidopis thaliana mixed structure cDNA library of using customization is (in MV Sport 6.0; Invitrogen, Paisley UK) increases as template.Under standard conditions, use Hifi Taq archaeal dna polymerase, utilize the 200ng template in 50 μ l PCR mixtures to carry out PCR.The primer is prm07277 (SEQ ID NO:280; 5 '-ggggacaagtttgtacaaaaaagcaggcttaaacaATGGAGTTGTTAATGTGTTCG 3 ') and prm07278 (SEQ ID NO:281 justice is arranged, and initiator codon is a boldface letter, and the AttB1 site is a small letters:; Antisense, complementary AttB2 site is a small letters: 5 ' ggggaccactttgtacaagaaagctgggtTGATGAAGATCTTAAAAGGTGA 3 '), it comprises the AttB site that is used for the Gateway reorganization.The amplification PCR fragment also uses standard method to give purifying.Implement the first step of Gateway method subsequently, i.e. BP reaction is recombinated in PCR fragment and the pDONR201 plasmid generation body during this period to produce " the entering the clone " according to the Gateway name, pSPL15.Plasmid pDONR201 conduct

The component part of technology is bought from Invitrogen.

Embodiment 44: expression vector establishment

Entering clone p13075 uses with p06659 (a kind of purpose carrier that is used for the rice conversion) in the LR reaction subsequently.This carrier contains as functional element on the T-DNA border: plant selectable marker; The selection markers expression cassette is with intention and cloned the Gateway box of recombinating in the described purpose nucleotide sequence generation LR body that enters in the clone.The rice HMGB promotor (SEQ ID NO:294) that is used for constitutive expression is positioned at the upstream of this Gateway box.Alternatively, it is same useful representing the HMGB promotor by SEQ IDNO:46.Similar construct is used in GOS2 promotor (SEQ ID NO:295) the control SPL15 encoding sequence generation down of composing type.

After the LR reconstitution steps, the expression vector pHMGB::SPL15 (Figure 26) that produces or pGOS2::SPL15 are converted into agrobacterium strains LBA4044 and subsequent transformation to rice plant.

Embodiment 45: Plant Transformation

Rice transforms

The Agrobacterium that contains the conversion of expression vector is used for transforming rice plant.Ripe dry seed shelling with the Japanese cultivar Nipponbare of rice.By incubation in 70% ethanol one minute, subsequently at 2%HgCl ₂In 30 minutes, subsequently with sterile distilled water washing 6 times 15 minutes and implement sterilization.The disinfectant seed is containing 2 subsequently, and the substratum of 4-D (callus inducing medium) is gone up and sprouted.Incubation will downcut from the callus of scutel and breed on a kind of substratum after 4 weeks in the dark.After 2 weeks, callus by breeding or breed uploading with a kind of substratum to be commissioned to train to support in other 2 weeks.The embryogenic callus sheet is uploaded to be commissioned to train at fresh culture and was supported 3, cultivates (to strengthen the cell fission activity) afterwards altogether.

The agrobacterium strains LBA4404 that contains expression vector is used for common cultivation.Agrobacterium is seeded in to contain on the suitable antibiotic AB substratum and at 28 ℃ and cultivated 3.Subsequently bacterium is collected and is resuspended in liquid and cultivate altogether in the substratum to density (OD ₆₀₀) about 1.Suspension is transferred to culture dish subsequently and callus was soaked 15 minutes in this suspension.Callus is organized subsequently and to be cultivated on the substratum altogether and in the dark in 25 ℃ of incubations 3 days blotting and be transferred to solidified on the filter paper.Altogether the callus of cultivating in the dark in 28 ℃ in the presence of selective agent in containing 2,4 weeks of cultivation on the substratum of 4-D.During the section, form mushroom resistant calli island at this moment.To regeneration culture medium and behind incubation under the light, release of embryo generation potentiality and seedling are in 4-5 week growth subsequently in this material transfer.Seedling is downcut from callus and, wherein seedling is transferred to soil from described substratum containing incubation 2-3 week on the substratum of plant hormone.The hardened seedling is cultivated in the greenhouse under high humidity and short day.

For a construct, produce about 35 independent T0 rice transformant.With former generation transformant be transferred to the greenhouse from incubator for tissue culture.Behind the copy number of quantitative PCR analysis with checking T-DNA inset, the single copy transgenic plant that only keep performance selective agent tolerance are used to gather in the crops the T1 seed.Seed is gathered in the crops after transplanting subsequently the 3-5 month.Present method produces term single gene seat transformant (Aldemita and Hodges1996, Chan etc. 1993, Hiei etc. 1994) to surpass 50% ratio.

Corn transforms

The conversion of corn is carried out according to the modification method to (1996.Nature Biotech 14745-50) described methods such as Ishida.Conversion in corn be that genotype relies on and only the special genes type can operate and be used for transforming and regeneration.Inbred lines A188 (University of Minnesota) or be the good source of the donor material that is used to transform with A188 as parent's hybrid, but other genotype also can successfully be used.Mealie from maize plant after pollination about 11 days (DAP) results, this moment, the length of immature embryos was about 1 to 1.2mm.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.The embryo that downcuts is on the callus inducing medium, cultivate on the corn regeneration culture medium subsequently, and wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to the maize rooting substratum and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Wheat transforms

The conversion of wheat is carried out with the method that (1996) Nature Biotech 14 (6): 745-50 such as Ishida such as Ishida describe.Usually in conversion, use (can obtain) Cultivar Bobwhite from Mexico CIMMYT.Immature embryos is cultivated altogether with the agrobacterium tumefaciens that contains expression vector and transgenic plant take place to recover by organ.With the Agrobacterium incubation after, embryo on the callus inducing medium, external cultivation on regeneration culture medium subsequently, wherein said regeneration culture medium contains selective agent (for example imidazolone, but can use the multiple choices mark).Culture plate is cultivated 2-3 week under illumination at 25 ℃, or grows until seedling.Green seedling is transferred to root media and cultivates 2-3 week at 25 ℃ from each embryo, until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant of performance selective agent T-DNA inset tolerance and that contain single copy, produce the T1 seed.

Soybean transforms

According to Texas A﹠amp; M United States Patent (USP) 5,164, the modification method soybean transformation of method described in 310.Several commercial soybean varieties are feasible for conversion by this method.Cultivar Jack (can be able to obtain from Illinois seed money) is generally used for transforming.Soybean seeds is sterilized so that external sowing.From 7 age in days seedling, downcut hypocotyl, radicle and a slice cotyledon.The cotyledon of further cultivating epicotyl and remainder is to grow the armpit tight knot.These armpit tight knots are downcut and with the agrobacterium tumefaciens incubation that contains expression vector.After cultivating processing altogether, explant is washed and is transferred to the selection substratum.The regenerated seedling is downcut and places on the seedling elongation medium.The seedling that length is no more than 1cm places on the root media until root development.The seedling that to take root migrates in the soil in greenhouse.From the plant tolerance of performance selective agent and that contain single copy T-DNA inset, produce the T1 seed.

Semen Brassicae campestris/canola oil dish transforms

Cotyledon petiole of use 5-6 age in days seedling and hypocotyl are as being used for the explant of tissue culture and transforming according to (1998, Plant Cell Rep 17:183-188) such as Babic.Commercial Cultivar Westar (Agriculture Canada) is the standard variety that is used to transform, but also can use other kind.Canola oil colza is done the surface sterilization so that external sowing.From external seedling, downcut and have the cotyledon petiole explant that adheres to cotyledon, and immerse bacterial suspension with the cut ends of (containing expression vector) Agrobacterium by petiole explant and inoculate.Explant subsequently on the MSBAP-3 substratum that contains 3mg/l BAP, 3% sucrose, 0.7% plant agar (Phytagar) at 23 ℃, illumination in 16 hours was cultivated 2 days down.After cultivating 2 altogether with Agrobacterium, petiole explant is transferred on the MSBAP-3 substratum of 3mg/l BAP, cefotaxime, Pyocianil or the Ticarcillin/Clavulanate Acid (300mg/l) that contain and continues 7, and cultivate containing on the MSBAP-3 substratum of cefotaxime, Pyocianil or Ticarcillin/Clavulanate Acid and selective agent subsequently, regenerate until seedling.When seedling has 5-10mm length, seedling is downcut and is transferred to seedling elongation medium (MSBAP-0.5 that contains 0.5mg/l BAP).The seedling of the about 2cm of length is transferred to the root media (MS0) that is used for root induction.The seedling that to take root migrates in the soil in greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Clover transforms

The reproducibility clone of clover uses the method for (McKersie etc., 1999 Plant Physiol 119:839-847) to be transformed.Regeneration of clover and conversion are that genotype is dependent and thereby need aftergrowth.The method that obtains the reproducibility plant has been described.For example, these reproducibility plants any other commercial alfalfa variety that can be selected from Cultivar Rangelander (Agriculture Canada) or describe as Brown DCW and AAtanassov (1985.Plant Cell Tissue Culture 4:111-112).Alternatively, RA3 kind (University of Wisconsin) has been selected for (Walker etc., 1978 Am J Bot 65:654-659) in the tissue culture.Petiole explant and the agrobacterium tumefaciens C58C1 pMP90 (McKersie etc., 1999 Plant Physiol 119:839-847) or the overnight culture of LBA4404 that contain expression vector are cultivated altogether.Explant is containing 288mg/L Pro, 53mg/L Thioproline, 4.35g/L K in the dark ₂SO ₄With cultivated altogether 3 days on the SH inducing culture of 100 μ m Syringylethanones. explant half concentrate in the Murashige-Skoog substratum (Murashige and Skoog, 1962) washing and plating contain not containing Syringylethanone suitable selective agent and suitable microbiotic with the identical SH inducing culture of restraining the Agrobacterium growth on.After several weeks, somatic embryo is transferred to do not contain growth regulator, do not contain microbiotic and the BOi2Y that contains 50g/L sucrose grows in the substratum.Somatic embryo concentrates on the Murashige-Skoog substratum half subsequently to be sprouted.The sprigging that to take root is cultivated to flowerpot and in the greenhouse.Produce the T1 seed the plant that singly copies the T-DNA inset from showing the selective agent tolerance and containing.

Embodiment 46: evaluation method

Prepare 46.1 estimate

Produce about 35 T0 rice transformant independently.Transformant was transferred to the greenhouse from incubator for tissue culture and was used for growth and results T1 seed former generation.Stay 7 incidents, the T1 offspring of wherein said incident is to genetically modified existence/do not exist with the 3:1 ratio and separate.For each incident in these incidents, select to contain genetically modified about 10 strain T1 seedling (heterozygote and homozygote) and lack genetically modified about 10 strain T1 seedling (inefficacy zygote) by monitoring visual marker expression.Transgenic plant and corresponding inefficacy zygote are cultivated on random site side by side.Greenhouse experiment is short day (illumination in 12 hours), 28 ℃ and 22 ℃ and relative humidity 70% in the dark under light.

5 T1 incidents T2 from generation to generation in according to as be used for T1 identical evaluation method from generation to generation and do further assessment, but that each incident adopts is more individual.Make plant pass through the digital imagery case for several times until the ripening stage from sowing time.On each time point, to every strain plant from least 6 different angles shooting digital pictures (2048 x, 1536 pixels, 1,600 ten thousand colors).

Early stage vigor is plant (seedling) over-ground part area of sprouting 3 weeks of back.The increase of root biomass is expressed as the increase of root total biomass (being measured as viewed maximum biomass during plant life); Or be expressed as the increase of root/seedling index (ratio when being measured as the active growth of root and seedling between interim quality and the seedling quality).

46.2 statistical study: F-check

Use double factor ANOVA (variance analysis) to be used for the overall evaluation of plant phenotype feature as statistical model.All measuring parameters with whole plants of whole incidents of gene transformation of the present invention are implemented F check.Implement the F check to check that gene is for the effect of whole transformation events and the mass action (being called overall gene action again) of checking gene.The threshold value that is used for the significance of true overall gene action is arranged on 5% probability level for F check.Significance F test value indicates gene action, means that the existence of gene not only or position just cause the difference on the phenotype.

For checking the effect of gene in incident, i.e. strain is a specific effect, uses the data set from the transgenic plant and the invalid plant of correspondence to carry out the t check in each incident." invalid plant " or " invalid segregant " or " inefficacy zygote " handled in the mode identical with transgenic plant, but isolating plant has therefrom taken place in transgenosis.The invalid plant negative plant that transforms that also can be described as isozygotying.The threshold value that is used for the significance of t check is arranged on 10% probability level.The result of some incidents can be higher or lower than this threshold value.This is based on hypothesis like this, and promptly gene may only have effect in some position in genome, and the appearance of this position dependence effect is not rare.This gene action is called " the strain system effect of gene " in this article again.The p-value distributes relatively by t-value and t-or alternatively distributes by F-value and F-and relatively obtains.It is correct probability that this p-value provides null hypothesis (being that genetically modified effect does not exist) subsequently.

Embodiment 47: evaluation result

Plant shoot divides area (or leaf biomass) to measure by counting is different from the pixel of background on the digital picture of dividing from plant shoot sum.This value changes into square physical surface value of mm expression to the averaging of picture of taking from different perspectives on identical time point and by correction.The over-ground part plant area that experiment confirm is measured by this way is relevant with the biomass of ground plant part.The over-ground part area is the time point that has reached its maximum leaf biomass plant.

With sophisticated main panicle gather in the crops, count, pack, add bar code label and subsequently in loft drier 37 ° of dryings 3 days.Subsequently with panicle threshing and collection and count whole seeds.Use blowing device to separate full grain and empty grain.Discarding empty grain also counts remainder once more.Full grain is weighed on analytical balance.The full grain number that the full seed number passes through behind the counting separating step is determined.The whole full grain that the seed ultimate production is gathered in the crops from plant by weighing is measured.The capsomere number that every strain plant seed sum is gathered in the crops from plant by counting is measured.Full seed number and the extrapolated thousand seed weight of gross weight (TKW) thereof from counting.Harvest index (HI) is defined as seed ultimate production and over-ground part area (mm in the present invention ²) between ratio, multiply by coefficient 10 ⁶As the every paniculiform number of always spending that defines among the present invention is seed sum and the ripe ratio of leading between the paniculiform number.As the full rate of the seed that defines among the present invention is the ratio (be expressed as %) of full seed number to seed (or Xiao Hua) sum.

As showing Q to showing as shown in the W, compare with suitable control plant, over-ground part biomass, each paniculiform number, seed production, seed sum, full seed number, thousand seed weight (TKW) and harvest index spent increase in the transgenic plant of the expression of nucleic acids increase of coding SPL15 transcription factor polypeptide.Shown from T1 and T2 result from generation to generation.

Table Q shows the percentage ratio of the number of the transgenic event with the increase of over-ground part biomass, this increase and the statistical correlation that this increase is checked according to F-.

Table Q: T1 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check of the transgenic event that the over-ground part biomass increases from generation to generation.

Table R shows that having each panicle spends the percentage ratio of the number of the transgenic event of sum increase, this increase and the statistical correlation that this increase is checked according to F-.

Table R: T1 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check that each panicle is spent the transgenic event that number increases from generation to generation.

Table S shows the percentage ratio of the number of the transgenic event with seed ultimate production (seed gross weight) increase, this increase and the statistical correlation that this increase is checked according to F-.

Table S: T1 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check of the transgenic event that seed production increases from generation to generation.

Table T shows the percentage ratio of the number of the transgenic event with the increase of seed sum, this increase and the statistical correlation that this increase is checked according to F-.

Table T: T1 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check of the transgenic event that the seed sum increases from generation to generation.

Table U shows the percentage ratio of the number of the transgenic event with the increase of full seed number, this increase and the statistical correlation that this increase is checked according to F-.

Table U: T1 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check of the transgenic event that the full seed number increases from generation to generation.

Table V shows the percentage ratio of the number of the transgenic event with harvest index increase, this increase and the statistical correlation that this increase is checked according to F-.

T1 of the transgenosis rice that Table V increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide and T2 have number, the percentage ratio of this increase and the P value of F-check of the transgenic event that harvest index increases from generation to generation.

Table W shows the percentage ratio of the number of the transgenic event with thousand seed weight (TKW) increase, this increase and the statistical correlation that this increase is checked according to F-.

Table W: T1 and the T2 of the transgenosis rice that increases at the expression of nucleic acid of coding SPL15 transcription factor polypeptide have transgenic event number, the percentage ratio of this increase and the P value of F-check that thousand seed weight (TKW) increases from generation to generation.

Embodiment 48: the phenotype evaluation result of constitutive promoter control being expressed the transgenic plant of SEQ ID NO:234 down

Provided in the Table X expressing the evaluation result of transgenosis rice plant that being used under the control of composing type GOS2 promotor implement the nucleotide sequence of the inventive method.Also show the difference percentage ratio between transgenic plant and the corresponding inefficacy zygote.

Compare with control plant (in this case, the inefficacy zygote), the transgenic plant of expressing the nucleotide sequence be used for implementing the inventive method have the early stage vigor of increase and the TKW of increase.

Table X: to expressing the evaluation result of T1 generation transgenosis rice plant that strong composing type GOS2 promotor control being used to down implement the nucleotide sequence of the inventive method.

Proterties	The increase % of T1 best incident in the generation
		The early stage vigor that increases	17％
Seed ultimate production (every strain plant)	18％
		The full seed sum	19％
The full rate of seed that increases	10％
		The harvest index that increases	15％

Sequence table

＜110〉Crow Pu Disai benefactor department

＜120〉has the plant of enhanced yield correlated character and be used to produce the method for this plant

<130>PF58328

<160>301

＜170〉PatentIn version 3 .3

<210>1

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＜213〉Arabidopis thaliana (Arabidopsis thaliana)

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＜213〉Arabidopis thaliana

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＜213〉artificial sequence

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＜223〉primer: prm00957

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<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: prm00958

<400>4

<210>5

<211>1243

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＜213〉rice (Oryza sativa)

<400>5

<210>6

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<220>

＜221〉variant

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<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(9)..(9)

＜223 〉/replace=" Thr "

<220>

＜221〉variant

<222>(14)..(14)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(15)..(15)

＜223 〉/replace=" Met "/replace=" Val "

<220>

＜221〉variant

<222>(18)..(18)

＜223 〉/replace=" Glu "/replace=" Ala "/replace=" Ser "

<220>

＜221〉variant

<222>(21)..(21)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(22)..(22)

＜223 〉/replace=" Arg "/replace=" Gly "

<220>

＜221〉variant

<222>(23)..(23)

＜223 〉/replace=" Ser "/replace=" Ile "

<220>

＜221〉variant

<222>(25)..(25)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(26)..(26)

＜223 〉/replace=" Asp "

<220>

＜221〉variant

<222>(29)..(29)

＜223 〉/replace=" Asp "

<220>

＜221〉variant

<222>(31)..(31)

＜223 〉/replace=" Lys "

<220>

＜221〉variant

<222>(34)..(34)

＜223 〉/replace=" Ala "

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<210>7

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<220>

＜221〉variant

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＜223 〉/replace=" Lys "/replace=" Gln "

<220>

＜221〉variant

<222>(12)..(12)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(13)..(13)

＜223 〉/replace=" Met "

<220>

＜221〉variant

<222>(14)..(14)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(24)..(24)

＜223 〉/replace=" Ala "

<220>

＜221〉variant

<222>(30)..(30)

＜223 〉/replace=" Met "

<220>

＜221〉variant

<222>(38)..(38)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(39)..(39)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(44)..(44)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(45)..(45)

＜223 〉/replace=" Ser "/replace=" Asn "/replace=" Asp "/replace=

″Lys″

<220>

＜221〉variant

<222>(48)..(48)

＜223 〉/replace=" Phe "/replace=" Met "/replace=" Ile "

<220>

＜221〉variant

<222>(50)..(50)

＜223 〉/replace=" Ala "

<220>

＜221〉variant

<222>(57)..(57)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(60)..(60)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(65)..(65)

＜223 〉/replace=" Ser "/replace=" Gln "/replace=" Ala "

<220>

＜221〉variant

<222>(66)..(66)

＜223 〉/replace=" Lys "

<220>

＜221〉variant

<222>(68)..(68)

＜223 〉/replace=" Phe "/replace=" Ile "

<220>

＜221〉uncertain

<222>(69)..(70)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉variant

<222>(71)..(71)

＜223 〉/replace=" Ile "/replace=" Met "

<220>

＜221〉variant

<222>(72)..(72)

＜223 〉/replace=" Asn "/replace=" Ser "

<220>

＜221〉variant

<222>(73)..(73)

＜223 〉/replace=" Leu "/replace=" Gln "

<220>

＜221〉variant

<222>(74)..(74)

＜223 〉/replace=" Met "

<220>

＜221〉variant

<222>(76)..(76)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(77)..(77)

＜223 〉/replace=" Ser "/replace=" Ala "/replace=" Ile "

<220>

＜221〉variant

<222>(79)..(79)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(82)..(82)

＜223 〉/replace=" Val "/replace=" Thr "

<220>

＜221〉variant

<222>(83)..(83)

＜223 〉/replace=" Thr "/replace=" Ser "

<220>

＜221〉variant

<222>(85)..(85)

＜223 〉/replace=" Thr "/replace=" Lys "

<220>

＜221〉variant

<222>(91)..(91)

＜223 〉/replace=" His "

<220>

＜221〉variant

<222>(93)..(93)

＜223 〉/replace=" Thr "

<220>

＜221〉variant

<222>(97)..(97)

＜223 〉/replace=" Ser "

<400>7

<210>8

<211>171

<212>PRT

＜213〉Arabidopis thaliana

<400>8

<210>9

<211>606

<212>DNA

＜213〉Arabidopis thaliana

<400>9

<210>10

<211>201

<212>PRT

＜213〉Arabidopis thaliana

<400>10

<210>11

<211>1128

<212>DNA

＜213〉rice

<400>11

<210>12

<211>220

<212>PRT

＜213〉rice

<400>12

<210>13

<211>1164

<212>DNA

＜213〉common wheat (Triticum aestivum)

<400>13

<210>14

<211>220

<212>PRT

＜213〉common wheat

<400>14

<210>15

<211>1165

<212>DNA

＜213〉Zea mays (Zea mays)

<400>15

<210>16

<211>220

<212>PRT

＜213〉Zea mays

<400>16

<210>17

<211>631

<212>DNA

＜213〉European spruce (Picea abies)

<220>

<221>misc_feature

<222>(546)..(546)

＜223〉n is a, c, g, or t

<400>17

<210>18

<211>120

<212>PRT

＜213〉European spruce

<400>18

<210>19

<211>769

<212>DNA

＜213〉colea (Brassica napus)

<400>19

<210>20

<211>209

<212>PRT

＜213〉colea

<400>20

<210>21

<211>636

<212>DNA

＜213〉cabbage mustard (Brassica oleracea var.alboglabra)

<220>

<221>misc_feature

<222>(622)..(622)

＜223〉n is a, c, g, or t

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<210>22

<211>198

<212>PRT

＜213〉cabbage mustard

<220>

＜221〉uncertain

<222>(194)..(194)

＜223〉Xaa can be any naturally occurring amino acid

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<210>23

<211>958

<212>DNA

＜213〉tobacco (Nicotiana tabacum)

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＜213〉potato (Solanum tuberosum)

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＜213〉potato

<400>26

<210>27

<211>967

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＜213〉soybean (Glycine max)

<400>27

<210>28

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＜213〉soybean

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<210>29

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<212>DNA

＜213〉grape (Vitis vinifera)

<400>29

<210>30

<211>214

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＜213〉grape

<400>30

<210>31

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＜213〉barley (Hordeum vulgare)

<400>31

<210>32

<211>215

<212>PRT

＜213〉barley

<400>32

<210>33

<211>1175

<212>DNA

＜213〉upland cotton (Gossypium hirsutum)

<400>33

<210>34

<211>208

<212>PRT

＜213〉upland cotton

<400>34

<210>35

<211>1119

<212>DNA

＜213〉dichromatism chinese sorghum (Sorghum bicolor)

<400>35

<210>36

<211>225

<212>PRT

＜213〉dichromatism chinese sorghum

<400>36

<210>37

<211>975

<212>DNA

＜213〉tomato (Lycopersicon esculentum)

<400>37

<210>38

<211>206

<212>PRT

＜213〉tomato

<220>

＜221〉uncertain

<222>(74)..(74)

＜223〉Xaa can be any naturally occurring amino acid

<400>38

<210>39

<211>1395

<212>DNA

＜213〉Arabidopis thaliana

<400>39

<210>40

<211>464

<212>PRT

＜213〉Arabidopis thaliana

<400>40

<210>41

<211>1182

<212>DNA

＜213〉Pinus (Pinus sp.)

<400>41

<210>42

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＜213〉Pinus

<400>42

<210>43

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<212>DNA

＜213〉rice

<400>43

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<211>267

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＜213〉rice

<400>44

<210>45

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>45

<210>46

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>46

<210>47

<211>2193

<212>DNA

＜213〉rice

<400>47

<210>48

<211>42

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Asn "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(12)..(12)

＜223 〉/replace=" Tyr "

<220>

＜221〉variant

<222>(16)..(16)

＜223 〉/replace=" Ile "/replace=" Leu "

<220>

＜221〉variant

<222>(17)..(17)

＜223 〉/replace=" Gly "

<220>

＜221〉variant

<222>(18)..(18)

＜223 〉/replace=" Ala "/replace=" Val "

<220>

＜221〉variant

<222>(19)..(19)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(20)..(20)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(23)..(23)

＜223 〉/replace=" Pro "/replace=" Ala "/replace=" Asn "

<220>

＜221〉variant

<222>(30)..(30)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(31)..(31)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(32)..(32)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(33)..(33)

＜223 〉/replace=" Asn "/replace=" Glu "

<220>

＜221〉variant

<222>(35)..(35)

＜223 〉/replace=" Cys "/replace=" Arg "/replace=" Ser "

<220>

＜221〉variant

<222>(37)..(37)

＜223 〉/replace=" Glu "

<220>

＜221〉variant

<222>(38)..(38)

＜223 〉/replace=" Ile "/replace=" Arg "/replace=" Lys "

<220>

＜221〉variant

<222>(41)..(41)

＜223 〉/replace=" Glu "

<400>48

<210>49

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(5)..(5)

＜223 〉/replace=" Arg "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(8)..(8)

＜223 〉/replace=" Thr "/replace=" Ser "

<220>

＜221〉variant

<222>(9)..(9)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Asn "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Arg "/replace=" Asn "

<220>

＜221〉variant

<222>(12)..(12)

＜223 〉/replace=" Cys "/replace=" Arg "

<220>

＜221〉variant

<222>(13)..(13)

＜223 〉/replace=" His "

<220>

＜221〉variant

<222>(14)..(14)

＜223 〉/replace=" Lys "

<400>49

<210>50

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Gln "/replace=" Val "/replace=" Ala "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(8)..(8)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(9)..(9)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Cys "/replace=" Phe "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Met "

<400>50

<210>51

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(1)..(1)

＜223 〉/replace=" Ala "/replace=" Val "/replace=" Leu "

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Pro "

<220>

＜221〉uncertain

<222>(3)..(3)

＜223〉Xaa can be any naturally occurring amino acid, preferred Leu,

Pro or His

<220>

＜221〉uncertain

<222>(3)..(3)

＜223〉Xaa can be any naturally occurring amino acid, preferred Leu,

Pro or His

<220>

＜221〉uncertain

<222>(6)..(6)

＜223〉Xaa can be any naturally occurring amino acid, preferred Leu,

Pro or His

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" His "

<400>51

<210>52

<211>735

<212>DNA

＜213〉barley

<400>52

<210>53

<211>244

<212>PRT

＜213〉barley

<400>53

<210>54

<211>1196

<212>DNA

＜213〉barley

<400>54

<210>55

<211>244

<212>PRT

＜213〉barley

<400>55

<210>56

<211>1161

<212>DNA

＜213〉common wheat

<400>56

<210>57

<211>244

<212>PRT

＜213〉common wheat

<400>57

<210>58

<211>1210

<212>DNA

＜213〉common wheat

<400>58

<210>59

<211>244

<212>PRT

＜213〉common wheat

<400>59

<210>60

<211>735

<212>DNA

＜213〉one grained wheat (Triticum monococcum)

<400>60

<210>61

<211>244

<212>PRT

＜213〉one grained wheat

<400>61

<210>62

<211>735

<212>DNA

＜213〉common wheat

<400>62

<210>63

<211>244

<212>PRT

＜213〉common wheat

<400>63

<210>64

<211>738

<212>DNA

＜213〉rye grass (Lolium perenne)

<400>64

<210>65

<211>245

<212>PRT

＜213〉rye grass

<400>65

<210>66

<211>738

<212>DNA

＜213〉lolium temulentum (Lolium temulentum)

<400>66

<210>67

<211>245

<212>PRT

＜213〉lolium temulentum

<400>67

<210>68

<211>738

<212>DNA

＜213〉Zea mays

<400>68

<210>69

<211>245

<212>PRT

＜213〉Zea mays

<400>69

<210>70

<211>738

<212>DNA

＜213〉Zea mays

<400>70

<210>71

<211>245

<212>PRT

＜213〉Zea mays

<400>71

<210>72

<211>762

<212>DNA

＜213〉rice

<400>72

<210>73

<211>253

<212>PRT

＜213〉rice

<400>73

<210>74

<211>741

<212>DNA

＜213〉rice

<400>74

<210>75

<211>246

<212>PRT

＜213〉rice

<400>75

<210>76

<211>735

<212>DNA

＜213〉sinocalamus latiflorus (Dendrocalamus latiflorus)

<400>76

<210>77

<211>244

<212>PRT

＜213〉sinocalamus latiflorus

<400>77

<210>78

<211>735

<212>DNA

＜213〉sinocalamus latiflorus

<400>78

<210>79

<211>244

<212>PRT

＜213〉sinocalamus latiflorus

<400>79

<210>80

<211>813

<212>DNA

＜213〉Zea mays

<400>80

<210>81

<211>270

<212>PRT

＜213〉Zea mays

<400>81

<210>82

<211>689

<212>DNA

＜213〉dichromatism chinese sorghum

<400>82

<210>83

<211>228

<212>PRT

＜213〉dichromatism chinese sorghum

<400>83

<210>84

<211>822

<212>DNA

＜213〉Zea mays

<400>84

<210>85

<211>273

<212>PRT

＜213〉Zea mays

<400>85

<210>86

<211>786

<212>DNA

＜213〉lolium temulentum

<400>86

<210>87

<211>261

<212>PRT

＜213〉lolium temulentum

<400>87

<210>88

<211>786

<212>DNA

＜213〉rye grass

<400>88

<210>89

<211>261

<212>PRT

＜213〉rye grass

<400>89

<210>90

<211>831

<212>DNA

＜213〉barley

<400>90

<210>91

<211>276

<212>PRT

＜213〉barley

<400>91

<210>92

<211>804

<212>DNA

＜213〉rice

<400>92

<210>93

<211>267

<212>PRT

＜213〉rice

<400>93

<210>94

<211>804

<212>DNA

＜213〉rice

<400>94

<210>95

<211>267

<212>PRT

＜213〉rice

<400>95

<210>96

<211>668

<212>DNA

＜213〉spidewort (Tradescantia virginiana)

<400>96

<210>97

<211>222

<212>PRT

＜213〉spidewort

<220>

＜221〉variant

<222>(11)..(11)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉variant

<222>(218)..(218)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉variant

<222>(221)..(221)

＜223〉Xaa can be any naturally occurring amino acid

<400>97

<210>98

<211>723

<212>DNA

＜213〉spidewort

<400>98

<210>99

<211>241

<212>PRT

＜213〉spidewort

<220>

＜221〉uncertain

<222>(228)..(228)

＜223〉Xaa can be any naturally occurring amino acid

<400>99

<210>100

<211>599

<212>DNA

＜213〉spidewort

<400>100

<210>101

<211>199

<212>PRT

＜213〉spidewort

<400>101

<210>102

<211>753

<212>DNA

＜213〉oil palm (Elaeis guineensis)

<400>102

<210>103

<211>250

<212>PRT

＜213〉oil palm

<400>103

<210>104

<211>699

<212>DNA

＜213〉allium (Allium sp.)

<400>104

<210>105

<211>233

<212>PRT

＜213〉allium

<400>105

<210>106

<211>744

<212>DNA

<213>Dendrobium grex Madame Thong-IN

<400>106

<210>107

<211>247

<212>PRT

<213>Dendrobium grex Madame Thong-IN

<400>107

<210>108

<211>2194

<212>DNA

＜213〉rice

<400>108

<210>109

<211>804

<212>DNA

＜213〉rice

<400>109

<210>110

<211>267

<212>PRT

＜213〉rice

<400>110

<210>111

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>111

<210>112

<211>47

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>112

<210>113

<211>2193

<212>DNA

＜213〉rice

<400>113

<210>114

<211>42

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Asn "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(12)..(12)

＜223 〉/replace=" Tyr "

<220>

＜221〉variant

<222>(16)..(16)

＜223 〉/replace=" Ile "/replace=" Leu "

<220>

＜221〉variant

<222>(17)..(17)

＜223 〉/replace=" Gly "

<220>

＜221〉variant

<222>(18)..(18)

＜223 〉/replace=" Ala "/replace=" Val "

<220>

＜221〉variant

<222>(19)..(19)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(20)..(20)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(23)..(23)

＜223 〉/replace=" Pro "/replace=" Ala "/replace=" Asn "

<220>

＜221〉variant

<222>(30)..(30)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(31)..(31)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(32)..(32)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(33)..(33)

＜223 〉/replace=" Asn "/replace=" Glu "

<220>

＜221〉variant

<222>(35)..(35)

＜223 〉/replace=" Cys/ replacement=" Arg "/replace=" Ser "

<220>

＜221〉variant

<222>(37)..(37)

＜223 〉/replace=" Glu "

<220>

＜221〉variant

<222>(38)..(38)

＜223 〉/replace=" Ile "/replace=" Arg "/replace=" Lys "

<220>

＜221〉variant

<222>(41)..(41)

＜223 〉/replace=" Asp "

<400>114

<210>115

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(5)..(5)

＜223 〉/replace=" Arg "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(8)..(8)

＜223 〉/replace=" Thr "/replace=" Ser "

<220>

＜221〉variant

<222>(9)..(9)

＜223 〉/replace=" Ile "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Asn "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Arg "/replace=" Asn "

<220>

＜221〉variant

<222>(12)..(12)

＜223 〉/replace=" Cys "/replace=" Arg "

<220>

＜221〉variant

<222>(13)..(13)

＜223 〉/replace=" His "

<220>

＜221〉variant

<222>(14)..(14)

＜223 〉/replace=" Lys "

<400>115

<210>116

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Gln "/replace=" Val "/replace=" Ala "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(8)..(8)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(9)..(9)

＜223 〉/replace=" Phe "

<220>

＜221〉variant

<222>(10)..(10)

＜223 〉/replace=" Cys "/replace=" Phe "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Met "

<400>116

<210>117

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence

<220>

＜221〉variant

<222>(1)..(1)

＜223 〉/replace=" Ala "/replace=" Val "/replace=" Leu "

<220>

＜221〉variant

<222>(2)..(2)

＜223 〉/replace=" Pro "

<220>

＜221〉uncertain

<222>(3)..(3)

＜223〉Xaa can be an arbitrary amino acid, preferred Leu, Phe or His

<220>

＜221〉uncertain

<222>(6)..(6)

＜223〉Xaa can be arbitrary naturally occurring amino acid, preferred Val or

Leu

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" His "

<400>117

<210>118

<211>735

<212>DNA

＜213〉barley

<400>118

<210>119

<211>244

<212>PRT

＜213〉barley

<400>119

<210>120

<211>1196

<212>DNA

＜213〉barley

<400>120

<210>121

<211>244

<212>PRT

＜213〉barley

<400>121

<210>122

<211>1161

<212>DNA

＜213〉common wheat

<400>122

<210>123

<211>244

<212>PRT

＜213〉common wheat

<400>123

<210>124

<211>1210

<212>DNA

＜213〉common wheat

<400>124

<210>125

<211>244

<212>PRT

＜213〉common wheat

<400>125

<210>126

<211>735

<212>DNA

＜213〉one grained wheat

<400>126

<210>127

<211>244

<212>PRT

＜213〉one grained wheat

<400>127

<210>128

<211>735

<212>DNA

＜213〉common wheat

<400>128

<210>129

<211>244

<212>PRT

＜213〉common wheat

<400>129

<210>130

<211>738

<212>DNA

＜213〉rye grass

<400>130

<210>131

<211>245

<212>PRT

＜213〉rye grass

<400>131

<210>132

<211>738

<212>DNA

＜213〉lolium temulentum

<400>132

<210>133

<211>245

<212>PRT

＜213〉lolium temulentum

<400>133

<210>134

<211>738

<212>DNA

＜213〉Zea mays

<400>134

<210>135

<211>245

<212>PRT

＜213〉Zea mays

<400>135

<210>136

<211>738

<212>DNA

＜213〉Zea mays

<400>136

<210>137

<211>245

<212>PRT

＜213〉Zea mays

<400>137

<210>138

<211>762

<212>DNA

＜213〉rice

<400>138

<210>139

<211>253

<212>PRT

＜213〉rice

<400>139

<210>140

<211>741

<212>DNA

＜213〉rice

<400>140

<210>141

<211>246

<212>PRT

＜213〉rice

<400>141

<210>142

<211>735

<212>DNA

＜213〉sinocalamus latiflorus

<400>142

<210>143

<211>244

<212>PRT

＜213〉sinocalamus latiflorus

<400>143

<210>144

<211>735

<212>DNA

＜213〉sinocalamus latiflorus

<400>144

<210>145

<211>244

<212>PRT

＜213〉sinocalamus latiflorus

<400>145

<210>146

<211>813

<212>DNA

＜213〉Zea mays

<400>146

<210>147

<211>270

<212>PRT

＜213〉Zea mays

<400>147

<210>148

<211>689

<212>DNA

＜213〉dichromatism chinese sorghum

<400>148

<210>149

<211>228

<212>PRT

＜213〉dichromatism chinese sorghum

<400>149

<210>150

<211>822

<212>DNA

＜213〉Zea mays

<400>150

<210>151

<211>273

<212>PRT

＜213〉Zea mays

<400>151

<210>152

<211>786

<212>DNA

＜213〉lolium temulentum

<400>152

<210>153

<211>261

<212>PRT

＜213〉lolium temulentum

<400>153

<210>154

<211>786

<212>DNA

＜213〉rye grass

<400>154

<210>155

<211>261

<212>PRT

＜213〉rye grass

<400>155

<210>156

<211>831

<212>DNA

＜213〉barley

<400>156

<210>157

<211>276

<212>PRT

＜213〉barley

<400>157

<210>158

<211>804

<212>DNA

＜213〉rice

<400>158

<210>159

<211>267

<212>PRT

＜213〉rice

<400>159

<210>160

<211>804

<212>DNA

＜213〉rice

<400>160

<210>161

<211>267

<212>PRT

＜213〉rice

<400>161

<210>162

<211>668

<212>DNA

＜213〉spidewort

<400>162

<210>163

<211>222

<212>PRT

＜213〉spidewort

<220>

＜221〉uncertain

<222>(11)..(11)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉uncertain

<222>(218)..(218)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉uncertain

<222>(221)..(221)

＜223〉Xaa can be any naturally occurring amino acid

<400>163

<210>164

<211>723

<212>DNA

＜213〉spidewort

<400>164

<210>165

<211>241

<212>PRT

＜213〉spidewort

<220>

＜221〉uncertain

<222>(228)..(228)

＜223〉Xaa can be any naturally occurring amino acid

<400>165

<210>166

<211>599

<212>DNA

＜213〉spidewort

<400>166

<210>167

<211>199

<212>PRT

＜213〉spidewort

<400>167

<210>168

<211>753

<212>DNA

＜213〉oil palm

<400>168

<210>169

<211>250

<212>PRT

＜213〉oil palm

<400>169

<210>170

<211>699

<212>DNA

＜213〉allium

<400>170

<210>171

<211>233

<212>PRT

＜213〉allium

<400>171

<210>172

<211>744

<212>DNA

<213>Dendrobium grex Madame Thong-IN

<400>172

<210>173

<211>247

<212>PRT

<213>Dendrobium grex Madame Thong-IN

<400>173

<210>174

<211>2194

<212>DNA

＜213〉rice

<400>174

<210>175

<211>1725

<212>DNA

＜213〉Arabidopis thaliana

<400>175

<210>176

<211>574

<212>PRT

＜213〉Arabidopis thaliana

<400>176

<210>177

<211>1707

<212>DNA

＜213〉Arabidopis thaliana

<400>177

<210>178

<211>568

<212>PRT

＜213〉Arabidopis thaliana

<400>178

<210>179

<211>1662

<212>DNA

＜213〉soybean

<400>179

<210>180

<211>553

<212>PRT

＜213〉soybean

<400>180

<210>181

<211>1674

<212>DNA

＜213〉soybean

<400>181

<210>182

<211>557

<212>PRT

＜213〉soybean

<400>182

<210>183

<211>1632

<212>DNA

<213>Medicago trunculata

<400>183

<210>184

<211>557

<212>PRT

<213>Medicago trunculata

<400>184

<210>185

<211>2079

<212>DNA

＜213〉rice

<400>185

<210>186

<211>692

<212>PRT

＜213〉rice

<400>186

<210>187

<211>2133

<212>DNA

＜213〉Zea mays

<400>187

<210>188

<211>710

<212>PRT

＜213〉Zea mays

<400>188

<210>189

<211>1374

<212>DNA

＜213〉Root or stem of Littleleaf Indianmulberry (Lotus corniculatus)

<400>189

<210>190

<211>457

<212>PRT

＜213〉Root or stem of Littleleaf Indianmulberry

<400>190

<210>191

<211>202

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the AP2 structural domain of Arath_PLT1 and Arath_PLT2

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Thr "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Glu "

<220>

＜221〉variant

<222>(57)..(57)

＜223 〉/replace=" Glu "

<220>

＜221〉variant

<222>(62)..(62)

＜223 〉/replace=" Ala "

<220>

＜221〉variant

<222>(93)..(93)

＜223 〉/replace=" Asn "

<220>

＜221〉variant

<222>(102)..(102)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(187)..(187)

＜223 〉/replace=" Asn "

<400>191

<210>192

<211>8

<212>PRT

＜213〉artificial sequence

<220>

＜223〉motif 1

<220>

＜221〉variant

<222>(3)..(3)

＜223 〉/replace=" Leu "

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Glu "

<400>192

<210>193

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉motif 2

<220>

＜221〉variant

<222>(1)..(1)

＜223 〉/replace=" Leu "

<220>

＜221〉variant

<222>(3)..(3)

＜223 〉/replace=" Ser "

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" Glu "

<400>193

<210>194

<211>2193

<212>DNA

＜213〉rice

<400>194

<210>195

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: PRM08180

<400>195

<210>196

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: PRM08181

<400>196

<210>197

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: PRM08182

<400>197

<210>198

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: PRM08183

<400>198

<210>199

<211>1638

<212>DNA

＜213〉Populus tremuloides (Populus tremuloides)

<400>199

<210>200

<211>545

<212>PRT

＜213〉Populus tremuloides

<400>200

<210>201

<211>1515

<212>DNA

＜213〉Populus tremuloides

<400>201

<210>202

<211>504

<212>PRT

＜213〉Populus tremuloides

<400>202

<210>203

<211>785

<212>DNA

＜213〉grape

<400>203

<210>204

<211>260

<212>PRT

＜213〉grape

<400>204

<210>205

<211>150

<212>DNA

＜213〉colea

<400>205

<210>206

<211>49

<212>PRT

＜213〉colea

<400>206

<210>207

<211>162

<212>DNA

＜213〉scarlet runner bean (Phaseolus coccineus)

<400>207

<210>208

<211>53

<212>PRT

＜213〉scarlet runner bean

<400>208

<210>209

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉motif 2

<220>

＜221〉variant

<222>(1)..(1)

＜223 〉/replace=" Leu "

<220>

＜221〉uncertain

<222>(3)..(3)

＜223〉Xaa can be any naturally occurring amino acid

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" Glu "

<400>209

<210>210

<211>2194

<212>DNA

＜213〉rice

<400>210

<210>211

<211>1245

<212>DNA

＜213〉rice

<400>211

<210>212

<211>675

<212>DNA

＜213〉rice

<400>212

<210>213

<211>224

<212>PRT

＜213〉rice

<400>213

<210>214

<211>696

<212>DNA

＜213〉rice

<400>214

<210>215

<211>231

<212>PRT

＜213〉rice

<400>215

<210>216

<211>633

<212>DNA

＜213〉rice

<400>216

<210>217

<211>210

<212>PRT

＜213〉rice

<400>217

<210>218

<211>546

<212>DNA

＜213〉Arabidopis thaliana

<400>218

<210>219

<211>188

<212>PRT

＜213〉Arabidopis thaliana

<400>219

<210>220

<211>525

<212>DNA

＜213〉Arabidopis thaliana

<400>220

<210>221

<211>174

<212>PRT

＜213〉Arabidopis thaliana

<400>221

<210>222

<211>369

<212>DNA

＜213〉Arabidopis thaliana

<400>222

<210>223

<211>122

<212>PRT

＜213〉Arabidopis thaliana

<400>223

<210>224

<211>573

<212>DNA

＜213〉Arabidopis thaliana

<400>224

<210>225

<211>190

<212>PRT

＜213〉Arabidopis thaliana

<400>225

<210>226

<211>423

<212>DNA

＜213〉puncture vine clover (Medicago truncatula)

<400>226

<210>227

<211>140

<212>PRT

＜213〉puncture vine clover

<400>227

<210>228

<211>882

<212>DNA

＜213〉barley

<400>228

<210>229

<211>293

<212>PRT

＜213〉barley

<400>229

<210>230

<211>2193

<212>DNA

＜213〉rice

<400>230

<210>231

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: prm06808

<400>231

<210>232

<211>50

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: prm06809

<400>232

<210>233

<211>2194

<212>DNA

＜213〉rice

<400>233

<210>234

<211>1065

<212>DNA

＜213〉Arabidopis thaliana

<400>234

<210>235

<211>354

<212>PRT

＜213〉Arabidopis thaliana

<400>235

<210>236

<211>1128

<212>DNA

＜213〉Arabidopis thaliana

<400>236

<210>237

<211>375

<212>PRT

＜213〉Arabidopis thaliana

<400>237

<210>238

<211>1149

<212>DNA

<213>Aquilegia formosa x Aquilegia pubescens

<400>238

<210>239

<211>382

<212>PRT

<213>Aquilegia formosa x Aquilegia pubescens

<400>239

<210>240

<211>1170

<212>DNA

＜213〉upland cotton

<400>240

<210>241

<211>389

<212>PRT

＜213〉upland cotton

<400>241

<210>242

<211>1017

<212>DNA

＜213〉(Ipomoea nil) leads a cow

<400>242

<210>243

<211>338

<212>PRT

＜213〉lead a cow

<400>243

<210>244

<211>1035

<212>DNA

＜213〉lettuce (Lactuca sativa)

<400>244

<210>245

<211>344

<212>PRT

＜213〉lettuce

<400>245

<210>246

<211>1137

<212>DNA

＜213〉apple (Malus domestica)

<400>246

<210>247

<211>378

<212>PRT

＜213〉apple

<400>247

<210>248

<211>1014

<212>DNA

<213>Medicago trunculata

<400>248

<210>249

<211>337

<212>PRT

<213>Medicago trunculata

<400>249

<210>250

<211>1143

<212>DNA

<213>Nicotiana bentamiana

<400>250

<210>251

<211>380

<212>PRT

<213>Nicotiana bentamiana

<400>251

<210>252

<211>1254

<212>DNA

＜213〉rice

<400>252

<210>253

<211>417

<212>PRT

＜213〉rice

<400>253

<210>254

<211>1182

<212>DNA

＜213〉rice

<400>254

<210>255

<211>393

<212>PRT

＜213〉rice

<400>255

<210>256

<211>1119

<212>DNA

＜213〉potato

<400>256

<210>257

<211>372

<212>PRT

＜213〉potato

<400>257

<210>258

<211>1140

<212>DNA

＜213〉grape

<400>258

<210>259

<211>379

<212>PRT

＜213〉grape

<400>259

<210>260

<211>1209

<212>DNA

＜213〉Zea mays

<400>260

<210>261

<211>402

<212>PRT

＜213〉Zea mays

<400>261

<210>262

<211>1137

<212>DNA

＜213〉Zea mays

<400>262

<210>263

<211>378

<212>PRT

＜213〉Zea mays

<400>263

<210>264

<211>403

<212>DNA

＜213〉intend Chinese sorghum (Sorghum propinquum)

<400>264

<210>265

<211>134

<212>PRT

＜213〉intend Chinese sorghum

<400>265

<210>266

<211>732

<212>DNA

＜213〉onion (Allium cepa)

<400>266

<210>267

<211>244

<212>PRT

＜213〉onion

<400>267

<210>268

<211>969

<212>DNA

＜213〉Common Snapdragon (Antirrhinum majus)

<400>268

<210>269

<211>323

<212>PRT

＜213〉Common Snapdragon

<400>269

<210>270

<211>633

<212>DNA

＜213〉colea

<400>270

<210>271

<211>211

<212>PRT

＜213〉colea

<400>271

<210>272

<211>852

<212>DNA

＜213〉sugarcane (Saccharum officinarum)

<400>272

<210>273

<211>283

<212>PRT

＜213〉sugarcane

<400>273

<210>274

<211>647

<212>DNA

＜213〉alta fascue

<400>274

<210>275

<211>215

<212>PRT

＜213〉alta fascue

<400>275

<210>276

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉motif 1

<220>

＜221〉variant

<222>(1)..(1)

＜223 〉/replace=" Thr "

<220>

＜221〉variant

<222>(3)..(3)

＜223 〉/replace=" Arg "/replace=" Met "/replace=" Gln "

<220>

＜221〉variant

<222>(6)..(6)

＜223 〉/replace=" Gln "/replace=" Arg "

<220>

＜221〉variant

<222>(7)..(7)

＜223 〉/replace=" Arg "/replace=" Glu "

<220>

＜221〉variant

<222>(8)..(8)

＜223 〉/replace=" Val "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Gly "

<400>276

<210>277

<211>78

<212>PRT

＜213〉artificial sequence

<220>

＜223〉the SPL DNA binding domains (DBD) of Arath_SPL15 transcription factor

<400>277

<210>278

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉motif 2

<220>

＜221〉variant

<222>(3)..(3)

＜223 〉/replace=" Asn "/replace=" Thr "

<220>

＜221〉variant

<222>(4)..(4)

＜223 〉/replace=" Gly "/replace=" Arg "

<220>

＜221〉variant

<222>(11)..(11)

＜223 〉/replace=" Thr "

<400>278

<210>279

<211>1130

<212>DNA

＜213〉rice

<400>279

<210>280

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: prm07277

<400>280

<210>281

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer: prm07278

<400>281

<210>282

<211>1110

<212>DNA

＜213〉overgrown with weeds blue or green

<400>282

<210>283

<211>369

<212>PRT

＜213〉overgrown with weeds blue or green

<400>283

<210>284

<211>1080

<212>DNA

＜213〉soybean

<400>284

<210>285

<211>359

<212>PRT

＜213〉soybean

<400>285

<210>286

<211>1128

<212>DNA

＜213〉Populus tremuloides

<400>286

<210>287

<211>375

<212>PRT

＜213〉Populus tremuloides

<400>287

<210>288

<211>1059

<212>DNA

<213>Citrus clementina

<400>288

<210>289

<211>352

<212>PRT

<213>Citrus clementina

<400>289

<210>290

<211>565

<212>DNA

＜213〉beet (Beta vulgaris)

<400>290

<210>291

<211>188

<212>PRT

＜213〉beet

<400>291

<210>292

<211>536

<212>DNA

＜213〉rubber tree (Hevea brasiliensis)

<400>292

<210>293

<211>177

<212>PRT

＜213〉rubber tree

<400>293

<210>294

<211>1130

<212>DNA

＜213〉rice

<400>294

<210>295

<211>2194

<212>DNA

＜213〉rice

<400>295

<210>296

<211>1149

<212>DNA

＜213〉Zea mays

<220>

<221>misc_feature

<222>(157)..(157)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(894)..(894)

＜223〉n is a, c, g, or t

<220>

<221>misc_feature

<222>(954)..(954)

＜223〉n is a, c, g, or t

<400>296

<210>297

<211>382

<212>PRT

＜213〉Zea mays

<220>

＜221〉uncertain

<222>(53)..(53)

＜223〉Xaa can be any naturally occurring amino acid

<400>297

<210>298

<211>660

<212>DNA

＜213〉Zea mays

<400>298

<210>299

<211>219

<212>PRT

＜213〉Zea mays

<400>299

<210>300

<211>669

<212>DNA

＜213〉soybean

<400>300

<210>301

<211>222

<212>PRT

＜213〉soybean

<400>301

Claims (139)

1. be used for strengthening the method for plant output correlated character, the plant that the expression and randomly select of it nucleic acid that comprises preferential increase coding HAL3 polypeptide in plantling has the output of increase with respect to control plant.

2. according to the process of claim 1 wherein the expression of described increase, preferably in the locus of the gene of coding HAL3 polypeptide, import genetic modification and realize by importing genetic modification.

3. according to the method for claim 2, wherein said genetic modification is by any or accomplished in many ways of T-DNA activation, TILLING and homologous recombination.

4. according to the method for claim 2, wherein said genetic modification is by any or accomplished in many ways of T-DNA activation, TILLING and homologous recombination.

5. according to the process of claim 1 wherein that the expression of described increase realizes by the nucleic acid that imports and express coding HAL3 polypeptide in plant.

6. according to the method for claim 5, the proteinic nucleic acid of wherein said coding HAL3 is by any nucleic acid SEQ ID NO or its part that provide in Table A, or can with the sequence representative of any nucleic acid SEQ ID NO hybridization that in Table A, provides.

7. according to the method for claim 5, arbitrary nucleic acid SEQ ID NO that the proteinic nucleic acid encoding of wherein said coding HAL3 provides in Table A directly to homologue or collateral line homologue.

8. according to each method of claim 1 to 7, wherein said enhanced yield correlated character comprises the output and/or the early stage vigor of increase.

9. method according to Claim 8, the output of wherein said increase are the seed productions that increases.

10. according to the method for claim 9, the seed production of wherein said increase be following one or more: a) the seed biomass of Zeng Jiaing; B) harvest index of Zeng Jiaing; And c) (full) seed number of Zeng Jiaing.

11. according to the method for claim 9, the increase of wherein said seed production is at least 10% with respect to control plant.

12. according to each method of claim 5 to 11, the proteinic nucleic acid of wherein said coding HAL3 effectively connects the seedling specificity promoter, the preferred β expansion protein promoter that effectively connects.

13. according to each method of claim 5 to 12, the proteinic nucleic acid of wherein said coding HAL3 is plant origin, preferably from dicotyledons, also preferably from cress, more preferably from Arabidopsis, most preferably from Arabidopis thaliana.

14. by according to the obtainable plant of the method for arbitrary aforementioned claim or its part, comprise seed, wherein said plant or its part comprise the recombinant nucleic acid of coding HAL3 polypeptide.

15. construct, it comprises:

(a) nucleic acid of coding HAL3 polypeptide;

(b) nucleotide sequence that can drive (a) one or more regulating and controlling sequences of preferentially in the seedling tissue of plant, expressing; Randomly

(c) transcription termination sequence.

16. according to the construct of claim 15, wherein said one or more regulating and controlling sequences are the seedling specificity promoter at least, preferably β expansion protein promoter.

17. according to the purposes of the construct of claim 15 or 16, be used for strengthening plant output correlated character, be preferably used for increasing output and/or vigor with respect to control plant.

18. according to the purposes of claim 17, wherein said output is the seed production that increases.

19. according to the purposes of claim 17, wherein said vigor is early stage vigor.

20. use according to claim 15 or 16 described construct plant transformed, plant part or vegetable cell.

21. be used to produce the method that has the transgenic plant of enhanced yield correlated character with respect to control plant, described method comprises:

(i) import the also preferential expression of nucleic acid in the seedling tissue of plant that increases coding HAL3 polypeptide; With

Cell (ii) cultivates plants under the condition that promotes plant-growth and growth.

22. with respect to the transgenic plant that control plant has the enhanced yield correlated character, described enhanced yield correlated character is owing to the expression of preferential increase HAL3 polypeptide in seedling produces.

23. according to claim 14,20 or 22 transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

24. according to claim 14,20,22 or 23 each the parts gathered in the crops of plant, the wherein said preferably seed of part of gathering in the crops.

25. from according to the plant of claim 23 and/or according to the product of the part gathered in the crops of the plant of claim 24.

26. the nucleic acid of coding HAL3 polypeptide is used for strengthening the purposes of plant output correlated character, wherein said nucleic acid effectively connects the seedling specificity promoter.

27. according to the purposes of claim 26, wherein said output correlated character comprises seed production and/or early stage vigor.

28. be used for strengthening the method for plant output correlated character, it comprises adjusting, the expression of the preferred increase coding proteinic nucleic acid of MADS15 in plant.

29. according to the method for claim 28, wherein said MADS15 protein comprises any or multiple following motif: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQID NO:51.

30. according to the method for claim 28 to 29, the wherein said expression of being regulated activates any or accomplished in many ways of label, TILLING, site-directed mutagenesis, orthogenesis or homologous recombination by T-DNA.

31. according to each method of claim 28 to 29, the wherein said expression of being regulated realizes by import and express the proteinic nucleic acid of coding MADS15 in plant, and the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51.

32. according to the method for claim 31, the proteinic nucleic acid of wherein said coding MADS15 is any nucleic acid SEQ ID NO or its part that provides among the D at table, or can with the sequence of any nucleic acid SEQ ID NO hybridization that in table D, provides.

33. according to the method for claim 31, arbitrary nucleic acid SEQ ID NO that the proteinic nucleic acid encoding of wherein said coding MADS15 provides among the D at table directly to homologue or collateral line homologue.

34. according to each method of claim 28 to 33, wherein said enhanced yield correlated character is the root biomass.

35. according to each method of claim 31 to 34, the proteinic nucleic acid of wherein said coding MADS15 effectively connects to form type promotor, preferred GOS2 promotor.

36. according to each method of claim 31 to 35, the proteinic nucleic acid of wherein said coding MADS15 is plant origin, preferably from monocotyledons, also preferably from Gramineae, more preferably from Oryza, most preferably from rice.

37. by according to each the obtainable plant of method or its part of claim 28 to 36, comprise seed, wherein said plant or its part comprise the proteinic recombinant nucleic acid of coding MADS15.

38. construct, it comprises:

(a) the proteinic nucleic acid of coding MADS15, the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51;

(b) can drive one or more regulating and controlling sequences that the nucleotide sequence of (a) is expressed; Randomly

(c) transcription termination sequence.

39. according to the construct of claim 38, wherein said one or more regulating and controlling sequences are constitutive promoter at least, preferred GOS2 promotor.

40. according to the purposes of the construct of claim 38 or 39, be used to produce with respect to control plant have the enhanced yield correlated character, the plant of the root output that especially increases.

41. use construct plant transformed, plant part or vegetable cell according to claim 38 or 39.

42. be used to produce the method that has the transgenic plant of enhanced yield correlated character with respect to control plant, described method comprises:

(i) import and express the proteinic nucleic acid of coding MADS15 in plant, the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ IDNO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; With

Cell (ii) cultivates plants under the condition that promotes plant-growth and growth.

43. have with respect to control plant increase output transgenic plant or from the transgenic plant cells of described transgenic plant, the output of described increase produces because of the expression of the increase of the proteinic nucleic acid of coding MADS15, and the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQID NO:51.

44. according to claim 37,41 or 43 transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat, or from the transgenic plant cells of described transgenic plant.

45. according to the part gathered in the crops of the plant of claim 44, the wherein said preferably root of part of gathering in the crops.

46. from according to the plant of claim 44 and/or according to the product of the part gathered in the crops of the plant of claim 45.

47. the proteinic nucleic acid of coding MADS15 purposes in the output correlated character in strengthening plant, the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51.

48. according to the purposes of claim 47, wherein said enhanced yield correlated character is the root biomass that increases with respect to control plant.

49. be used for strengthening with respect to suitable control plant the method for plant output correlated character, it comprises the expression of preferential reduction endogenous MADS15 gene in described plant.

50. according to the method for claim 49, wherein said MADS15 protein comprises any or multiple following motif: SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117.

51. according to the method for claim 49 or 50, the expression of wherein said reduction realizes by the downward modulation of the RNA mediation of genetic expression.

52. according to the method for claim 51, the downward modulation of wherein said RNA mediation is by suppressing realization altogether.

53. according to the method for claim 51, the downward modulation of wherein said RNA mediation realizes by using antisense MADS15 nucleotide sequence.

54. according to the method for claim 49 or 50, the expression of wherein said reduction uses MADS15 gene or its segmental inverted repeats to realize that described inverted repeats preferably can form hairpin structure.

55. according to the method for claim 49 or 50, wherein using has specific ribozyme to realize the expression of described reduction to MADS15 nucleic acid.

56., wherein realize the expression of described reduction by insertion mutagenesis according to the method for claim 49 or 50.

57. according to each method of claim 49 to 56, wherein said endogenous MADS15 gene is as the MADS15 gene of finding with its natural form in the plant or imports the isolated M ADS15 nucleic acid of plant subsequently.

58. according to the method for claim 57, the MADS15 nucleic acid of wherein said importing effectively connects to form the type promotor, preferred GOS2 promotor.

59. method according to claim 57 or 58, the MADS15 nucleic acid of wherein said importing is from plant origin or artificial source, preferably wherein use MADS15 nucleic acid transformed plant with endogenous MADS15 dna homolog, more preferably wherein be used for transforming monocots from monocotyledonous MADS15 nucleotide sequence, also more preferably wherein be used to be converted in the grass, most preferably wherein be used to transform rice plant from the MADS15 nucleotide sequence of rice from MADS15 sequence gramineous.

60. method according to claim 59, wherein said MADS15 nucleotide sequence from rice comprises the Nucleotide of successive basically of the sufficient length of SEQ ID NO:109 (OsMADS15), or comprise coding OsMADS15 (SEQ ID NO:110) directly to the sufficient length of the nucleotide sequence of homologue or collateral line homologue successive Nucleotide basically.

61. according to the method for claim 60, wherein the described of OsMADS15 directly represented by SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ IDNO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159 and SEQ IDNO:161 to homologue or collateral line homologue.

62. according to the method for claim 60 or 61, the Nucleotide of successive basically that directly comes the nucleotide sequence of free SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ IDNO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158 and SEQ IDNO:160 representative of the OsMADS15 that wherein encodes (SEQ IDNO:110) to the described Nucleotide of successive basically of the nucleotide sequence of homologue or collateral line homologue.

63. according to each method of claim 49 to 62, wherein said enhanced yield correlated character comprises the output of increase, the preferred seed production that increases and/or the biomass of increase.

64. according to the method for claim 63, the seed production of wherein said increase be selected from following one or more: a) the seed biomass (seed weight) of Zeng Jiaing; B) every strain plant flowers number of Zeng Jiaing; And c) (full) seed number of Zeng Jiaing.

65. by according to each the obtainable plant of method or its part of claim 49 to 64, comprise seed, wherein said plant or its part comprise reorganization MADS15 nucleic acid.

66. construct, it comprises:

(i) can make the MADS15 nucleic acid of endogenous MADS15 gene silencing;

(ii) can drive one or more regulating and controlling sequences of the nucleotide sequence expression of (a); Randomly

(iii) transcription termination sequence.

67. according to the construct of claim 66, wherein said one or more regulating and controlling sequences are constitutive promoter at least, preferred GOS2 promotor.

68., be used to produce and have enhanced yield correlated character, the seed production that especially increases and/or the plant of biomass with respect to control plant according to the purposes of the construct of claim 66 or 67.

69. use construct plant transformed, plant part or vegetable cell according to claim 66 or 67.

70. be used to produce the method that has the transgenic plant of enhanced yield correlated character with respect to control plant, described method comprises:

(i) import and in plant, express the MADS15 nucleic acid that can make endogenous MADS15 gene silencing; With

Cell (ii) cultivates plants under the condition that promotes plant-growth and growth.

71. have the transgenic plant of enhanced yield correlated character or from the transgenic plant cells of described transgenic plant with respect to control plant, described enhanced yield correlated character causes that because of the expression of the reduction of the proteinic nucleic acid of coding MADS15 the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQ ID NO:114, SEQ IDNO:115, SEQ ID NO:116, SEQ ID NO:117.

72. according to claim 65,69 or 71 transgenic plant, or from the transgenic plant cells of described transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

73. according to the part gathered in the crops of the plant of claim 72, the wherein said preferably seed of part of gathering in the crops.

74. from according to the plant of claim 72 and/or according to the product of the part gathered in the crops of the plant of claim 73.

75. the nucleic acid of coding MADS15 is used for strengthening the purposes of plant output correlated character, the proteinic aminoacid sequence of wherein said MADS15 comprises any or multiple following motif: SEQID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117.

76. according to the purposes of claim 75, wherein said enhanced yield correlated character comprises the seed production of increase.

77. be used for increasing the method for plant biomass, the plant that the expression and randomly select of nucleic acid in plant that it comprises increases coding PLT transcription factor polypeptide has the output of increase with respect to suitable control plant.

78. be used for increasing with respect to control plant the method for plant abiotic stress resistance, it comprises that the expression and randomly select of nucleic acid in plant that increases coding PLT transcription factor polypeptide has the plant of the abiotic stress resistance of increase.

79. according to the method for claim 78, the abiotic stress resistance of wherein said increase is the nutrition intake efficient that increases with respect to control plant.

80., wherein realize the expression of described increase by the nucleic acid that imports and in plant, plant part or vegetable cell, express coding PLT transcription factor polypeptide according to each method of claim 77 to 79.

81. 0 method according to Claim 8, the nucleic acid of wherein said coding PLT transcription factor be any nucleic acid SEQ ID NO of in table 1, providing or its part or can with the sequence of any nucleic acid SEQ ID NO hybridization that in table 1, provides.

82. 0 method according to Claim 8, arbitrary nucleic acid SEQ ID NO that the nucleic acid encoding of wherein said coding PLT transcription factor provides in table D directly to homologue or collateral line homologue.

83. according to claim 77,80,81 and 82 each methods, the output of wherein said increase is the seed production that increases.

84. 3 method according to Claim 8, wherein said seed production be selected from following one or more: a) the seed biomass of Zeng Jiaing; B) every strain plant flowers number of Zeng Jiaing; C) (full) seed number of Zeng Jiaing; D) the full rate of the seed of Zeng Jiaing; E) harvest index of Zeng Jiaing; F) thousand seed weight of Zeng Jiaing (TKW); G) the seed area of Zeng Jiaing; H) seed length of Zeng Jiaing; And i) the seed width of Zeng Jiaing.

85. 0 to 84 each method according to Claim 8, wherein said nucleic acid effectively connects to form type promotor, preferred GOS2 promotor.

86. 0 to 84 each method according to Claim 8, the nucleic acid of wherein said coding PLT transcription factor polypeptide is plant origin, preferably from dicotyledons, also preferably from Cruciferae, more preferably from Arabidopsis, most preferably from Arabidopis thaliana.

87. by according to each the obtainable plant of method or its part of claim 77 to 86, comprise seed, wherein said plant or its part comprise the nucleic acid of coding PLT transcription factor polypeptide, wherein said nucleic acid effectively connects to form the type promotor.

88. construct, it comprises:

(i) nucleic acid of coding PLT transcription factor polypeptide;

(ii) can drive one or more regulating and controlling sequences of the nucleotide sequence expression of (i); Randomly

(iii) transcription termination sequence.

89. 8 construct according to Claim 8, wherein said constitutive promoter is the GOS2 promotor.

90. the purposes of 8 or 89 construct is used to produce the plant that has the abiotic stress resistance of the output of increase, the seed production that especially increases and/or increase with respect to control plant according to Claim 8.

91. with 8 or 89 construct plant transformed, plant part or vegetable cell according to Claim 8.

92. be used to produce the method for transgenic plant, described transgenic plant have the output of increase and/or the abiotic stress resistance of increase, wherein said method comprises:

(i) import and in vegetable cell, express the nucleic acid of coding PLT transcription factor polypeptide; With

Cell (ii) cultivates plants under the condition that promotes plant-growth and growth.

93. have with respect to control plant the output of increase and/or increase abiotic stress resistance transgenic plant or from the vegetable cells of described transgenic plant, the output of described increase and/or the abiotic stress resistance of increase are produced by the expression of the increase of the nucleic acid of coding PLT transcription factor polypeptide.

94. 7,91 or 93 transgenic plant according to Claim 8, or from the transgenic plant cells of described transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

95. according to the part gathered in the crops of the plant of claim 94, the wherein said preferably seed of part of gathering in the crops.

96. from according to the plant of claim 94 and/or according to the product of the part gathered in the crops of the plant of claim 93.

97. the purposes of the nucleic acid of coding PLT transcription factor polypeptide, or the purposes of PLT transcription factor polypeptide are used for increasing abiotic stress resistance and/or being used to improve plant biomass, seed production especially with respect to control plant.

98. be used for strengthening the method for plant output correlated character, it comprises the expression of nucleic acids of regulating coding bHLH transcription factor in the plant, wherein said bHLH transcription factor by SEQ ID NO213, SEQ ID NO 215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 any or aforementioned any directly to homologue or the representative of collateral line homologue.

99. according to the method for claim 98, the wherein said expression of being regulated activates any or accomplished in many ways of label, TILLING or homologous recombination by T-DNA.

100. method according to claim 98, the wherein said expression of being regulated realizes by the nucleic acid that imports and express coding bHLH transcription factor in plant, wherein said bHLH transcription factor by any or the aforementioned SEQ ID NO of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 any directly to homologue or the representative of collateral line homologue.

According to each method of claim 98 to 100, wherein said enhanced yield correlated character is early stage vigor.

According to the method for claim 100, wherein said nucleic acid effectively connects to form type promotor, preferred GOS2 promotor.

According to each method of claim 100 to 102, wherein said nucleic acid is according to SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226 and SEQ ID NO:228, SEQ ID NO:296, SEQ ID NO:298, the part of any nucleotide sequence of SEQ ID NO:300 or allelic variant or splice variant or can with according to SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ IDNO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226 and SEQ IDNO:228, SEQ ID NO:296, SEQ ID NO:298, the sequence of the nucleic acid array hybridizing of any of SEQ ID NO:300, wherein said part, allelic variant, splice variant or hybridization sequences coding bHLH transcription factor.

According to each method of claim 98 to 103, the nucleic acid of wherein said coding bHLH transcription factor is plant origin, preferably from monocotyledons, also preferably from Gramineae, more preferably from Oryza, most preferably from rice.

By according to each the obtainable plant of method or its part of claim 98 to 104, comprise seed, wherein said plant or its part comprise the nucleic acid of coding bHLH transcription factor.

Isolated polypeptide, it comprises:

(i) by the aminoacid sequence of any representative of SEQ ID NO:297, SEQ ID NO:299 and SEQ ID NO:301;

(ii) aminoacid sequence, its (a) has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity and (b) have a bHLH structural domain to increase progressively preferred sequence and any aminoacid sequence by SEQ ID NO:297, SEQ IDNO:299 and SEQ ID NO:301 representative;

(iii) at above (i) or the derivative of the arbitrary aminoacid sequence that provides (ii).

Isolated nucleic acid molecule, it comprises:

(i) by the nucleic acid of any representative of SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300;

(ii) by the complementary sequence of the nucleic acid of any representative of SEQ ID NO:296, SEQ ID NO:298 and SEQ ID NO:300;

The nucleic acid of the bHLH transcription factor of (iii) encoding, described bHLH transcription factor (a) has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to increase progressively preferred order and aminoacid sequence by any representative of SEQ ID NO:297, SEQ ID NO:299 and SEQ ID NO:301, and has (b) bHLH structural domain.

Construct, it comprises:

(a) nucleic acid of coding bHLH transcription factor, described bHLH transcription factor by any or the arbitrary aforementioned SEQ ID NO of SEQ ID NO213, SEQ ID NO 215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301 directly to homologue or the representative of collateral line homologue;

(b) can drive one or more regulating and controlling sequences that the nucleotide sequence of (a) is expressed; Randomly

(c) transcription termination sequence.

According to the construct of claim 108, wherein said one or more regulating and controlling sequences are constitutive promoter at least, preferred GOS2 promotor.

110., be used to produce with respect to control plant and have enhanced yield correlated character, the plant of the early stage vigor of enhanced especially according to the purposes of the construct of claim 108 or 109.

111. use construct plant transformed, plant part or vegetable cell according to claim 108 or 109.

112. be used to produce the method that has the transgenic plant of enhanced yield correlated character with respect to control plant, described method comprises:

(a) import and in plant, express the nucleic acid of coding bHLH transcription factor, described bHLH transcription factor by any or the arbitrary aforementioned SEQ ID NO of SEQ ID NO 213, SEQ ID NO 215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO 225, SEQ ID NO:297, SEQ ID NO:299, SEQ IDNO:301 directly to homologue or the representative of collateral line homologue; With

(b) cell that under the condition that promotes plant-growth and growth, cultivates plants.

113. have the transgenic plant of the output of increase with respect to control plant, or from the transgenic plant cells of described transgenic plant, the output of described increase is produced by the expression of the increase of the nucleic acid of coding bHLH transcription factor, wherein said bHLH transcription factor by any or the arbitrary aforementioned SEQ IDNO of SEQ ID NO 213, SEQ IDNO215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO 225, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301 directly to homologue or the representative of collateral line homologue.

114. according to claim 105,111 or 113 transgenic plant, or from the transgenic plant cells of described transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

115. according to the part gathered in the crops of the plant of claim 114, the wherein said preferably seed of part of gathering in the crops.

116. from according to the plant of claim 114 and/or according to the product of the part gathered in the crops of the plant of claim 115.

117. the nucleic acid of coding bHLH transcription factor purposes in the output correlated character in strengthening plant, wherein said bHLH transcription factor by any or the arbitrary aforementioned SEQ ID NO of SEQ ID NO 213, SEQ ID NO 215, SEQID NO:217, SEQ ID NO:219, SEQ ID NO 225, SEQ ID NO:297, SEQ IDNO:299, SEQ ID NO:301 directly to homologue or the representative of collateral line homologue.

118. according to the purposes of claim 117, wherein said enhanced yield correlated character is early stage vigor.

119. be used for increasing the method for plant output, the plant that the expression and randomly select of nucleic acid in plant that it comprises increases coding SPL15 transcription factor polypeptide has the output of increase with respect to suitable control plant.

120. according to the method for claim 119, the wherein expression of any or described increase of accomplished in many ways by T-DNA activation, TILLING, site-directed mutagenesis, orthogenesis and homologous recombination.

121., wherein realize the expression of described increase by the nucleic acid that imports and in plant, express coding SPL15 transcription factor polypeptide according to the method for claim 119.

122. according to the method for claim 121, the nucleic acid of wherein said coding SPL15 transcription factor is any nucleic acid SEQ ID NO or its part that provides among the N at table, or can with the sequence of any nucleic acid SEQ ID NO hybridization that in table N, provides.

123. according to the method for claim 121, arbitrary nucleic acid SEQ ID NO that the nucleic acid encoding of wherein said coding SPL15 transcription factor provides among the N at table directly to homologue or collateral line homologue.

124. according to each method of claim 119 to 123, the output of wherein said increase is the seed production that increases.

125. the method for claim 124, the seed production of wherein said increase comprise following one or more: every strain plant flowers number of Zeng Jiaing a) the over-ground part biomass b of Zeng Jiaing); C) seed production of Zeng Jiaing; D) (full) seed number of Zeng Jiaing; E) thousand seed weight of Zeng Jiaing (TKW); And f) harvest index of Zeng Jiaing.

126. according to each method of claim 121 to 125, wherein said nucleic acid effectively connects to form the type promotor, preferably connects HMGB (the high speed swimming B of family) promotor or GOS2 promotor, further preferably connects rice HMGB promotor or rice GOS2 promotor.

127. according to each method of claim 121 to 126, the nucleic acid of wherein said coding SPL15 transcription factor polypeptide is plant origin, preferably from dicotyledons, further preferably from Cruciferae, most preferably from Arabidopis thaliana.

128. by according to each the obtainable plant of method or its part of claim 119 to 127, comprise seed, wherein said plant or its part comprise the recombinant nucleic acid of coding SPL15 transcription factor.

129. construct, it comprises:

(a) nucleic acid of coding SPL15 transcription factor polypeptide;

(b) can drive one or more regulating and controlling sequences that the nucleotide sequence of (a) is expressed; Randomly

(c) transcription termination sequence.

130. according to the construct of claim 129, wherein said one or more regulating and controlling sequences are constitutive promoter at least, preferred HMGB promotor or GOS2 promotor.

131. the purposes according to the construct of claim 129 or 130 is used to produce the plant that has the output of increase with respect to control plant.

132. use construct plant transformed, plant part or vegetable cell according to claim 129 or 130.

133. be used to produce the method for transgenic plant that has the output of increase with respect to control plant, described method comprises:

(a) import and in vegetable cell, express the nucleic acid of coding SPL15 transcription factor polypeptide; With

(b) cell that under the condition that promotes plant-growth and growth, cultivates plants.

134. with respect to the transgenic plant that control plant has the output of increase, the output of described increase is produced by the expression of the increase of the nucleic acid of coding SPL15 transcription factor.

135. according to claim 128,132 or 134 transgenic plant, or from the transgenic plant cells of described transgenic plant, wherein said plant is crop plants or monocotyledons or cereal, as rice, corn, wheat, barley, grain, rye, triticale, Chinese sorghum and oat.

136. according to the part gathered in the crops of the plant of claim 135, the wherein said preferably seed of part of gathering in the crops.

137. from according to the plant of claim 135 and/or according to the product of the part gathered in the crops of the plant of claim 136.

138. the purposes of the nucleic acid of coding SPL15 transcription factor polypeptide is used for the output with respect to control plant increase plant.

139. according to the purposes of claim 138, the output of wherein said increase is the seed production that increases.

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