CN103998611A

CN103998611A - Polynucleotides, polypeptides and methods for enhancing photossimilation in plants

Info

Publication number: CN103998611A
Application number: CN201280053974.5A
Authority: CN
Inventors: M·努奇奥; L·波特; J·科恩
Original assignee: Syngenta Participations AG
Current assignee: Syngenta Participations AG
Priority date: 2011-11-03
Filing date: 2012-11-02
Publication date: 2014-08-20
Also published as: WO2013067259A2; WO2013067259A3; HUP1400505A2; CA2853490A1; WO2013067252A1; AU2012332343A1; MX2014005375A

Abstract

The present invention relates generally to the field of molecular biology and regards various polynucleotides, polypeptides and methods that may be employed to enhance yield in transgenic plants. Specifically the transgenic plants may exhibit increased yield, increased biomass or increased photoassimilation.

Description

For strengthening polynucleotide, polypeptide and the method for plant photo-assimilation

Invention field

This disclosure relates generally to biology field and about being used for strengthening photo-assimilation in transgenic plant and increasing different polynucleotide, polypeptide and the using method of its output.The transgenic plant that comprise any polynucleotide described here or polypeptide can represent any one in the proterties being made up of the photo-assimilation of the biomass that increase, enhancing or the output of increase.

Background of invention

The world population increasing and the atrophy supply in available arable land that can be used for agricultural have stimulated for the needs that increase farm efficiency area research.The conventional means utilization of crop and gardening improvement selects breeding technique to identify the plant with desirable feature.But such selection breeding technique has some shortcomings, these technology are often labour-intensive and cause plant conventionally to contain heterology hereditary component, and they may always not cause the desirable proterties of transmitting from mother plant.Molecular biological progress has allowed the mankind to improve the germplasm of animal and plant.The genetic engineering of plant makes to need to separate and operation genetic material (being typically DNA or rna form) and subsequently by the genome of this genetic material introduced plant.This technology has the ability of giving crop or plant and have the proterties of economic, the agronomic or Horticulture of different improvement.

Summary of the invention

One embodiment of the present of invention are a kind of expression cassettes, this expression cassette comprises that at least three kinds are selected from the polynucleotide of lower group, this group is made up of the following: a kind of polynucleotide of a kind of polynucleotide of a kind of Phosphoenolpyruvate carboxylase of encoding, a kind of polynucleotide of a kind of fructose-1,6-diphosphate Phosphoric acid esterase of encoding, a kind of polynucleotide of a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) of encoding, a kind of phosphoribulokinase of encoding and the two kinase whose a kind of polynucleotide of a kind of pyruvate phosphate of encoding.This expression cassette can comprise a kind of fructose-1 of coding, a kind of polynucleotide of a kind of polynucleotide of a kind of polynucleotide of a kind of polynucleotide of 6-bisphosphate Phosphoric acid esterase, a kind of polynucleotide of a kind of phosphoribulokinase of encoding and a kind of Phosphoenolpyruvate carboxylase of encoding or a kind of polynucleotide of a kind of fructose-1,6-diphosphate Phosphoric acid esterase of encoding, a kind of phosphoribulokinase of encoding, the two kinase whose a kind of polynucleotide of a kind of pyruvate phosphate of encoding and a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) of encoding.

This expression cassette can contain coding and SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO:3, SEQ ID NO.4 or SEQ ID NO:5 and have the polynucleotide of at least 70%, 80%, 90% or 95% conforming polypeptide.Alternately, this expression cassette can comprise the polynucleotide of coding containing the polypeptide of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 or SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.These polynucleotide of this expression cassette can may be operably coupled to one or more photoinduction type promotors.The polynucleotide of this expression cassette can also comprise the polynucleotide described in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 or SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

Extra embodiment comprises a kind of method for increasing biomass, and the method comprises: by any introduced plant cell in described expression cassette; Plant cell growth is become to plant; And select the transgenic plant of the biomass with increase.This plant can be a kind of C4 plant, and can be selected from lower group, and this group is made up of the following: sugarcane, corn and Chinese sorghum.Alternately, this plant can be corn.

Another embodiment comprises a kind of method of preparing transgenic plant, and the method comprises: by any introduced plant in described expression cassette; Plant cell growth is become to plant; And select to comprise the plant of this expression cassette.This plant can be a kind of C4 plant, and can be selected from lower group, and this group is made up of the following: sugarcane, corn and Chinese sorghum.Alternately, this plant can be corn.

Brief Description Of Drawings

Fig. 1 is the plasmid figure of 19862, has shown SoFBP, SoPRK and ZmPEPC expression cassette in a binary vector.Promotor of " pr-" prefix designates; Intron of " i-" prefix designates; Enhanser of " e-" prefix designates; Encoding sequence of " c-" prefix designates; Terminator of " t-" prefix designates.

Fig. 2 is the plasmid figure of 19863, has shown SoFBP, SbPPDK and SbNADP-MD expression cassette in a binary vector.Promotor of " pr-" prefix designates; Intron of " i-" prefix designates; Enhanser of " e-" prefix designates; Encoding sequence of " c-" prefix designates; Terminator of " t-" prefix designates.

Fig. 3 has described the photo-assimilation and night respiration effect every day in B027A F1.(A) steady-state light assimilation speed of cultivating under closed chamber condition and (B) night respiration effect.Plant experience 25 DEG C 16 hours and 8 hours of 20 DEG C of nights by day.Relative humidity is 60%.By maintaining Atmospheric CO at 400ppm meter injection by day ₂.Photo-assimilation is the CO injecting for maintaining this 400ppm set point ₂rate of chronometer.Night respiration effect is the CO discharging night ₂, be absorbed CO the day before yesterday ₂function.Data are about 40 strain plants.

Detailed description of the invention

Unless otherwise indicated, practice of the present invention adopts the routine techniques of phytology, microbiology, tissue culture, molecular biology, chemistry, biological chemistry, number of plant genetics, statistics and recombinant DNA technology in the skill of this area.This class technology is fully explained in the literature.Referring to uncommon graceful (Thimann) (1982) phytology of Li sea as precious in Nancy (Langenheim) &: plant biology and with the relation (Botany:Plant Biology and Its Relation to Human Affairs) of mankind's affairs, John Willie (John Wiley); The somatic cell genetics (Cell Culture and Somatic Cell Genetics of Plants) of cell cultures and plant, the 1st volume, Wa Xier (Vasil) edits (1984); People (1986) microbial worlds (The Microbial World) such as Stanier (Stanier), the 5th edition, Prentice-Hall (Prentice-Hall); The basic plant pathology method (Basic Plant Pathology Methods) of Ding Gela (Dhringra) & Xin Kelai (Sinclair) (1985), CRC press; People (1982) molecular clonings such as the Germania base of a fruit this (Maniatis): experiment guide (Molecular Cloning:A Laboratory Manual); DNA clone (DNA Cloning), I volume and II volume, Danny Glover (Glover) editor (1985); Oligonucleotide synthesizes (Oligonucleotide Synthesis), and Gai Te (Gait) edits (1984); Nucleic acid hybridization (Nucleic Acid Hybridization), Hei Musi (Hames) & John Higgins (Higgins) editor (1984); And book series Enzymology method (Methods in Enzymology), section's Lip river Brunswick (Colowick) & Kapp orchid (Kaplan) editor, academic press (Academic Press, Inc.), San Diego (San Diego), California (Calif).

Unit, prefix and symbol can the generally acknowledged form of its SI represent.Unless otherwise indicated, respectively with 5 ' to 3 ' direction write from left to right nucleic acid; Write from left to right aminoacid sequence with amino to the direction of carboxyl.Digital scope comprises the numeral of confining spectrum.Amino acid can be mentioned by its common known trigram symbol or the one-letter symbol of being recommended by IUPAC-IUB biochemical nomenclature commission at this.Similarly, Nucleotide can be mentioned by its generally accepted single-letter code.More completely define with reference to specification sheets generally with undefined term.

Unless otherwise defined, all technical terms used herein and scientific terminology have with belong to the field of the invention within common the understood identical implication of those of ordinary skill.

It should be understood that, the present invention is not limited to concrete grammar, experimental program, clone, the floristics of description like this or belongs to class, construct and reagent.It is to be further understood that term is only the object in order to describe specific embodiment as used herein, and be not intended to limit the scope of the invention.

As used herein, singulative " (kind) " and " being somebody's turn to do " comprise multiple indicators, unless context separately has clearly regulation.Therefore, for example, mention that " carrier " is to mention one or more carriers and comprise its equivalent well known by persons skilled in the art.

Term " about " be used herein to expression approximately, substantially, left and right or in its vicinity.In the time that term " about " is combined with a numerical range, the upper and lower bound of the numerical value that it is enumerated by expansion is modified this scope.Conventionally, term " about " be used herein to by numerical value modify to 20% quantity variance higher than and lower than prescribed value.

As used herein, word "or" means any one member of concrete inventory and comprises any combination of the member on this inventory.

Term " comprises (comprises) ", " comprising (comprising) ", " comprising (includes) ", " comprising (including) ", " thering is (having) " with and cognate mean " including, but not limited to ".Term " consisting of " mean " comprise and be confined to ".

Term " mainly consisting of " means composition, method or structure and can comprise other composition, step and/or part, but condition composition, step and/or partial sterility matter that to be these other change essential characteristic and the novel feature of composition required for protection, method or structure.

When in this instruction numerical range, mean any reference numerals (mark or integer) comprising in indicated scope." mobility scale/scope " of phrase the first designation number and the second designation number " between mobility scale/scope " and the first designation number " extremely " the second designation number used interchangeably and means at this and comprise the first and second designation numbers and all marks and integer therebetween.As used herein, term " method " refers to mode, means, technology and the program for completing Given task, including, but not limited to the practitioner of chemistry, pharmacology, biology, biological chemistry and medical field known or those modes, means, technology and the program easily developed by known way, means, technology and program.Should be understood that some feature of the present invention being described in the situation of embodiment separately for object clearly can also be provided in single embodiment by array configuration.On the contrary, for simplicity, the different characteristics of the present invention of describing under the background of single embodiment also can be individually or with any applicable sub-portfolio or be provided in of the present invention any other describe in embodiment in suitable situation.Some feature of describing under the background of different embodiment is not thought the essential feature of those embodiment, unless embodiment is invalid in the situation that there is no those key elements.

" microorganism (microbe) " refers to any microorganism (microorganism) (comprising eucaryon and prokaryotic micro-organisms), for example fungi, yeast, bacterium, ray fungi, algae and protozoon, together with other unicellular structures.

Term " the conservative variant of modifying " is applicable to amino acid and nucleotide sequence.About concrete nucleotide sequence, conservative modification variant refers to those nucleic acid of the conservative modification variant of coding same acid sequence or aminoacid sequence.Due to the degeneracy of genetic code, any given protein of a lot of function identical nucleic acid coding.For example, all coded amino acid L-Ala of codon GCA, GCC, GCG and GCU.Therefore, each position of being specified by a codon at L-Ala, this codon can change over described any corresponding codon and not change coded polypeptide.The variation of this class nucleic acid is " silent variant " and represent that a kind of conservative modification makes a variation.Also describing the each of this nucleic acid at each nucleotide sequence of this coded polypeptide may silent variant.(except AUG, it is unique password of methionine(Met) normally to persons of ordinary skill in the art will recognize that each codon in nucleic acid; An exception is micrococcus rubens (Micrococcus rubens), for it, GTG is that Methionine codon (people (1993) general microbiology magazine (J.Gen.Microbiol.) 139:425-32 such as stoneman (Ishizuka)) can be modified to produce the molecule that function is identical.Therefore, each silent variant of the nucleic acid of code book invention polypeptide is implied in each described peptide sequence and is combined in by reference this.

As used herein, " control plant " or " contrast " can be the non-transgenic plant for generation of the parental line of the transgenic plant at this.In some cases, control plant can be transgenic plant strain, and it comprises empty carrier or marker gene, but does not contain the recombination of polynucleotide of the present invention of expressing in assessed transgenic plant.In other cases, control plant is the transgenic plant that carry out expressing gene by composition promotor.In general, control plant is the plant of strain identical with tested transgenic plant or kind, and it does not characterize the recombinant DNAs of giving specific trait of transgenic plant.This plant for generations that there is no this recombinant DNA of giving specific trait can be (elite) non-transgenic plant of natural wild-type plant, original seed or the transgenic plant that do not characterize the recombinant DNA of giving specific trait of transgenic plant.The plant for generations of specifically not giving the recombinant DNA of proterties can be sisters' strain (sibling) with the transgenic plant of the recombinant DNA of specifically giving proterties.This strain of sisters for generations plant can comprise other recombinant DNAs.

About aminoacid sequence, those skilled in the art will recognize that, when change cause amino acid by chemofacies when the aminoacid replacement, the single amino acid or amino acid whose indivedual replacements, disappearance or the interpolation for nucleic acid, peptide, polypeptide or protein sequence of little percentage that change, add or lack in coded sequence are " the conservative variants of modifying ".Therefore, the amino-acid residue of any number of the integer group of free from 1 to 15 composition of choosing can so change.Therefore, for example, can carry out 1,2,3,4,5,7 or 10 change.The conservative variant of modifying typically provides the unmodified peptide sequence of originating to it similar biological activity.For example, substrate specificity, enzymic activity or ligand/receptor are in conjunction with being generally natural protein at least 30%, 40%, 50%, 60%, 70%, 80% or 90% of its natural substrate, preferably 60%-90%.Provide amino acid whose conservative property replacement table similar in function to know in the art.

Six groups contain the amino acid for conservative replacement each other separately below:

L-Ala (A), Serine (S), Threonine (T);

Aspartic acid (D), L-glutamic acid (E);

L-asparagine (N), glutamine (Q);

Arginine (R), Methionin (K);

Isoleucine (I), leucine (L), methionine(Met) (M), α-amino-isovaleric acid (V) and

Phenylalanine (F), tyrosine (Y), tryptophane (W).

Also pause (Creighton) referring to crith, protein (Proteins), W.H. freeman publishing company (W.H.Freeman and Co.) (1984).

Refer to and comprise the information of specifying protein for translating into about " coding (encoding) " or " coding (encoded) " of specifying nucleic acid.A kind of nucleic acid of coded protein can comprise that within the translation district of this nucleic acid non-translated sequence (for example intron) maybe can lack the non-translated sequence (for example, in cDNA) of this type of insertion.A kind of information exchange of protein of being used for encoding is crossed and is accessed to your password son and describe in detail.Typically, carry out encoding amino acid sequence by the nucleic acid that uses " general " genetic code.But, can use the variant of universal code, for example be present in the universal code in some plants, animal and fungi plastosome, mycoplasma capri (Mycoplasma capricolum) periodical (Proc.Natl.Acad.Sci.USA) 82:2306-9 of institute of people (1985) NAS such as () mountain tails (Yamao) or ciliate macronucleus (ciliate Macronucleus), now nucleic acid is expressed with these organisms.

In the time that nucleic acid is prepared with synthesis mode or changed, can utilize nucleic acid will be expressed in predetermined host's wherein known codon preference.For example, although nucleotide sequence of the present invention all can be expressed in unifacial leaf and dicotyledons kind, but can modify to consider the sub-preference of the specific cryptosystem of monocotyledons or dicotyledons and GC content preference sequence, be different (in silent people (1989) nucleic acids research (the Nucleic Acids Res.) 17:477-98 such as (Murray) and be combined in by reference this) because these preferences have been illustrated.Therefore, concrete amino acid whose Zea mays preference codon may be derived from: from the known sequence in Zea mays.Use above-mentioned and list in the people's such as (Murray) table 4 in silent about the Zea mays codon of 28 kinds of genes from Zea mays plant.

As used herein, " allos " of mentioning a kind of nucleic acid means the nucleic acid that is derived from a kind of alien species, if or were derived from same species, would be that the human intervention by having a mind to is carrying out from its natural form the nucleic acid of modifying in fact aspect formation and/or locus.For example, the promotor that may be operably coupled to allos structure gene be from from structure gene derived from the different species of species wherein, if or from same species, one or both is in fact to modify from their primitive form.Heterologous protein can be derived from alien species, if or from same species, be that the human intervention by having a mind to is modified in fact from its primitive form.

" host cell " refers to the cell that comprises heterologous nucleic acid sequence of the present invention, and it contains carrier and supports copying and/or expressing of expression vector.Host cell can be prokaryotic cell prokaryocyte, for example intestinal bacteria, or eukaryotic cell, cell for example yeast, insect, plant, Amphibians or mammiferous.Preferably, host cell is unifacial leaf or dicotyledons cell, including, but not limited to Zea mays, Chinese sorghum, Sunflower Receptacle, soybean, wheat, clover, paddy rice, cotton, canola oil dish (canola), barley, grain (millet) and tomato.Particularly preferred monocotyledons host cell is Zea mays host cell.

Term " hybridization complex " comprises and referring to by two double-strandednucleic acid structures that the single-chain nucleic acid sequence of selective cross forms each other.

Nucleic acid being inserted under the situation in cell, term " introducing " is by any means, for example " transfection ", " conversion " or " transduction " and comprise refer to nucleic acid is attached in cell eucaryon or protokaryon, the genome that its amplifying nucleic acid can be attached to cell (for example, karyomit(e), plasmid, plastid or Mitochondrial DNA) in, change into as the part of minichromosome or for example, by self-replicating of transient expression (, the mRNA of transfection).

As used herein, " gene stacking " refers to two or more genes introduced in a kind of organic genome.May make us it is desirable for for example, the gene stacking with any other useful proterties (increasing plant height etc.) of giving anti-insect property, disease resistance, increase output or be known in the art by gene as described in this.Alternately, can and give other proterties by the transgenic plant that comprise gene as described herein, polypeptide or polynucleotide, such as the natural character allelotrope stack of improved water utilization, increase disease resistance etc.The expression cassette that can have multiple genes by introducing is maybe by the breeding/hybridization and proterties is superposeed together with containing the other plant of one or more other proterties of the plant with one or more proterties.

Term " separation " refers to the material of for example nucleic acid or protein, and it does not have in fact or substantially as conventionally following with it or component interactional with it of finding in its naturally occurring environment.The material separating is optionally included in its physical environment does not find material therewith.As defined herein, " separation " nucleic acid is also referred to as " allos " nucleic acid.Unless otherwise indicated, term " NUE nucleic acid " refers to the nucleic acid of the polynucleotide (" NUE polynucleotide ") that comprise coding total length or partial-length NUE polypeptide.

As used herein, " nucleic acid " comprises and referring in strand or the polymkeric substance deoxyribonucleotide of double chain form or ribonucleotide, unless and limit in addition, contain the known analogue of the essential property in the following areas with natural nucleotide: it is hybridized to single-chain nucleic acid in the mode for example, to naturally occurring Nucleotide (peptide nucleic acid(PNA)) similar.

" nucleic acid library " refers to the set of DNA isolation or RNA molecule, and in one case, these molecules comprise specifies organic genomic whole substance of transcribing part to represent.Exemplary nucleic acid library, being structured in standard molecular biology reference of for example genomic library and cDNA library taught, as uncle outstanding (Berger) and Ji Meier (Kimmel) (1987) molecule clone technology guide (Guide To Molecular Cloning Techniques), from book series Enzymology method (Methods in Enzymology), the 152nd volume, academic press, San Diego, California; People (1989) molecular clonings such as Pehanorm Brooker (Sambrook): experiment guide (Molecular Cloning:A Laboratory Manual), the 2nd edition, 1-3 volume; And modern molecular biology experimental technique (Current Protocols in Molecular Biology), the modern experimental technique (Current Protocols) that the people such as Su Beier difficult to understand (Ausubel) edit, Green publishes (the Greene Publishing Associates of affiliated company, and co-partnership company's (1994 supplementary issue) of John Willie father and son company (John Wiley & Sons, Inc.) Inc.); Pehanorm Brooker (Sambrook) & Russell (Russell) (2001) molecular cloning: laboratory manual (Molecular Cloning:ALaboratory Manual), the third edition, press of cold spring harbor laboratory (Cold Spring Harbor Laboratory Press), cold spring port (Cold Spring Harbor), New York, the U.S..In another example, as defined herein, " nucleic acid library " also can be regarded as representative and comprises and specify organic established part or be actually the not essence previous generation and show to specify organic whole genomic library.For example, little RNA, mRNA and methylate DNA.As defined herein, nucleic acid library also may contain the variant (for example, the set of the variant of concrete protein) of concrete molecule.

As used herein, " being operably connected " comprise and refer to that First ray (for example promotor) is connected with the function between the second sequence, and wherein this promoter sequence is initial and mediate transcribing corresponding to the DNA of this second sequence.In general, being operably connected and referring to that connected nucleotide sequence is continuous, and in the time that needs engage two protein coding regions, is continuous and in same reading frame.

As used herein, term " plant " comprises and refers to whole strain plant, plant organ (such as leaf, stem, root, etc.), seed and vegetable cell and its filial generation.As used herein, vegetable cell is including, but not limited to the cell in seed, suspension culture, embryo, meristem zone, callus, leaf, root, branch, gametophyte, sporophyte, pollen and sporule.Can be conventionally equally extensive with the higher plant classification that is suitable for transformation technology for the plant classification in the inventive method, comprise unifacial leaf and dicotyledons, comprise from the kind with subordinate's class: Cucurbita (Cucurbita), rose belongs to (Rosa), Vitis (Vitis), white walnut (Juglans), Fragaria (Fragaria), Lotus (Lotus), Medicago (Medicago), donkey food grass belongs to (Onobrychis), Trifolium (Trifolium), Trigonella (Trigonella), Vigna (Vigna), Citrus (Citrus), linum (Linum), heroubill belongs to (Geranium), cassava (Manihot), Daucus (Daucus), Arabidopsis (Arabidopsis), rue and beat genus (Brassica) with a stick, Rhaphanus (Raphanus), sinapsis alba belongs to (Sinapis), Atropa (Atropa), Capsicum (Capsicum), Datura (Datura), poison tobacco (Hyoscyamus), tomato belongs to (Lycopersicon), Nicotiana (Nicotiana), Solanum (Solanum), green winter Solanum (Petunia), Digitalis (Digitalis), Rana, Maastricht Treaty belongs to (Majorana), Cichorium (Ciahorium), Helianthus (Helianthus), Lactuca (Lactuca), Brome (Bromus), Asparagus (Asparagus), antirrhinum (Antirrhinum), hemerocallis (Heterocallis), Nemesis (Nemesis), Pelargonium (Pelargonium), Panicum (Panieum), Pennisetum (Pennisetum), Ranunculus (Ranunculus), Senecio (Senecio), salpiglossis belongs to (Salpiglossis), Cucumis (Cucumis), the magnificent genus in cloth Lip river (Browaalia), Glycine (Glycine), Pisum (Pisum), Phaseolus (Phaseolus), lolium (Lolium), Oryza (Oryza), Avena (Avena), Hordeum (Hordeum), Secale (Secale), allium (Allium) and Triticum (Triticum).Particularly preferred plant is Zea mays (Zeamays).

C4 plant as defined herein, is to utilize C ₄carbon fixation approach makes CO ₂first be bonded to the phosphoric acid enol form propanone in mesophyll cell, cause the formation of C4 compound, this C4 compound shuttles back and forth to vascular bundle sheath cell, and here its decarboxylation is with by CO ₂discharge, be ready to use in C ₃in approach.The example of C4 plant includes but not limited to the member of Gramineae (Poaceae family, also referred to as Gramineae or true grasses), for example sugarcane, corn, Chinese sorghum, three-coloured amaranth, grain; The member of Cyperaceae; And many sections of Dicotyledoneae, comprise composite family daisy; Cruciferae Caulis et Folium Brassicae capitatae; And the Euphorbiaceae root of Beijing euphorbia.

As used herein, " output " can comprise the bushel number (as for example, adjusted for Grain water (zeistic moisture is generally 15%)) that refers to every acre of cereal crop in the time of results, and the volume of the biomass that produce is (for fodder crop, for example clover, and the roots of plants size of various crop).Grain water in seed is measured in the time of results.Grain test weight after adjustment is defined as weight taking pound/bushel as unit (level of Grain water is adjusted during to results).Biomass are measured as the weight of the produced vegetable material of gathering in the crops.Output can be subject to numerous characteristics impact, comprises the efficiency of the incidence, seed size, dross and the fixed nitrogen that are not limited to position on plant of plant height, pod number, pod, internode number, pod fragmentation, efficiency, carbon assimilation, plant structure, seed germination per-cent, seedling gesture and the juvenile character of nutrient assimilation.Output is affected by following factor also can: number seeds, seed size, the composition (starch, oil, protein) of seed and the feature of seed filling of germination efficiency (being included in the germination under stress conditions), growth velocity (being included in the growth velocity under stress conditions), spike number order, each fringe.Plant biomass can be measured by several different methods, comprises number seeds, the seed weight of unit weight, every strain plant, the number seeds of per unit area (be the seed of every acre, or seed weight), bushel/acre, ton/acre or kg/ha.For example, the output of the corn grain that shells that corn yield can be measured as per unit produces area, for example, for example, taking bushel/acre or ton/hectare as unit, often based on moisture, adjustment is reported, 15.5% moisture.In addition, corn bushel is 56 pounds by weight in Iowa by legal definition, and a useful reduction factor of corn yield is: 100 bushels/acre equal 6.272 tons/hectare.Other measurements of output are convention in the art.In certain embodiments of the present invention, output can coerce and/or non-stress condition under increase.

As used herein, " polynucleotide " comprise the ribodesose polynucleotide, ribose polynucleotide or its analogue that refer to the essential attributes with natural nucleotide, this is because they hybridize to the substantially the same nucleotide sequence of nucleotide sequence of hybridizing with naturally occurring Nucleotide under stringent hybridization condition, and/or allows to translate into one or more identical amino acid of one or more amino acid of translating into naturally occurring one or more Nucleotide.Polynucleotide can be natural or full length sequence or the subsequence of allos structure gene or regulatory gene.Unless otherwise instructed, otherwise this term comprises finger: the sequence of appointment is together with its complementary sequence.Therefore, for stability or other reasons, main chain having been carried out to the DNA that modifies or RNA is if this term is at " polynucleotide " that this meant.In addition,, only for two examples, the DNA or the RNA that comprise rare base (for example inosine) or modified base (base of for example tritylation) are as this term polynucleotide as used herein.Will appreciate that, the DNA and the RNA that play many useful targets well known by persons skilled in the art have been carried out to a variety of modifications.As used herein, term polynucleotide are contained this class chemically modified, enzyme modification or the metabolism modified forms of polynucleotide, together with the chemical species of virus and the peculiar DNA of cell (especially comprising simple cell and complex cell) and RNA.

Term " polypeptide ", " peptide " and " protein " are used interchangeably at this, to refer to the polymkeric substance of amino-acid residue.These terms are applicable to the aminoacid polymers that wherein one or more amino-acid residues are corresponding naturally occurring amino acid whose artificial chemistry analogue, together with being applicable to naturally occurring aminoacid polymers.

As used herein, " promotor " comprises and refers to transcribing the upstream of beginning and relating to RNA polymerase and the identification of other protein and in conjunction with initial region of transcribing of DNA." plant promoter " is the promotor of transcribing in can initial vegetable cell.Exemplary plant promoter including, but not limited to from plant, plant virus and be included in the gene of expressing vegetable cell bacterium (for example Agrobacterium (Agrobacterium) or rhizobium (Rhizobium)) obtain those promotors.Example is preferential initial some tissue, for example, the promotor of transcribing in leaf, root, seed, fiber, xylem vessel, test-tube baby or sclerenchyma.These promotors are called as " tissue is preferred "." cell type " specificity promoter mainly drives for example, expression in some cell type (, the dimension tube cell in root or leaf) in one or more organs." can induce " or " adjustable " promotor is the promotor under environment control.Can affect the existence that the envrionment conditions example of transcribing comprises anaerobic condition or light by inducible promoter.Another kind of type promotor is to grow to regulate promotor, for example, drives the promotor of expressing during pollen development.Organize preferred promoter, cell type specificity promotor, grow adjusting promotor and inducible promoter formation " non-composition " promotor classification." composition " promotor is the promotor working in most cells under most of envrionment conditionss.

Any suitable promoter sequence can be used by nucleic acid construct of the present invention.According to some embodiments of the present invention, promotor is composition promotor, tissue-specific promoter or photoinduction type promotor.

Applicable composition promotor comprises such as CaMV35S promotor (people such as Mark Odell (Odell), nature (Nature) 313:810-812,1985); Arabidopis thaliana At6669 promotor (referring to PCT publication No. WO04081173A2); Zea mays Ubi1 (people such as Mark Odell (Christensen), molecular biology of plants (Plant Mol.Biol.) 18:675-689,1992); Rice actin (people such as McElroy (McElroy), vegetable cell (Plant Cell) 2:163-171,1990); PEMU (people such as Lars spy (Last), theoretical with applied genetics (Theor.Appl.Genet.) 81:581-588,1991); CaMV19S (people such as Nelson (Nilsson), plant physiology (Physiol.Plant) 100:456-462,1997); GOS2 (people such as De Peite (de Pater), plant magazine (Plant J) November; 2 (6): 837-44,1992); Ubiquitin (people such as Harald Christensen (Christensen), molecular biology of plants 18:675-689,1992); Paddy rice cyclophilin (people such as Bu Huoerci (Bucholz), molecular biology of plants 25 (5): 837-43,1994); Zea mays H3 histone (people such as Lai Beiji (Lepetit), molecular gene group and genetics (Mol.Gen.Genet.) 231:276-285,1992); Muscle fibrin 2 (is pacified people such as (An), plant magazine 10 (1); 107-121,1996), composition tip of a root CT2 promotor (SEQ ID NO:1535; Also referring to PCT application number IL/2005/000627) and synthetic super MAS people such as (, plant magazine (The Plant Journal) 7:661-76,1995) Nie (Ni).Other composition promotors comprise U.S. Patent number 5,659, those in 026,5,608,149,5,608,144,5,604,121,5,569,597,5,466,785,5,399,680,5,268,463 and 5,608,142.

Applicable tissue-specific promoter [as is described in the people such as such as Yamamoto (Yamamoto), plant magazine (Plant J.) 12:255-265,1997 including, but not limited to leaf specificity promoter, the people such as power (Kwon), plant physiology (Plant Physiol.) 105:357-67,1994, the people such as Yamamoto (Yamamoto), plant cell physiology (Plant Cell Physiol.) 35:773-778,1994, the people such as dagger-axe Bristol (Gotor), plant magazine (Plant J.) 3:509-18,1993, the people such as Rothko difficult to understand (Orozco), molecular biology of plants 23:1129-1138,1993, and people such as loose ridge (Matsuoka), periodical (Proc.Natl.Acad.Sci.USA) 90:9586-9590 of institute of NAS, 1993], [for example from seed-specific gene, (people such as (Simon) is covered in west to select seeds promotor, molecular biology of plants (Plant Mol.Biol.) 5.191,1985, the people such as Scholfield (Scofield), journal of biological chemistry (J.Biol.Chem.) 262:12202,1987, the people such as Bath Zi Nisiji (Baszczynski), molecular biology of plants (Plant Mol.Biol.) 14:633, 1990), Bertholletia excelsa albumin (is trained people such as raw (Pearson'), molecular biology of plants (Plant Mol.Biol.) 18:235-245, 1992), legumin (the people such as Ellis, molecular biology of plants (Plant Mol.Biol.) 10:203-214, 1988), gluten (the paddy rice) (people such as Gao Yan (Takaiwa), molecular gene group and genetics (Mol.Gen.Genet.) 208:15-22, 1986, the people such as Gao Yan (Takaiwa), the biochemical meeting in Europe federation's wall bulletin (FEBS Letts.) 221:43-47, 1987), zein (the people such as Max Metzker (Matzke), molecular biology of plants (Plant Mol Biol), 143:323-321990), napA (executes the people such as tal fibre shellfish lattice (Stalberg), plant (Planta) 199:515-519, 1996), wheat SPA (Albany tal fibre (Albanietal), vegetable cell (Plant Cell), 9:171-184, 1997), sunflower oil body protein (the oleosin) (people such as comings (Cummins), molecular biology of plants (Plant Mol.Biol.) 19:873-876, 1992)], endosperm specificity promoter [for example wheat LMW and HMW, glutenin-1 (molecular gene group and genetics (Mol Gen Genet) 216:81-90, 1989, NAR17:461-2), wheat a, b and g gliadine (EMBO3:1409-15,1984), barley ltrl promotor, barley B1, C, D hordein (theoretical and applied genetics (Theor Appl Gen) 98:1253-62,1999, plant magazine 4:343-55,1993, molecular gene group and genetics (Mol Gen Genet) 250:750-60, 1996), barley DOF (the people such as meter Na (Mena), plant magazine (The Plant Journal), 116 (1): 53-62, 1998), Biz2 (EP99106056.7), synthetic promoter (the people such as about Sa of Vicente (Vicente)-kappa (Carbajosa), plant magazine (Plant J) 13:629-640, 1998), paddy rice prolamine NRP33, paddy rice-Lysozyme lb-1 (people such as Wu (Wu), plant cell physiology (Plant Cell Physiology) 39 (8) 885-889, 1998), the paddy rice alpha-globulin REB/OHP-1 (people such as middle shallow (Nakase), molecular biology of plants (Plant Mol.Biol.) 33:513-S22, 1997), paddy rice ADP-glucose PP (Study on Transformation (Trans Res) 6:157-68, 1997), Zea mays ESR gene family (plant magazine (Plant J) 12:235-46, 1997), Chinese sorghum γ-kafirin (molecular biology of plants (Plant Mol.Biol.) 32:1029-35, 1996)], embryo-specific promoter [the such as paddy rice OSH1 (people such as Sa Tuo (Sato), periodical (Proc.Nat.Acad.Sci.USA) 93:8117-8122 of institute of NAS), KNOX (the special horse in Persian (Postma)-people such as Harris agate (Haarsma), molecular biology of plants (Plant Mol.Biol.) 39:257-71, 1999), paddy rice oil body protein (the people such as Wu (Wu), journal of biological chemistry (J.Biochem.), 123:386, 1998)], and flower specific promoter [for example AtPRP4, chalcone synthase (chalene synthase, chsA) (the people such as model Mahmut Demir (Van der Meer), molecular biology of plants (Plant Mol.Biol.) 15,95-109,1990), LAT52 (people such as day Wei (Twell), molecular gene group and genetics (Mol.Gen Genet.) 217:240-245, 1989), without lobe (apetala)-3, plant reproductive tissue [for example OsMADS promotor (U.S. Patent application 2007/0006344)].

Applicable abiotic stress inducible promoter including, but not limited to salt inducible promoter as the RD29A (people such as mountain pass (Yamaguchi)-Xiao rugged (Shinozalei), molecular gene group and genetics (Mol.Gen.Genet.) 236:331-340,1993); As Zea mays rab17 gene promoter, (ripple draws people such as (Pla) to drought-inducible promoter, molecular biology of plants (Plant Mol.Biol.) 21:259-266,1993), the Zea mays rab28 gene promoter (people such as Basque (Busk), plant magazine 11:1285-1295,1997) and the Zea mays Ivr2 gene promoter (people such as Pei Erlaisiji (Pelleschi), molecular biology of plants (Plant Mol.Biol.) 39:373-380,1999); Thermal induction type promotor is as the hot tomato hsp80 promotor (U.S. Patent number 5,187,267) from tomato.

Photoinduction type promotor has the expression of enhancing between with the light light period, and in the situation that there is no light, substantially reduced express or without expression.The example of photoinduction type promotor includes but not limited to (Berry)-La Wei (Lowe) in SSU small ylidene gene promotor shellfish, (1982) molecule and applied genetics magazine (J.Mol.Appl.Gen.) 1:483-498; Pea ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase promotor, Broglie (Broglie), the people such as R., (1984) science (Science) 224:838-843; The people such as Fa Ciqiaodi (Facciotti), (1985) " with the photoinduction type expression (Light-inducible Expression of a Chimeric Gene in Soybean Tissue Transformed with Agrobacterium) of the mosaic gene in the soyabean tissue of Agrobacterium transformation ", biotechnology (Biotechnology), 3:241-246; The people such as Fu Lvhe (Fluhr), " organ specificity of plant gene and photoinduction type are expressed (Organ-Specific and Light-Induced Expression of Plant Genes) ", science (Science) (1986) 232:1106-1112; La Mupa (Lamppa), G. wait people (1985) " the light adjustment type of the wheat Cab gene in transgene tobacco and organ specificity are expressed (Light-regulated and organ-specific expression of a wheat Cab gene in transgenic tobacco) ", nature (Nature) the 316th volume: 750-752; Simpson (Simpson), J., Deng people, (1985) " the light inducibility of a kind of mosaic gene under the control of the 5'-of pea chlorophyll a/b-binding-protein gene flanking sequence and tissue specific expression (Light-inducible and tissue-specific expression of a chimeric gene under control of the5'-flanking sequence of a pea chlorophyll a/b-binding protein gene) ", EMBO magazine, the 4th volume, o. 11th: 2723-2729; PSSU gene promoter, the people such as He Leila (Herrera)-Ai Siteleina (Estrella), nature (Nature) (1984) 310:115-120; U.S. Patent number 5,750,385 etc.

Term " enzymic activity " is intended to comprise demethylation, hydroxylation, epoxidation, N-oxidation, sulphur oxidation (sulfooxidation), N-, S-and O-dealkylation, devulcanization, deaminizating, and the reduction of azo-group, nitro and N-oxide groups.Term " nucleic acid " refers to and is strand or double chain form, or the deoxyribonucleotide of sense or antisense or ribonucleoside acid polymer, unless and restriction in addition, otherwise contained to hybridize to the known analogue of the natural nucleotide of nucleic acid with mode like naturally occurring ucleotides.Unless otherwise instructed, otherwise concrete nucleotide sequence comprises its complementary sequence.

" structure gene " is a part for gene, comprises coded protein, polypeptide or its a part of DNA section, and does not comprise the 5 ' sequence that drives transcription initiation.The structure gene not interpretable product of alternately encoding.Structure gene can be the gene conventionally existing in cell, or common non-existent gene in its cell of introducing or cell position, and in a rear situation, it is called as " heterologous gene ".Heterologous gene can, whole or in part from any source known in the art, comprise the DNA of bacterial genomes or episome, eukaryote, nucleus or plasmid DNA, cDNA, viral DNA or chemosynthesis.Structure gene can contain can affect biological activity or its feature, the biological activity of expression product or one or more modifications of chemical structure, expression rate or expression control mode.These modify sudden change, insertion, disappearance and replacement including, but not limited to one or more Nucleotide.Structure gene can form a continual encoding sequence, or it can comprise the one or more introns that join by suitable splice site.Structure gene can be interpretable or not interpretable, comprises and is antisense orientation.Structure gene can be derived from multiple sources and be derived from the mixture of the section of multiple gene orders (natural existence or synthetic, the wherein synthetic DNA that refers to chemosynthesis).

" being derived from " and being used in reference to from a source (chemistry and/or biological) obtains, obtains, receives, reviews, copies or originate from.Derivative can produce by the chemistry to primary source or biological operation (including, but not limited to replacing, add, insert, lack, extract, separate, suddenly change and copying).

As referring to a part for component Nucleotide, " chemosynthesis " relevant to DNA sequence dna assemble in vitro.The artificial chemistry of DNA synthesizes can complete by program used for a long time (Ed Caruthers (Caruthers), the method (Methodology of DNA and RNA Sequencing) of DNA and RNA order-checking, (1983), Wei Siman (Weissman) (editor), Prey lattice press (Praeger Publishers), New York, the 1st chapter); Can carry out automatic chemosynthesis with one of multiple commercially available machine.

As used herein, " restructuring " comprises cell or the carrier that finger has been modified by introducing heterologous nucleic acids, or this cell source is from the cell of so modifying.Therefore, for example, as having a mind to the result of human intervention, reconstitution cell is expressed the gene of undiscovered same form in the cell of natural (non-restructuring) form, or the natural gene of expressing unconventionality expression, low expression in other cases or not expressing, maybe can there is expression minimizing or that eliminate of natural gene.As used herein, term " restructuring " is not for example contained, by natural event (, spontaneous mutation, Natural Transformation/transduction/swivel base), for example without the need for meaning human intervention with regard to event, and the change of the cell causing or carrier.

As used herein, " expression cassette " is restructuring or the synthetic nucleic acid construct producing with a series of appointment nucleic acid elements, allows concrete nucleic acid at target cell transcription.Expression cassette can be incorporated in plasmid, karyomit(e), Mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.Typically, except other sequences, the expression cassette part of expression vector comprises the nucleic acid and the promotor that need to be transcribed.

Term " residue " or " amino-acid residue " or " amino acid " are used interchangeably at this, to refer to be incorporated to the amino acid in protein, polypeptide or peptide (being jointly called " protein ").Amino acid can be naturally occurring amino acid and, unless restriction in addition can be contained the known analogue of the natural amino acid that can work with mode like naturally occurring amino acids.

The degree that term " selective cross " comprises the nucleic acid target sequence hybridization that refers to nucleotide sequence and appointment under stringent hybridization condition than the degree of itself and non-target nucleic acid sequence hybridization can detect higher (for example, at least 2 times to background), and refer to substantially get rid of non-target nucleic acid.Selective cross sequence typically has approximately at least 40% sequence identity, preferably 60%-90% sequence identity and 100% sequence identity (, complementation) most preferably to each other.

Term " stringent condition " or " stringent hybridization condition " comprise refer to probe by with the condition of the degree of its target sequence hybridization higher by detecting than the degree of it and other sequence hybridizations (for example, at least 2 times to background).Stringent condition be sequence dependent and will be different under different situations.Hybridize and/or the stringency of wash conditions by control, can identify and this probe to the target sequence (homology detection) of 100% complementation nearly.Alternately, can adjust stringent condition to allow some mispairing in sequence, make like this to detect the more similarity of low degree (allos detection).Preferably, probe is about 500 length of nucleotides, but length can alter a great deal, from being less than 500 Nucleotide to the whole length that equals target sequence.

Typically, stringent condition will be these: wherein salt concn is less than about 1.5M Na ion, typically is approximately 0.01 to 1.0M Na ionic concn (or other salt), pH is 7.0 to 8.3, and for short probe (for example, 10 to 50 Nucleotide) temperature at least 30 DEG C for example, for long probe (, being greater than 50 Nucleotide) at least about 60 DEG C.Stringent condition also can for example, by adding destabilizing agent, methane amide or step on Ha Teshi reagent (Denhardt's) and realize.Exemplary low stringency is included at 37 DEG C hybridizes with the buffered soln of 30% to 35% methane amide, 1M NaCl, 1%SDS (sodium lauryl sulphate), and at 50 DEG C to 55 DEG C 1 × to washing in 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrate).Exemplary medium stringent condition is included at 37 DEG C hybridizes in 40% to 45% methane amide, 1M NaCl, 1%SDS, and at 55 DEG C to 60 DEG C 0.5 × to 1 × SSC, wash.Exemplary high stringent condition is included at 37 DEG C hybridizes in 50% methane amide, 1M NaCl, 1%SDS, and in 0.1 × SSC, washs at 65 DEG C at 60 DEG C.Specificity depends on the washing after hybridization conventionally, and key factor is ionic strength and the temperature of final washing soln.For DNA-DNA crossbred, can be from rice grace Cowes (Meinkoth) and Wal (Wahl) (1984) analytical biochemistry (Anal.Biochem.), the equation guestimate T of 138:267-84 _m: T _m=81.5 DEG C of .+16.6 (log M)+0.41 (%GC)-0.61 (%form)--500/L; Wherein M is the volumetric molar concentration of monovalent cation, and %GC is guanosine in DNA and the percentage of cytidylic acid(CMP), and %form is the per-cent of methane amide in hybridization solution, and L is the length (unit is base pair) of crossbred.T _mtemperature (under the ionic strength limiting and pH) while being the probe hybridization of 50% complementary target sequence and Perfect Matchings.For every 1% mispairing, T _mreduce approximately 1 DEG C; Therefore, capable of regulating T _m, condition hybridization and/or washing so as hybridization to desirable conforming sequence.For example, there is the conforming sequence of >90% if found, can be by T _mreduce by 10 DEG C.In general, stringent condition is chosen as to the heat fusion joint (T under the ionic strength and the pH that limit than particular sequence and complement thereof _m) low approximately 5 DEG C.But utmost point stringent condition can utilize lower than heat fusion joint (T _m) hybridization and/or the washing of 1 DEG C, 2 DEG C, 3 DEG C or 4 DEG C; Medium stringent condition can utilize lower than heat fusion joint (T _m) hybridization and/or the washing of 6 DEG C, 7 DEG C, 8 DEG C, 9 DEG C or 10 DEG C; Low stringency condition can utilize lower than heat fusion joint (T _m) hybridization and/or the washing of 11 DEG C, 12 DEG C, 13 DEG C, 14 DEG C, 15 DEG C or 20 DEG C.Use this equation, hybridization and washing composition and desirable T _m, those of ordinary skill in the art should be understood that the variation of solution severity hybridization and/or washing has obtained description inherently.If desirable mispairing degree causes T _mbe less than 45 DEG C (aqueous solution) or 32 DEG C (formamide soln), preferably increase SSC concentration to make using higher temperature.The detailed guide of related nucleic acid hybridization sees with Publication about Document: carry Ji Sen (Tijssen), Biochemistry and Molecular Biology laboratory technique-with nucleic acid probe hybridization (Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes), part i, the 2nd chapter, " Hybridization principle general introduction and nucleic acid probe determining strategy (Overview of principles of hybridization and the strategy of nucleic acid probe assays) " likes to think only your (Elsevier), New York (1993), and modern molecular biology experimental technique (Current Protocols in Molecular Biology), the 2nd chapter, the people such as Su Beier difficult to understand (Ausubel) edit, Green publishes and Willie interdisciplinary science press (Greene Publishing and Wiley-Interscience), New York (1995).Unless otherwise indicated, otherwise high severity is defined as at 65 DEG C at 4 × SSC, 5 × step on Ha Teshi reagent (the 5g ficoll (Ficoll) in 500ml water, 5g polyvinylpyrrolidone, 5g bovine serum albumin) in this application, 0.1mg/ml boils salmon sperm dna, and hybridize in 25mM Na phosphoric acid salt, and in 0.1 × SSC, 0.1%SDS, wash at 65 DEG C.

As used herein, " transgenic plant " comprise the plant that finger comprises heterologous polynucleotide in its genome.In general, heterologous polynucleotide is stably integrated in genome, makes like this polynucleotide be passed to the successive generation.Heterologous polynucleotide can be integrated in genome separately or as a part for recombinant expression cassettes.Use " transgenosis " to comprise at this: any cell, clone, callus, tissue, plant part or plant that genotype has changed because of the existence of heterologous nucleic acids, comprise those transgenosiss of so being changed at first together with those those by carrying out from initial transgenosis that sexual hybridization or vegetative propagation produced.As used herein, term " transgenosis " is not contained by conventional plant breeding method or by natural event, the change of the genome (chromogene group or karyomit(e) alia gene group) that for example random cross fertilization, non-recombinant virus infection, the conversion of non-recombinant bacteria, non-restructuring swivel base or spontaneous mutation cause.

As used herein, " carrier " comprises and refers to for transfection host cell and can insert therein the nucleic acid of polynucleotide.Carrier is replicon normally.Expression vector allows transcribing of insertion nucleic acid wherein.

" cross and express " and refer to that the expression level in transgenosis organism exceedes the expression level in normal or unconverted organism.

" plant tissue " comprise broken up with undifferentiated tissue or plant, multi-form including, but not limited to root, stem, branch, leaf, pollen, seed, tumor tissues and cell and culture, for example single cell, protoplastis, embryo and callus.Plant tissue can be in plant or in organ, tissue or cell culture.

" preferred expression ", " preferentially transcribing " or " preferably transcribing " refer to the following expression of gene product interchangeably, these gene products are in one or several plant tissue (space constraint) and/or express with higher level in one or several development of plants stage (time limitation) preferably, and in its hetero-organization/etap, there is relatively low-level expression.

Term " conversion " refers to nucleic acid fragment to transfer in the genome of host cell, causes heredity stable on gene." instantaneous conversion " refers to that transgenosis and foreign DNA introduced wherein (for example as by as the method for agrobacterium-mediated conversion or particle gun bombardment), but the cell of not selected for stable maintenance.The cell that " stable conversion " selected and regenerated after referring to and transforming on selection substratum.

" conversion/genetically modified/restructuring " refer to that heterologous nucleic acids molecule introduced host's organism wherein, for example bacterium or plant.This nucleic acid molecule can be stably incorporated in host's genome, or this nucleic acid molecule can also be served as a kind of extrachromosomal molecule existence.So a kind of extrachromosomal molecule can self-replicating.The cell, tissue or the plant that transform should be understood to not only contain the end product of conversion process, and contain its transgenosis filial generation." non-transformed ", " non-transgenic " or " non-restructuring " host refer to the wild-type organism that does not contain heterologous nucleic acids molecule, for example bacterium or plant.

Term " translational enhancer sequence " refers to the DNA sequence dna part between promotor and encoding sequence of a gene, and it is transcribed into RNA and is present in the mRNA of processing completely (5') of translation initiation codon upstream.Translational enhancer sequence can affect processing, mRNA stability or the translation efficiency of primary transcript to mRNA.But " witness marking " refers to that it is expressed and do not give transformant advantage can become and can detect or visible gene.The example of witness marking is including, but not limited to beta-Glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP).

" wild-type " refers to the normal gene, virus or the organism that do not carry out any sudden change or modification found at occurring in nature.

As used herein, " vegetable material ", " plant part " or " plant tissue " refer to vegetable cell, plant protoplast, plant can by the Plant cell and tissue culture thing of its regeneration, plant callus (plant calli), vegetation bed (plant clump) and in plant or plant part complete vegetable cell, these plant parts are for example embryo, pollen, ovule, seed, leaf, flower, branch, fruit, grain, fringe, cob, shell, stem, root, the tip of a root, flower pesticide, stem tuber, rhizome etc.

As used herein, " protein extract " refers to the partial protein or the gross protein that extract from plant part.Plant protein extracting method is known in the art.

As used herein, " plant sample " refers to the plant tissue of complete or imperfect (seed for example grinding or plant tissue, the plant tissue of chopping, the tissue of freeze-drying).It can also be the extract that comprises complete or imperfect seed or plant tissue.

Following term is for describing sequence relation between two or more nucleic acid or polynucleotide or polypeptide: (a) " reference sequences ", (b) " comparison window ", (c) " sequence identity ", (d) " sequence identity percentage " and (e) " essence consistence ".

As used herein, " reference sequences " is the defined nucleotide sequence as sequence comparison basis.Reference sequences can be the subset of specified sequence or all; For example, the section of full-length cDNA or gene order or this complete cDNA or gene order.

As used herein, " comparison window " represents to comprise the section of the continuous and appointment that refers to polynucleotide sequence, wherein can be by this polynucleotide sequence and reference sequences comparison, and wherein for the best of two sequences is compared, the part in comparison window of this polynucleotide sequence is than this reference sequences (it does not comprise interpolation or disappearance), can comprise and add or disappearance (, room).In general, the length that comparison window is at least 20 continuous nucleotides, and can be optionally 30,40,50 and 100 or longer.Those of ordinary skill in the art should be understood that for fear of causing and the high similarity of reference sequences owing to comprising room in polynucleotide sequence, typically introduce gap penalty and it is deducted from matching number.

Nucleotide and aminoacid sequence are compared and known in the art for method relatively.Local homology's algorithm (BESTFIT) of Smith (Smith) & water graceful (Waterman) (1981) high applied mathematics (Adv.Appl.Math) 2:482 can carry out the best comparison for comparing to sequence; The sequence analysis algorithm (GAP) of interior moral Leman (Needleman) & Wen Sike (Wunsc) (1970) molecular biology magazine 48:443-53; The similarity searching method (special fasta (Tfasta) & fasta (Fasta)) of the periodical 85:2444 of institute of Pearson (Pearson) & Lippmann (Lipman) (1988) NAS; The computerize of these algorithms is implemented, including, but not limited to CLUSTAL, Wisconsin Genetics software package in the PC/Gene program of mountain scene city, California (Mountain View) Intelligenetics company, the 8th edition (can derive from Genetics Computer Group (genetics computer group) ( program (Accelrys company, San Diego, California)) in GAP, BESTFIT, BLAST, FASTA and TFASTA.As Publication about Document is described CLUSTAL program in detail: John Higgins (Higgins) & Sharp (Sharp) (1988) gene (Gene) 73:237-44; The CABIOS5:151-3 of John Higgins (Higgins) & Sharp (Sharp) (1989); People (1988) nucleic acids research (the Nucleic Acids Res.) 16:10881-90 such as Cole's pendant (Corpet); People (1994) molecular biology method (Meth.Mol.Biol.) 24:307-31 such as application (Computer Applications in the Biosciences) 8:155-65 and Pearson (Pearson) of people (1992) computer in bio-science such as yellow (Huang).The preferable procedure that is used for the best overall comparison of multiple sequences is PileUp (Feng (Feng) & Doolittle (Doolittle) (1987) molecular evolution magazine (J.Mol.Evol.), 25:351-60, it is similar to the method for being described by the CABIOS5:151-53 of John Higgins (Higgins) & Sharp (Sharp) (1989), and is combined in by reference this).Can comprise for the blast program family of database similarity search: the BLASTN for nucleotide query sequence to RiboaptDB sequence; For the BLASTX of nucleotide query sequence to Protein Data Bank sequence; For the BLASTP of protein search sequence to Protein Data Bank sequence; For the TBLASTN of protein search sequence to RiboaptDB sequence; And for nucleotide query sequence the TBLASTX to RiboaptDB sequence.Referring to modern molecular biology experimental technique (Current Protocols in Molecular Biology), the 19th chapter, the people such as Su Beier difficult to understand (Ausubel) edit, Green publishes and Willie interdisciplinary science press (Greene Publishing and Wiley-Interscience), New York (1995).

GAP uses the algorithm of above-mentioned interior moral Leman (Needleman) & Wen Sike (Wunsc) h to find the comparison of two complete sequence, and this comparison makes matching number maximize and room number is minimized.GAP considers that all possible comparison and null position and generation have the coupling base of maximum number and the comparison in minimum room.It allows to mate base and produces point penalty and room extension point penalty for unit provides room.GAP is necessary for each room that its inserts and obtains room and produce the income of point penalty matching number.If select to be greater than zero room extension point penalty, GAP must be additionally by each room of being inserted of being multiplied by the room length of extending point penalty in room produces income.Acquiescence room in Wisconsin Genetics software package the 10th edition produces point penalty value and room and extends point penalty value and be respectively 8 and 2.Room produces point penalty and room extension point penalty can be expressed as the integer in the integer group of selecting free 0 to 100 composition.Therefore, for example, room produces point penalty and room extension point penalty can be 0,1,2,3,4,5,6,7,8,9,10,15,20,30,40 and 50 or larger.

GAP represents a best member who compares family.Can have many members of this family, but other members do not have preferable quality.GAP shows four advantage feature for comparing: quality, ratio, consistence and similarity.Quality is the tolerance being maximized for aligned sequences.Ratio is that quality is divided by the base number in shorter section.Consistence per-cent is the per-cent of the symbol that in fact mates.Similarity per-cent is the per-cent of similar symbol.Symbol on opposite, room is ignored.In the time that the marking matrix value of pair of symbols is more than or equal to similarity threshold value 0.50, similarity is given a mark.The marking matrix using in Wisconsin Genetics (Wisconsin State genetics) software package the 10th edition is BLOSUM62 (referring to periodical (Proc.Natl.Acad.Sci.USA) 89:10915 of institute of Hei Nigaofu (Henikoff) & Hei Nigaofu (Henikoff) (1989) NAS).

Unless otherwise indicated, otherwise the sequence identity/similarity providing at this refers to and use BLAST2.0 routine package, use the value that default parameters obtains people (1997) nucleic acids research (Nucleic Acids Res.) 25:3389-402 such as () A Erqiuer (Altschul).

As it will be appreciated by the skilled addressee that, blast search hypothetical protein matter can be modeled as stochastic sequence.But much actual protein comprises nonrandom sequence area, they can be with poly-tract (homopolymeric tracts), short period tumor-necrosis factor glycoproteins or be rich in one or more amino acid whose regions.These low-complexity regions can be compared between incoherent protein, even if other regions of protein are completely different.Multiple low-complexity filter can be used to reduce this class low-complexity comparison.For example, can be used alone or in combination SEG (5 Tengs (Wooten) & Fidel's henries (Federhen) (1993) calculational chemistries (Comput.Chem.) 17:149-63) and (carat not in (Claverie) & Shi Daisi (States) (1993) calculational chemistry 17:191-201) low-complexity filters.

Under two nucleic acid or peptide sequence background, as used herein, " sequence identity " or " consistence " comprise finger when in the comparison window of specifying, compare to obtain maximum to seasonable two sequences in identical residue.In the time that reference protein uses sequence identity per-cent, recognize that the common difference in inconsistent residue position is conservative aminoacid replacement, wherein amino-acid residue (is for example had similar chemical property by other, electric charge and hydrophobicity) amino-acid residue replace, and therefore do not change the functional performance of molecule.In the time that sequence is different aspect conservative replacement, can adjust upward sequence identity per-cent to proofread and correct the conservative character of this replacement.Because being said to be, the different sequence of so conservative replacement there is " sequence similarity " or " similarity ".The method of carrying out this adjustment is well known to those skilled in the art.Typically, this relates to and replaces marking for part mispairing instead of mispairing completely by conservative, improves thus sequence identity percentage.Therefore, for example, if give 1 point of same amino acid and give non-conservative replacement zero, guard the mark replacing between zero and 1.For example according to this (Meyers) & Miller (Miller) (1988) computer of Meyer, the algorithm of application (Computer Applic.Biol.Sci.) 4:11-17 in bio-science calculates the conservative mark replacing, for example, at program PC/GENE (intelligent genetics (Intelligenetics), mountain scene city, California, U.S.) in implement.

As used herein, " sequence identity percentage " represents the definite values of sequence by a comparison window being compared to two best comparisons, wherein compared with reference sequences (it does not comprise interpolation or disappearance), polynucleotide sequence part in this comparison window can comprise adds or lacks (, room), for the best comparison of these two sequences.Calculate by the following method this percentage: determine that the positional number at two identical nucleic acid bases in sequence or amino-acid residue place is to produce matched position number, matched position number is multiplied by 100 and produce sequence identity percentage divided by the total positional number in comparison window and by result.

" the essence consistence " of term polynucleotide sequence refers to and uses the one in described comparison program and use canonical parameter, compared with the sequence that polynucleotide comprise and reference sequences, there is the sequence identity between 50%-100%, for example at least 50% sequence identity, at least 60% sequence identity, at least 70%, at least 80%, more preferably at least 90% and at least 95%.Those skilled in the art will recognize that, can suitably adjust these values by consideration codon degeneracy, amino acid similarity, reading frame location etc., to determine the corresponding consistence by two nucleotide sequence coded protein.For these objects, between the essence consistence ordinary representation 55%-100% of aminoacid sequence, for example at least 55%, at least 60%, at least 70%, 80%, 90% and at least 95% sequence identity.

Another consistent in fact instruction of nucleotide sequence is whether two molecules hybridize each other under stringent condition.The degeneracy of genetic code allows a lot of aminoacid replacement, thus the variation of the nucleotide sequence of the same amino acid that causes encoding, therefore likely DNA sequence dna codified phase homopolypeptide but not hybridization each other under stringent condition.For example, while producing the copy of nucleic acid when the maximum codon degeneracy that uses genetic code to allow, this thing happens.A two nucleotide sequences substantially consistent instruction are, the polypeptide of the first nucleic acid encoding and polypeptide generation immunological cross-reaction by the second nucleic acid encoding.

As used herein, phrase " plant biomass " refers to the amount (grams with air-dry or dry tissue is measured) of the tissue being produced in the season of growth by plant, and it also can determine or affect the output of plant biomass or every cultivated area.

The crop yield increasing is the proterties all over the world with considerable economic interests.Output is normally defined the measured production with economic worth from crop.This can define aspect quantity and/or quality.Output directly depends on a number of factors, and for example, the number of organ and size, plant structure (for example, branch number), seed are produced, leaf is aging etc.Root development, nutrition absorption, stress tolerance and early growth gesture can be also the important factors that determines output.In addition, in agricultural, wish very much exploitation can optimal growth condition together with non-optimal growth condition under (for example arid, under abiotic stress condition) demonstrate the crop that increases output.Therefore, optimize above-mentioned factor and can contribute to increase crop yield.

Seed production is the proterties of particularly important, because the seed of many plants is important for human and animal's nutrition.Crop, for example corn, paddy rice, wheat, canola oil dish and soybean account for the over half of mankind's total heat picked-up, no matter be via direct consumption seed itself, or the domestic animal of raising through the seed of processing via consumption.Plant seed is also sugar, oil and the source for the numerous species metabolite of different commercial runs.Seed is made up of embryo (source of new branch and root) and endosperm (the nutrient source of the embryonic development during duration of germination and early stage growth of seedling).The growth of seed relates to many genes, and need to will be sent in the seed of just growing from the metabolite of root, leaf and stem.Endosperm assimilates the metabolic precursor thereof of carbohydrate, oil and protein and they is synthesized and store macromole to make full seed.

In some cases, plant biomass is for the amount of the plant biomass that can produce for concrete plant.Compared with plantlet more, the larger plant with larger leaf area can typically absorb more light, nutrient and carbonic acid gas, and therefore may during the phase contemporaneously, increase larger weight (Fasoula (Fa Suola) & Tollenaar (holder Jules Renard) 2005Maydica50:39).For example, for example biomass (corn, grass, Chinese sorghum, sugarcane) being changed in the process of fuel (for example, as ethanol or butanols), the plant biomass of increase can be also to make us very much wishing.

The ability that increases plant biomass has many application in following field, and the production of for example decorative plant, agricultural, gardening, biofuel production, medicine, use plant are as enzyme industry and the forestry of the manufacturing site location of these molecules.Increasing output can also produce for being used for bio-reactor (for producing material with biotechnology, for example medicine, antibody, vaccine and fuel, or for the bio-transformation of organic waste materials) microorganism or algae and other such fields be applied.

Plant breeder is conventionally interested in the particular aspects of improvement output, and this depends on discussed crop or plant, and the part with relatively economical value of this plant or crop.For example, plant breeder is the improvement of the plant biomass of one or more parts of looking for plant (weight) especially, and these parts can comprise (can gather in the crops) part and/or the underground part gathered in the crops on the ground.If the over-ground part of plant or underground part are that this is correlated with especially for consumption.For many crops, special cereal (cereal), the improvement of seed production is to make us very much wishing.The seed production increasing can embody in many aspects, wherein depends on discussed crop or plant and end-use thereof, and the each independent aspect of seed production has different importance for plant breeder.

For plant breeder, can select and select the output aspect that needs to be changed is great advantage.The gene that can select the concrete aspect (for example, output under seed production, biomass weight, water use efficiency and stress conditions) that is suitable for changing output can be also to make us very much wishing.For example, the increase of filling ratio the thousand seed weight in conjunction with increase for example, make us wishing by being for crop (corn) very much.For paddy rice and wheat, the combination that increases filling ratio, harvest index and increase thousand seed weight is made us wishing by being very much.

The different system, computer program and the method that are used for the model that uses a kind of bioprocess can be predicted the component of candidate that strengthens this bioprocess, for example gene and/or the assortment of genes.For example, refer to as the method disclosed on May 10th, 2012 disclosed WO2012/061585, and be combined in by reference this.Show maybe and will represent a kind of object of biological product of this phenotype result for production, people can select a kind of component of candidate based on this phenotype result and definite sensitivity.For example, can phenotype result and the sensitivity based on definite based on wherein predicting that a kind of candidate gene can produce select this gene.In this way, even in the time failing to realize optimum expression level in this biological product at for example laboratory experiment and/or during manufacturing, a kind of single candidate gene of the variation relative insensitivity to optimum expression level also can produce predicted phenotype result or approach acceptably (based on a predefined difference) in a phenotype result of predicted phenotype result.

In one embodiment, can synthesize or separate the polynucleotide sequence of selected this or these candidate gene of identifying by the present invention and be incorporated in expression cassette, these expression cassettes contain genetic regulatory element with targeted expression level and one or more cell type.In one embodiment, at least one expression cassette can be incorporated in a binary vector and be transformed in plant.Then can determine sensitivity and actual phenotype result.As described in following instance, an embodiment identifies and uses method known to persons of ordinary skill in the art be incorporated in expression cassette and be transformed into three kinds or four kinds of candidate genes in plant with the present invention.These examples have also been described the currently known methods of the phenotype result for measuring these genetically modified plants.

One embodiment of the present of invention comprise containing independent or with a kind of Phosphoenolpyruvate carboxylase (PEPC of arbitrary combination, EC4.1.1.31), a kind of fructose-1,6-bisphosphate Phosphoric acid esterase (FBP, EC3.1.3.11), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADPMD, EC1.1.1.82), a kind of phosphoribulokinase (PRK, and a kind of expression cassette, cell or the plant of the two kinases (PPDK, EC2.7.9.1) of a kind of pyruvate phosphate EC2.7.1.19).Can find the sequence information of many PEPC, FBP, NADPMD, PRK or PPDK gene at document or for example, by the different obtainable databases of inquiry (BRENDA database (brenda.enzymes.org)).

An alternative embodiment of the invention comprises a kind of expression cassette, cell or the plant containing the combination of any two following genes, these genes comprise the two kinases (PPDK) of a kind of Phosphoenolpyruvate carboxylase (PEPC), a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADPMD), a kind of phosphoribulokinase (PRK) and a kind of pyruvate phosphate.

Still another embodiment of the present invention comprises a kind of expression cassette, cell or the plant containing the combination of any three following genes, these genes comprise the two kinases (PPDK) of a kind of Phosphoenolpyruvate carboxylase (PEPC), a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADPMD), a kind of phosphoribulokinase (PRK) and a kind of pyruvate phosphate.In a specific embodiment, expression cassette, cell or plant comprise a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of phosphoribulokinase (PRK) and a kind of Phosphoenolpyruvate carboxylase (PEPC).

Still another embodiment of the present invention comprises a kind of expression cassette, cell or the plant containing the combination of any four following genes, these genes comprise the two kinases (PPDK) of a kind of Phosphoenolpyruvate carboxylase (PEPC), a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADP-MD), phosphoribulokinase (PRK) and a kind of pyruvate phosphate.In a specific embodiment, expression cassette, cell or plant comprise a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of phosphoribulokinase (PRK), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADP-MD) and a kind of Phosphoenolpyruvate carboxylase (PEPC).

Still another embodiment of the present invention comprises a kind of expression cassette, cell or the plant containing the two kinases (PPDK) of a kind of Phosphoenolpyruvate carboxylase (PEPC), a kind of fructose-1,6-diphosphate Phosphoric acid esterase (FBP), a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) (NADP-MD), phosphoribulokinase (PRK) and a kind of pyruvate phosphate.

One embodiment of the present of invention can also comprise containing SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.6 to SEQ ID, SEQ ID NO.7, and a kind of expression cassette of the polynucleotide of SEQ ID NO.8, cell or plant.

An alternative embodiment of the invention comprises containing sequence SEQ ID NO.6, SEQ ID NO.7, and in SEQ ID NO.8 any two or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.6 to SEQ ID, SEQ ID NO.7, and a kind of expression cassette of the polynucleotide of SEQ ID NO.8, cell or plant.

Still another embodiment of the present invention comprises containing sequence SEQ ID NO.6, SEQ ID NO.7, and in SEQ ID NO.8 one or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.6 to SEQ ID, SEQ ID NO.7, and a kind of expression cassette of the polynucleotide of SEQ ID NO.8, cell or plant.

The present invention includes containing sequence SEQ ID NO.6, SEQ ID NO.7, in SEQ ID NO.8 at least one or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.6 to SEQ ID, SEQ ID NO.7, and a kind of expression cassette of the polynucleotide of SEQ ID NO.8, cell or plant.

Still another embodiment of the present invention comprises containing sequence SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, and SEQ ID NO.12 or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.9 to SEQ ID, SEQ ID NO.10, a kind of expression cassette of the polynucleotide of SEQ ID NO.11 and SEQ ID NO.12, cell or plant.

An alternative embodiment of the invention comprises containing sequence SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, and in SEQ ID NO.12 two or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.9 to SEQ ID, SEQ ID NO.10, a kind of expression cassette of the polynucleotide of SEQ ID NO.11 and SEQ ID NO.12, cell, plant, or Mammals.

One embodiment of the present of invention also comprise containing sequence SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, and in SEQ ID NO.12 one or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.9 to SEQ ID, SEQ ID NO.10, a kind of expression cassette of the polynucleotide of SEQ ID NO.11 and SEQ ID NO.12, cell, plant, or Mammals.

One embodiment of the present of invention comprise containing sequence SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11, and in SEQ ID NO.12 at least one or have 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% conforming polynucleotide or can be low, in or under high stringent hybridization condition, hybridize the NO.9 to SEQ ID, SEQ ID NO.10, a kind of expression cassette of the polynucleotide of SEQ ID NO.11 and SEQ ID NO.12, cell, plant, or Mammals.

Previous examples described here is only for schematic object, and is not intended to have restricted.Implementation of the present invention can be carried out in hardware, firmware, software or its any suitable combination.Implementation of the present invention can also be embodied as and be stored in an instruction on machine-readable medium, and it can be read and be carried out by one or more treaters.A tangible machine-readable medium can comprise the mechanism of any tangible nonvolatile of the information of the form in for example, being can read by a machine (calculating device) for storage or transmission.For example, a tangible machine-readable storage media can comprise read-only storage, random access memory, magnetic disc storage media, optic storage medium, flash memory device and other tangible storage medias.Invisible machine readable transmission medium can comprise the transmitting signal of invisible form, for example carrier wave, infrared signal, numerary signal and other invisible transmission mediums.In addition, firmware, software, routine or instruction can be described in above disclosure content with regard to particular exemplary implementation of the present invention, and carry out some effect.But, will be apparent that, only for simplicity, and this class effect is in fact produced by calculating device, treater, controller or other devices of carrying out firmware, software, routine or instruction in this class description.

Implementation of the present invention can be described as comprising concrete characteristic, structure or a feature, but every aspect or implementation can not necessarily comprise this concrete characteristic, structure or feature.In addition, when be combined concrete characteristic of description, structure or feature with an aspect or implementation, whether describe clearly, this characteristic, structure or feature can be combined and be included with other implementations no matter should be understood that.Therefore, can in the situation that not departing from scope and spirit of the present invention, make various changes and modifications provided description.Therefore, specification sheets and graphic should being regarded as are only exemplary, and scope of the present invention is only determined by appended claims.

Following instance provides multiple illustrative examples.General state of the art according to the present invention and in this area, those of ordinary skill should be appreciated that it is exemplary that following instance is only intended to, and can in the case of the scope of theme that does not depart from current requirement, adopt many changes, amendment and change.

Except as otherwise noted, otherwise clone's step of carrying out for purposes of the present invention (for example, as restriction cracking, agarose gel electrophoresis, the purifying of DNA fragmentation, connection, intestinal bacteria (E.coli) transformation, the growth of bacterium and the sequential analysis of recombinant DNA of DNA fragmentation) carry out as the people such as Pehanorm Brooker (Sambrook) (the same document) describe.

Sequence table general introduction

SEQ ID NO:1 has described a kind of peptide sequence, corn (Zea mays) phosphoric acid enol pyruvic acid carboxylase

SEQ ID NO:2 has described a kind of peptide sequence, spinach (Spinacia oleracea) fructose-1,6-diphosphate esterase

SEQ ID NO:3 has described a kind of peptide sequence, spinach phosphoribulokinase

SEQ ID NO:4 has described a kind of peptide sequence, spinach NADP-malate dehydrogenase (malic acid dehydrogenase)

SEQ ID NO:5 has described a kind of peptide sequence, the engineered pyruvate orthophosphate dikinase of Chinese sorghum (Sorghum bicolor)

SEQ ID NO:6 has described a kind of polynucleotide sequence, the SoFBP in expression cassette ZmPRK-1

SEQ ID NO:7 has described a kind of polynucleotide sequence, the SoPRK in expression cassette ZmSBP

SEQ ID NO:8 has described a kind of polynucleotide sequence, the ZmPEPC in expression cassette ZmPGK

SEQ ID NO:9 has described a kind of polynucleotide sequence, the SoFBP in expression cassette ZmPRK-2

SEQ ID NO:10 has described a kind of polynucleotide sequence, the SoPRK in expression cassette ZmNADPME

SEQ ID NO:11 has described a kind of polynucleotide sequence, the SbPPDK in expression cassette ZmPEPC

SEQ ID NO:12 has described a kind of polynucleotide sequence, the SbNADP-MD in expression cassette ZmPGK

Example 1: determine candidate

This example has been described a kind of genetic engineering strategy that promotes the photo-assimilation in corn and other NADP apple acid types C4 species.A kind of computer model output organization is become to the combination solution of 3 and 4 genes.Select separately 3 genes and combinations 4 genes for proterties exploitation.In order to implement this proterties, reach (BRENDA) database (brenda.enzymes.org) to Boulogne and inquire about relevant phosphoric acid enol pyruvic acid carboxylase (PEPC, EC4.1.1.31), fructose-1,6-bisphosphatase (FBPase, EC3.1.3.11), phosphoribulokinase (PRK, EC2.7.1.19), NADP-malate dehydrogenase (malic acid dehydrogenase) (NADPME, and the sequence information of pyruvate orthophosphate dikinase (PPDK, EC2.7.9.1) EC1.1.1.82).This analysis provides the protein sequence of the enzyme having characterized in function.Be used for obtaining from the PEPC of corn, from the FBPase of spinach, from the phosphoribulokinase of spinach and from the protein sequence of the NADP-malate dehydrogenase (malic acid dehydrogenase) of Chinese sorghum from the information of this database.In brief, reference information is used for identifying the candidate that is characterized Data support by function.Each sequence all must be supported by enzymic activity evidence.Protein sequence data (SEQ ID NO1-4) is provided.Although have available information and the announcement of certain number, find that the open sequence data of corn PPDK is incomplete.Therefore, define Chinese sorghum PPDK gDNA sequence with public data.Use corn PPDK cDNA and protein sequence to obtain Chinese sorghum gDNA and cDNA sequence as inquiry from Chinese sorghum genome database.Via with the corresponding EST Chinese sorghum cDNA that compares to increase.These sequences are pooled in a contig, this contig are divided into exon and compare with gDNA.There are 19 exons, and except one all the other all restrictions with GT ... AG sequence is the intron on border.There are some positions, wherein Chinese sorghum PPDK gDNA and cDNA sequence divergence; In most of the cases, cDNA sequence is substituted by gDNA sequence.Also corn and sorghum gluten matter sequence are compared and be used for further optimizing this gDNA sequence.Finally, introducing the yellow top of Blang chrysanthemum (Flaveria brownie) PPDK residue replaces.Result is the engineered sequence of SbPPDK, SEQ ID NO5.Also replacing gDNA sequence modification by base is reticent XhoI, SanDI, NcoI, SacI, RsrII and XmaI limiting acid endo enzyme site.Add a NcoI site at translation initiation codon place, and after translation stop codon, add a SacI site.

Example 2: the adjusting sequence that target candidate gene is expressed

Once after having identified candidate gene, regulate sequence so that the expression of these candidate genes is targeted to suitable cell type with regard to selecting.Design a series of expression of plants boxes to send the robust proterties genetic expression in mesophyll or vascular bundle sheath cell.Protein group data (the outstanding benevolence of agate (Majeran), W. wait people (2005) vegetable cell (Plant Cell), 17:3111-3140) be used for identifying that based on the expression pattern of interested gene candidate regulates sequence with the combination of express spectra data, and six new expression cassettes (Koln Buddhist (Coneva) V waits people (2007) experimental botany magazine (J of Exp Botany) 58:3679-3693) are identified.Each box is made up of promotor and terminator sequence.This promotor is made up of the sequence (being made up of First Exon and part Second Exon) of the non transcribed sequence of 5'-(First Intron) and a 5'-untranslated.In addition, this promotor is to be derived from tobacco mosaic virus (TMV) sequence (markon (Gallie), D.R., Wal Bart (Walbot), V (1992) nucleic acids research (Nucleic Acids Res), 20 (17): 4631-4638) and a translational enhancer of the sequence (Ke Zhake (Kozak), M. (2002) gene (Gene) 299:1-34) optimized of corn finish.This terminator by just after translation stop codon the sequence of initial 3'-untranslated and the non transcribed sequence of 3'-form.

Carry out particular bases replacement to eliminate inner XhoI, SanDI, NcoI, SacI, RsrII and XmaI limiting acid endo enzyme site.In addition, base replaces the ATG in the sequence that is used for eliminating 5'-untranslated and inserts terminator codon.These promotors 5' end place and XhoI/SanDI side joint and on 3' end with NcoI side joint.These terminators 5' end place and SacI side joint and on 3' end with RsrII/XmaI side joint.Box is sequentially cloned in the binary vector with RsrII cutting with RsrII/SanDI pieces.Box is summarized in following table, and it comprises the reference to relevant SEQ ID NO.

Table 1.

Example 3: expression cassette and combination

Contain the candidate gene selected by a method of the present invention one trigenic and tetragenic expression cassette binary vector will be used for separately reducing C4 photosynthesis model and export to put into practice.This trigenic C4 light compositing strengthens construct and is shown in table 2; This tetragenic C4 light compositing strengthens construct and is shown in table 3.Gene numbering has been indicated order, initial and extend to left margin at the right margin place of T-DNA.This trigenic binary vector is 19862 and is shown in Fig. 1.This tetragenic binary vector is 19863 and is shown in Fig. 2.

Table 2.

Table 3.

Example 4: Plant Transformation

Construct 19862 and 19863 transforms for the corn of Agrobacterium (Agrobacterium) mediation.The conversion of immature maize is substantially as people such as negro holders (Negrotto), and 2000, in vegetable cell report (Plant Cell Reports) 19:798-803, describe and carry out.For this example, all medium components are described substantially as in the people such as negro holder (Negrotto) (the same document).But various medium components known in the art can be replaced.

By the gene clone for transforming to be applicable to corn transform a carrier.Select the transgenic lines (people such as negro holder (Negrotto) for the carrier of this example containing being useful on, the same document) phosphomannose isomerase (PMI) gene together with can selective marker glufosinates Transacetylase (PAT) (U.S. Patent number 5,637,489).In brief, make the agrobacterium strains LBA4404 (pSB1) that contains a kind of Plant Transformation plasmid be grown in YEP (yeast extract (5g/L), protein peptone (10g/L), NaCl (5g/L), 15g/l agar, pH6.8) on solid medium, 28 DEG C of growths 2 to 4 days.By about 0.8X10 ⁹individual Agrobacterium is suspended in the LS-inf substratum that is supplemented with 100 μ M As people such as (, the same document) negro holders (Negrotto).Pre-induction bacterium 30-60 minute in this substratum.

To from the largest fringe of 8-12, be resected in liquid LS-inf+100 μ M As from A188 or other suitable genotypic immature embryos.With fresh these embryos of infection substratum rinsing.Then add Agrobacterium solution, and by these embryo vortexs 30 seconds and allow its sedimentation 5 minutes together with bacterium.Then these embryonic shield sheet side direction Shangdis are transferred in LSA substratum, and in the dark cultivated two to three days.Subsequently, the embryo between 20 and 25 of each Petri plates is transferred in the LSDc substratum that is supplemented with cefotaxime (250mg/l) and Silver Nitrate (1.6mg/l), and in the dark, cultivated 10 days at 28 DEG C.

The immature embryo that produces embryo callus is transferred in LSD1M0.5S substratum.On this substratum, select culture approximately 6 weeks, culturing step went down to posterity in the time of approximately 3 weeks.The callus of surviving (calli) is transferred in the Reg1 substratum that is supplemented with seminose.Cultivate (16 hours bright/8 hour dark schemes) at bright place and afterwards, then chlorenchyma is transferred in the Reg2 substratum without growth regulator, and hatch about 1-2 week.These plantlets are transferred in Ma Zhenta (Magenta) the GA-7 box (the horse Genta Incorporated (Magenta Corp, Chicago Ill.) in Chicago, Illinois) that contains Reg3 substratum, and in the growth of bright place.

Analyze PMI, PAT, a kind of candidate gene encoding sequence and the carrier main chain of plant by Plutarch graceful (TaqMan).By for PMI, PAT and this candidate gene encoding sequence is positive and transfer in greenhouse for the negative plant of carrier main chain.Analyze the expression of all trait expression boxes by qRT-PCR.Single copy event that qualification can be hatched, and transferred in greenhouse.

Example 5: express the assessment of the transgenic plant of candidate gene

Can assess Plant Light assimilation by some methods.Below the example of indication has been described and will how to have been measured the variation of Plant Light assimilation of above-mentioned transgenic plant.Can in V3 seedling, compare the first plant-growth between the hemizygote proterties positive and invalid seedling.In this is analyzed, about 60 B1 plants germinate and carry out gene type in 4.5 inches of basins.Germinate after approximately 17 days, water makes basin soil saturated, and by native face seal to avoid evaporating.At time zero, some seedlings are killed to determine bud quality (being fresh weight and dry weight form).Record basin quality every day with assessment vegetation water demand.After 7 days, results bud and weigh (fresh weight and dry weight).Proofread and correct vegetation water utilization with a basin without plant and report Natural Water loss.This scheme makes plant-growth and water conservancy with obtaining comparison between the proterties positive and invalid group.Improved photo-assimilation can make proterties sun plant can accumulate with respect to the more air biological quality of invalid plant.

A kind of the second method is to use an infrared gas analysis (IRGA) instrument to measure photo-assimilation.For example, a CIRAS-2IRGA device can be fixed on a trivet, gaseous interchange cuvette is clipped to gently to leaf and the noise data being produced by plant treatment is minimized.Pore aperture is moved very responsive to touch and plant.Can be by the environment programming that is applied to blade to simulate growth room's environment (400 μ mol mol ^-1cO ₂; 26 DEG C; Ambient moisture), thus it is synthetic under standard growth conditions, to assess steady-state light.In this way, can directly compare the photo-assimilation between the proterties positive and invalid plant.

For example, although IRGA is a kind of strong and common instrument of assessment light compositing activity (A/Ci curve), it has some warnings.First, it is only analyzed a vanelets and is not provided about whole strain plant and photosynthetic information Canopy, and these information determine that under a kind of agronomy background proterties function is final required.The second, in whole development of plants, need many measurements to determine A.The 3rd, the general state of this light compositing utensil depends on to measure which kind of leaf, and in the time that this leaf is measured, in whole plant, exists and change.Finally, it is to need directly a kind of invasive technique of contact leaf.A kind of data component producing is the reaction of leaf to instrument.Comprehensive, this has produced the variation factor of high (10%-15%).Therefore, can not detect little but significant photo-assimilation with this device changes.

In order to walk around these restrictions, can use large hypobaric chamber, (the University of Guelph of University of Guelph of for example Ontario, Ontario) the plant CO of the colony of a 30-40 plant is monitored in the cabin (Wheeler (Wheeler), the people such as R.M. (2011) Advances in Space Research (Adv Space Res) 47:1600-1607) in controlled ambient system research institution (Controlled Environment Systems Research Facility) accurately ₂demand, night respiration and transpiration last the period that lasts up to several weeks.

Example 6: with construct 19862 and 19863 production transgenic corns

Transgenic corns event is used binary vector 19862 and 19863 to produce according to example 4.Altogether identified 32 single copies without main chain 19862 events.Altogether identified 22 single copies without main chain 19863 events.In seedling leaf texture, measure the messenger RNA(mRNA) producing from each transgenosis by qRT-PCR.These qRT-PCR data are reported as the ratio of gene specific (encoding sequence) signal and endogenous control signal number of times 1000.The all trait expression boxes of data presentation in following table are brought into play as expected function and are produced the proterties transcript in leaf.Comprising the data for constitutive expression box, is a benchmark for strength of signal.Be noted that with than being limited to compared with the proterties box of mesophyll or vascular bundle sheath cell, this composition flask has activity far away on more leaf cell.

Table 4.

Be transferred to soil (V3) about two weeks afterwards, analyzing TO seedling leaf texture is sampled for qRT-PCR.Determine transcript abundance with gene specific TaqMan probe.Report is with respect to the data of EF1A transcript (internal reference).To measure in quadruplicate each event.For each construct, data are mean+SD.

Example 7: the seedling Biomass accumulation in growth room

Can use growth of seedling to determine whether proterties is potential and cause that output ties down.We determine whether that by this mensuration 19862 or 19863 proterties have reduced plant-growth.According to example 5, seed germination and evaluate seedling in growth room makes to backcross.Seedling for each event is carried out to gene type sets up proterties and separates and organize transgenosis group and invalid group.For each event, as expected, proterties separate be proved to be 1 invalid: 1 hemizygote.Data in following table have been summarized the result of some mensuration.For each event, the growth of transgenosis seedling can not be distinguished with invalid seedling.This shows that this proterties does not hinder growth.These wild-type plants are included in a benchmark.It should be noted that a plant generation of removing the parent from regenerating by tissue culture tends to slower than plant-growth unconverted or wild-type.Average data prompting, these 19862 plants can be slower than these wild-type plant growths, but this difference is not statistically significant.

Table 5.

The B1 seed germination of render transgenic carry out gene type in 4.5 inches of basins.By become transgenosis group and invalid group for the plant tissue of each event, they are grown in growth room.24 days results buds after plantation.Bud is dried to 5 days at 89 DEG C in baking oven, then weighs.For each construct, data report mean+SD.

Example 8: evaluate 19862 events in confined chamber

F1 hybrid seed is germinateed and carry out gene type.Plant tissue is become to transgenosis group and invalid group.To in the large hypobaric chamber in every group of controlled ambient system research institution at University of Guelph (University of Guelph) (Controlled Environment Systems Research Facility), cultivate.Results bud, is dried and weighs.After gene type, determine soon for the initial biomass of seedling, and the bud quality of these initial biomass while representing to begin one's study.For each group, data are mean+SD.

These data show that the strategy that this mathematical modeling improves plant performance for exploitation is useful instrument altogether.

All reference cited herein, (for example include but not limited to article on all patents, patent application and publication thereof, Scientific Magazine and data base entries data base entries and all obtainable annotations therein), its full content is combined in to this by reference, and its combination degree is that they supplement, explain, provide a kind of background or teach the method in this employing, technology and/or composition.

Claims

1. an expression cassette, this expression cassette comprises that at least three are selected from the polynucleotide of lower group, this group is made up of the following: the polynucleotide of the polynucleotide of a kind of Phosphoenolpyruvate carboxylase of encoding, the polynucleotide of a kind of fructose-1,6-diphosphate Phosphoric acid esterase of encoding, the polynucleotide of a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) of encoding, a kind of phosphoribulokinase of encoding and the two kinase whose polynucleotide of a kind of pyruvate phosphate of encoding.

2. expression cassette as claimed in claim 1, wherein this expression cassette comprises the encode polynucleotide of a kind of polynucleotide of fructose-1,6-diphosphate Phosphoric acid esterase, a kind of phosphoribulokinase of encoding and the polynucleotide of a kind of Phosphoenolpyruvate carboxylase of encoding.

3. expression cassette as claimed in claim 1, wherein this expression cassette comprises the polynucleotide of the polynucleotide of encode a kind of polynucleotide of fructose-1,6-diphosphate Phosphoric acid esterase, a kind of phosphoribulokinase of encoding, the two kinase whose polynucleotide of a kind of pyruvate phosphate of encoding and a kind of NADP-malate dehydrogenase (malic acid dehydrogenase) of encoding.

4. expression cassette as claimed in claim 1, wherein this polynucleotide encoding and SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO:3, SEQ ID NO.4 or SEQ ID NO:5 have at least 70%, 80%, 90% or 95% conforming polypeptide.

5. expression cassette as claimed in claim 1, wherein a kind of polypeptide that comprises SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 of this polynucleotide encoding.

6. expression cassette as claimed in claim 1, wherein this expression cassette comprise there is SEQ ID NO.2, the polypeptide of SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5.

7. expression cassette as claimed in claim 1, wherein these polynucleotide may be operably coupled to one or more photoinduction type promotors.

8. expression cassette as claimed in claim 1, wherein these polynucleotide comprise SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

9. expression cassette as claimed in claim 1, wherein these polynucleotide comprise SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.

10. for increasing a method for biomass, the method comprises

A. incite somebody to action expression cassette introduced plant cell as claimed in any one of claims 1-9 wherein;

B. this plant cell growth is become to plant; And

C. select the transgenic plant of the biomass with increase.

11. method as claimed in claim 10, wherein this plant is a kind of C4 plant.

12. methods as claimed in claim 11, wherein this plant is selected from lower group, and this group is made up of the following: sugarcane, corn and Chinese sorghum.

13. methods as claimed in claim 12, wherein this plant is corn.

14. prepare a method for transgenic plant, the method comprises:

B. this plant cell growth is become to plant; And

C. select to comprise the plant of this expression cassette.

15. method as claimed in claim 14, wherein this plant is a kind of C4 plant.

16. methods as claimed in claim 15, wherein this plant is selected from lower group, and this group is made up of the following: sugarcane, corn and Chinese sorghum.

17. methods as claimed in claim 16, wherein this plant is corn.

18. a kind of plant or plant part, comprise expression cassette as claimed in claim 1.

19. plant as claimed in claim 18 or plant part, wherein this plant part is vegetable cell.

20. plant as claimed in claim 18 or plant part, wherein this plant part is seed.

21. a kind of plant or plant part, prepared by method as claimed in claim 14.