CN101479294A - Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use - Google Patents

Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use Download PDF

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CN101479294A
CN101479294A CNA2007800224528A CN200780022452A CN101479294A CN 101479294 A CN101479294 A CN 101479294A CN A2007800224528 A CNA2007800224528 A CN A2007800224528A CN 200780022452 A CN200780022452 A CN 200780022452A CN 101479294 A CN101479294 A CN 101479294A
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S·拉维特
S·昆杜
A·G·劳
D·T·托姆斯
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Pioneer Hi Bred International Inc
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Abstract

The invention provides isolated polynucleotide molecules encoding mutant and wild-type alleles of the maize D9 gene. The invention further provides methods for modifying the growth of plants involving the use of these isolated polynucleotide molecules, isolated polypeptides, and transformed plants, seeds, and cells.

Description

Isolating polynucleotide molecule and using method corresponding to the mutation allele and the wild-type allele of corn D9 gene
Invention field
The present invention relates to carry out genetic manipulation with the gene pairs organism of control growing and growth especially plant.The invention further relates to the gene of control growing, comprise homologue and mutant forms, by its encoded protein with the plant of these gene transformation.
Background of invention
Dwarf plant has produced great effect to agricultural.The dwarfing mutation of wheat is extensive use of in the North America owing to lodging possibility decline and high yield.Can be by using other interests of downgrading the crop realization to comprise the higher and labor cost reduction of the amount, the planting density that reduce required sterilant and fertilizer.
In view of present population increases and is suitable for the trend that the land area of farming reduces, improve agricultural productivity is a very important challenge always.Downgrade crop is the important component part of our system agricultural industry always.Increase the use of downgrading crop and can help to satisfy following agriculture production requirement.Yet all crops all do not have the acceptable dwarfing mutation of available commercialization.
Except using dwarf plant control plant height, routinely synthesis of chemicals is applied to some important economically plant species, to reduce growth.The plant-growth regulator that is called as growth inhibitor is used for reducing the stem elongation in various crop, comprises cotton, grape vine, fruit tree, peanut, wheat and ornamental plant (such as rhododendron, chrysanthemum, laurustinus, poinsettia and many bed plants).All growth inhibitors commonly used all are the biosynthetic inhibitor of Plant hormones regulators,gibberellins, by reducing elongation restriction stem or shoot growth.In the U.S., the most widely used growth inhibitor is the first piperazine, and its registration is used for cotton.The benefit of using the first piperazine to obtain to cotton comprises that output increases, fallen leaves improve, stress tolerance improves, crop the ripening degree more homogeneous and the ability of early harvest more.In the past, the growth inhibitor daminozide was used for apple, grape and peanut in the U.S. with trade mark ALAR and KYLAR registration, but for it is forbidden in alimentary crop to the consideration of human health.Although the agricultural producer need substitute the product of daminozide, also there is not registration to be used for the growth inhibitor of grape, fruit tree and peanut in the U.S..Yet daminozide still is widely used in some non-edible plant species.
The molecular mechanism that discloses controlling plant process of growth such as cell fission and cell elongation might help to develop the new plant mutation of highly reduction and the novel method of the plant-growth of slowing down.These new plant mutation and method can be the peasant and the horticulturist provides using favourable alternative on the synthetic growth-inhibiting chemical environment.
The elongation of vegetable cell and organ is one of most critical parameters of plant-growth and growth.Yet the adjusting of this proterties in plant is a quite complicated process, because external factor and internal factor all influence it.Most important outside stimulus is a light, and its general pair cell elongation has inhibition or negative effect (Quail, P.H. (1995) Science 268:675-680; Kende etc., (1997) Plant Cell 9:1197-1210).The internal control of cell elongation is mediated by numerous chemical, and these chemical are commonly referred to as plant-growth regulator or hormone (Kende etc., (1997) PlantCell 9:1197-1210).In the plant hormone of classics, plant hormone and Plant hormones regulators,gibberellins (GA) all promote cell elongation, and phytokinin and dormin separately pair cell elongation all show negative effect (Kende etc., (1997) Plant Cell 9:1197-1210).Recently, identified the another kind of plant-growth regulator that is called as brassinolide, it also significantly promotes plant-growth (Yokota, T. (1997) Trends Plant Sci.2:137-143; Azpiroz etc., (1998) PlantCell 10:219-230; Choe etc., (1998) Plant Cell 10:231-243).Yet, still do not know plant hormone do individually or synergistically in order to control cell elongation mechanism.
A kind of mode of the mediated cell of promoting understanding elongation mechanism is to study the wherein damaged mutant of plant-growth this respect (Klee etc., (1991) Annu.Rev.Plant Physiol.PlantMol.Biol.42:529-551).Between most of plant species (comprising corn), identified numerous such mutant, wherein characterized the single gene mutation that influences plant height more than 25 kinds (Coe etc., (1988) are stated from: Corn ﹠amp; Corn Improvement, G.F.Sprague (editor) Madison, WI; Sheridan, W.F. (1988) Annu.Rev.Genet.22:353-385).It is relevant that these Dwarf Mutant are considered to GA, this mainly is because GA is unique plant hormone, its effect of regulating the corn height is confirmed (Phinney etc., (1985) Curr.Top.Plant Biochem.Physiol.4:67-74 convincingly; Fujioka etc., (1988) Proc.Natl.Acad.Sci.USA 85:9031-9035).Reactive and the reactive two class mutant of non-GA of GA in such maize mutant body, have been found.Although cloned the gene of the reactive mutant of numerous GA, and find to relate to GA biosynthesizing (Bensen etc., (1995) Plant Cell 7:75-84; Winkler etc., (1995) Plant Cell 7:1307-1317), but know little about it about defects property in the reactive maize mutant body of non-GA.
DELLA albumen is the main idea of Plant hormones regulators,gibberellins (GA) signal transduction cascade, as the negative conditioning agent (Silverstone etc., (2001) PlantCell 10:155-169) of the GA reaction of degrading in the presence of the GA concentration that promotes.DELLA territory albumen is interesting especially, reason is the Plant hormones regulators,gibberellins insensitivity dwarfing phenotype of their gain-of-function mutation body, these mutant partly cause " Green Revolution " (Peng etc., (1999) Nature400:256-261) by their harvesting wheat exponential increase.Sudden change in the terminal DELLA structural domain of N-often causes dominance GA-insensitivity phenotype (Silverstone etc., (2001) Plant Cell 10:155-169 by the stability that should bear conditioning agent that greatly increases the GA signal transduction; Gubler etc., (2002) PlantPhysiol.129:191-200; Itoh etc., (2002) Plant Cell 14:57-70).Recently, people such as Griffiths ((2006) Plant Cell 18:3399-3414) show that terminal I district of N-and II district are that DELLA albumen and Arabidopis thaliana (Arabidopsis) GID1a interaction are necessary.The terminal sudden change of C-in conservative GRAS structural domain causes afunction (Dill etc. usually, (2004) Plant Cell 16:1392-1405), composing type GA growth response phenotype be it should be noted that, overgrown with weeds blue or green (Brassica rapa) mutant Brrgal-d (Muangprom etc. that identify recently, (2005) Plant Physiol.137:931-938) and except the barley slnlc mutant (Gubler etc., (2002) PlantPhysiol.129:191-200).
For catching up with the demand that agriculture production increases,, need improve the fresh target of agronomy feature for the agricultural plants of genetic engineering.Relate in plant control cell fission and elongation proteic encoding gene separate and characterization of molecules will provide the fresh target of operation for the Agricultural Scientist.
Summary of the invention
Be provided for expressing in plant the composition and the method for the proteic gene of DELLA of encoding wild type and variant form, described DELLA albumen is by corn (Zea mays) D9 (Zm-D9) genes encoding.Said composition contains the proteic isolating polynucleotide molecule of Zm-D9 of encoding wild type and variant form.Said composition also comprises the isolating polynucleotide molecule of corn D9 gene.Polynucleotide molecule of the present invention for example can be used in transforming plant that the Zm-D9 of wild-type and variant form is proteic to be organized preferred expression or constitutive expression, be used for Antisense Suppression Zm-D9 gene and be used to separate the proteic homology polynucleotide molecule of encoding D ELLA.These polynucleotide molecules are used for changing the method for plant-growth (especially stem of plant and root growth), more particularly, are used for reducing or increasing the method for plant height.In one embodiment of the invention, described polynucleotide molecule is used to produce dwarf plant.
The expression cassette that contains polynucleotide molecule of the present invention is provided.Provide in addition and transform plant, its plant tissue, vegetable cell and seed.Isolating albumen by polynucleotide molecule coding of the present invention is provided.
The accompanying drawing summary
Fig. 1 illustrates isolating non-GA3 response type plant of Zm-D9MUT1 allelotrope (left side) and response type plant (right side).
Fig. 2 illustrates corn dwarfing 8 and downgrades the chromosomal localization of 9 genes.The analysis PCR of oat addition line shows, with predict by genetic mapping the same, in fact the Zm-D9 gene of inferring is positioned on the maize chromosome 5.This gene is found and is in and the different position of positive control Zm-D8PCR product (known its is positioned on the karyomit(e) 1).
Fig. 3 A-3F illustrates the proteic Subcellular Localization of corn DELLA that merges with AC-GFP1 (cyan multitube jellyfish (Aequoreacoerulescens) GFP).Fig. 3 A is that DSRED (Coral (Discosoma sp.) red fluorescent protein) expresses contrast.Fig. 3 B is Zm-D8:ACGFP1.Fig. 3 C be A and B merge.Fig. 3 D is that DSRED expresses contrast.Fig. 3 E is Zm-D9:ACGFP1.Fig. 3 F is the merging of D and E.The green bar rod is indicated 10 μ m in the drawings.
Fig. 4 illustrates the environmental T2 plant of back 56 days Arabidopis thaliana (Arabidopsis thaliana) Columbia that germinates, and it contains the corn DELLA cDNA by the MS-S2a promoters driven.From left to right: MS-S2A PRO::GUS; MS-S2A PRO::ZM-D8; MS-S2APRO::ZM-D9; MS-S2A PRO::MUT1ZM-D9; MS-S2A PRO::ZM-D8MPL; With MS-S2A PRO::ZM-D8MUT.
The representativeness that Fig. 5 illustrates Arabidopis thaliana T1 plant is split flower, and it contains the corn DELLA cDNA by the MS-S2a promoters driven.By removing two petals and two sepals in above the spending.
Fig. 6 is the aminoacid sequence comparison of Zm-D9 (SEQ ID NO:2) and the proteic aminoacid sequence of MUT1Zm-D9 (SEQ ID NO:4).
Fig. 7 be corn (ZM), Arabidopis thaliana (AT), turnip (BR), barley (Hordeumvulgare) (HV), rice (Oryza sativa) (OS) and the proteic multiple aminoacid sequence comparison of the DELLA of wheat (rht-D1a/b).
Fig. 8 is the nucleotide sequence comparison of the nucleotide sequence of Zm-D9 (SEQ ID NO:1) and MUT1Zm-D9 (SEQ ID NO:3).
Fig. 9 for coding corn (ZM), Arabidopis thaliana (AT), turnip (BR), barley (Hordeumvulgare) (HV), rice (Oryza sativa) (OS) and the multiple nucleotide sequence of the proteic nucleotide sequence of DELLA of wheat (rht-D1a/b) compare.
Figure 10 provides d8 and d9 gene relative expression's level (representing with the ppm) (Brenner etc. in 32 different tissues that obtain through Lynx MPSS system and the corn of etap, (2000) PNAS 97:1665-1670 and Brenner etc., (2000) Nat Biotechnol18:630-634).Vertical line is divided chart according to the organ that obtains sample.
Figure 11 provides part d9 and D9 to cross the threshold and has cloned (entry clone) collection of illustrative plates, and this collection of illustrative plates has shown the structural domain exchange mosaic of being produced.A-has shown amino acids coding difference in d9 and the ABC of part collection of illustrative plates of cloning of D9 and each indicating area.The aminoacid sequence of d9 INDEL is SGSGSGQPTDASPPA (SEQ ID NO:7).MUT1 D9 INDEL aminoacid sequence is QPTDASSPAAG (SEQ ID NO:8).B-has shown the chimeric part collection of illustrative plates based on d9 allelotrope (white portion), and MUT1 D9 section is a grey.C-has shown the chimeric part collection of illustrative plates based on MUT1 D9 allelotrope (gray area), and the d9 section is a white.
Figure 12 has described the T2 arabidopsis thaliana in detail in the morphological data (Boyes etc., (2001) Plant Cell 13:1499-1510) in vegetative period 8.00, and this plant is by natural d8 of MS-S2A promoter expression and the allelic cDNA of d9.It is not remarkable different group each other that the subscript letter representation is analyzed in 95% confidence level according to LSD.Data are the mean value of 8 replicate(determination)s (replicates) of 4 independent transformation events.
Figure 13 provides the data of the relevant transition of blooming in Arabidopis thaliana T2 and GS3xGaspe Flint corn T0 plant.It is not remarkable different group each other that the subscript letter representation is analyzed in 95% confidence level according to LSD.These data are the mean value of 8 replicate(determination)s of 4 independent eventss.The corn data are obtained by 1 replicate(determination) of 25 independent transformation events of each construct.
Figure 14 has described the morphology and the flowering time data of d9/D9 structural domain exchange T1 Arabidopis thaliana in detail.Notice that it is not remarkable different group each other that the subscript letter representation is analyzed in 95% confidence level according to LSD.ALT=changes; The polymorphism that allelic phenotype relation is exchanged changes, and makes otherness no longer remarkable.REV=reverses; Polymorphism makes allelic phenotype relation produce statistics and reverses significantly.The MS-S2A promotor is used to drive all above encoding sequences (CDS).Data are the mean value of 16.3 independent transformation events of each construct.Main 8.00 o'clock vegetative period detect the lotus throne diameter, highly, silique length and silique width (Boyes etc., (2001) Plant Cell 13:1499-1510).The lotus throne number of sheets when detecting to the fate of blooming and blooming main 5.10 o'clock vegetative period.
Sequence table
List in standard alphabet abbreviation and the demonstration of amino acid whose 3 alpha codes that nucleotides in the subsidiary sequence table and amino acid sequence use nucleotide base. Nucleotide sequence is followed conventional criteria, (namely arranges from left to right) also forward from 5 of sequence ' end beginning and advances to 3 ' end. Only shown a chain of each nucleotide sequence, but be appreciated that any mentioning shows that the chain part includes complementary strand. Amino acid sequence is followed conventional criteria, from the amino terminal of sequence begin and forward (be every row from left to right) advance to carboxyl terminal.
List in standard alphabet abbreviation and the demonstration of amino acid whose 3 alpha codes that nucleotides in the subsidiary sequence table and amino acid sequence use nucleotide base. Nucleotide sequence is followed conventional criteria, and from the beginning of 5 of sequence ' end and forward (be every row from left to right) advances to 3 ' end. Only shown a chain of each nucleotide sequence, but be appreciated that any mentioning shows that the chain part includes complementary strand. Amino acid sequence is followed conventional criteria, from the amino terminal of sequence begin and forward (be every row from left to right) advance to carboxyl terminal.
SEQ ID NO:1 has shown the complete encoding sequence of the wild-type allele of Zm-D9 gene.
SEQ ID NO:2 has shown the Zm-D9 aminoacid sequence by SEQ ID NO:1 coding.
SEQ ID NO:3 has shown the complete encoding sequence of the wild-type allele that does not have the Zm-D9 of terminator codon gene.The Nucleotide 1-1875 of SEQ ID NO:3 is corresponding to the Nucleotide 1-1875 of SEQ IDNO:1.If needed, nucleotide sequence or any other that terminator codon can be added SEQ ID NO:3 do not have 3 ' end of the encoding sequence of terminator codon.Such terminator codon comprises for example TAA, TAG and TGA.
SEQ ID NO:4 has shown the complete encoding sequence of the mutation allele (MUT1) of Zm-D9 gene.
SEQ ID NO:5 has shown the Zm-D9 aminoacid sequence by SEQ ID NO:4 coding.
SEQ ID NO:6 has shown the complete encoding sequence of the mutation allele (MUT1) that does not have the Zm-D9 of terminator codon gene.The Nucleotide 1-1866 of SEQ ID NO:6 is corresponding to the Nucleotide 1-1866 of SEQ ID NO:4.
The aminoacid sequence of d9 INDEL is SEQ ID NO:7.The aminoacid sequence of D9 INDEL is SEQ ID NO:8.
Detailed Description Of The Invention
The present invention relates to change the composition and the method for plant-growth.Described composition comprises the isolating polynucleotide molecule of the complete encoding sequence of the wild-type that contains corn D9 gene (it is called as the Zm-D9 gene at this paper) and mutation allele.Although on genetics, described Zm-D9 (Winkler and Freeling (1994) Planta 193:341-348), also on molecular level, do not characterize this gene in the past.The present invention also provides the proteic aminoacid sequence of DELLA by the wild-type of Zm-D9 and mutation allele coding.Method of the present invention relates to uses the proteic polynucleotide molecule of corn DELLA encoding wild type and variant form, that encoded by Zm-D9 to transform plant.
Polynucleotide molecule of the present invention is used in the growth that changes stem or bar (stalk) in the plant, so that produce the conversion plant of stem or bar change.More particularly, described polynucleotide molecule can be used for reducing or increasing the height of stem or bar, so that produce the plant of plant height or plant type reduction or raising.Polynucleotide molecule also is used for transforming structure and other agronomic traits that plant changes root in the mode of needs.Such agronomic traits include but not limited to set seeds (seed set), seed number, the output that can gather in the crops, spike length degree, drought tolerance, water application efficiency, nitrogen use efficiency, lodging resistance, leaf area, nitrogen accumulation, photosynthetic capacity and carbon and nitrogen partition.Therefore, the invention provides conversion plant, vegetable cell, plant tissue and seed.Described polynucleotide molecule also be used for making up subsequent transformation go into the expression cassette of target plant and vegetable cell, as the probe that separates other D9 sample gene, be used as molecule marker etc.
Composition of the present invention comprises natural wild-type and MUT1 Zm-D9 polynucleotide molecule and variant and fragment.Said composition also comprises the corresponding aminoacid sequence of natural wild-type and MUT1 Zm-D9 polynucleotide molecule, and the fragment of these aminoacid sequences and variant.The Zm-D9 sequence is shown in SEQ ID NO:1-6.Nucleotide sequence or corresponding antisense sequences are used for regulating the proteic expression of Zm-D9 of plant or vegetable cell.That is to say that described encoding sequence can be used for increasing expresses, and antisense sequences can be used for reducing expression.
Known DELLA albumen is regulated the vegetable cell elongation, can be used for Change Example such as vegetable cell elongation, plant height and root elongation.Referring to Itoh etc., (2002) Plant Cell 14:57-70; Achard etc., (2003) Plant Cell 15:2816-2825; And Fu and Harberd (2003) Nature421:740-743; All these documents all are hereby incorporated by.
Therefore, polynucleotide molecule of the present invention is used to change the method for plant-growth.In one embodiment of the invention, polynucleotide molecule of the present invention is used for changing the method for plant-growth.For this reason, polynucleotide molecule of the present invention can be used for numerous plant promoters in any expression cassette or polynucleotide constructs that effectively is connected.Can be included but not limited to following one or multinomial by the plant-growth aspect that the inventive method influences: plant height; Stem or bar height; One or more aspects of axis or bar metabolic activity, root architecture (for example the root degree of depth, root angle, root branch, tip of a root quantity, joint position root diameter, joint position root volume, root metabolic activity); The size of cell and organ, shape and quantity; Cell fission speed; Cell elongation speed; The growth velocity of plant, its organ, tissue and cell; The time and the position of organ growth (organ initiation); Life-span or the like.
Method of the present invention comprises with polynucleotide molecule conversion plant of the present invention, to reduce plant-growth.In one embodiment of the invention, use and in plant, drive the effective MUT1 Zm-D9 polynucleotide molecule that is connected of expression promoter and transform plant.This polynucleotide molecule contains the nucleotide sequence that is shown in SEQ ID NO:4 or 6, the nucleotide sequence that coding is shown in the polypeptide of SEQ ID NO:5, the perhaps fragment of any of these polynucleotide molecule or variant, described fragment or reservation of variant encoded polypeptides and the essentially identical biological activity of natural MUT1 Zm-D9 polypeptide.By in plant, expressing this MUT1 Zm-D9 polynucleotide molecule, can produce plant-dwarf plant that plant type reduces.
Therefore, method of the present invention is used to produce the dwarfing mutation of crop.Obtain to have the dwarfing crop of the agronomic traits of improvement by these methods, the agronomic traits of described improvement is for example for the lodging possibility descends, water application efficiency increases, life cycle descends, results efficient increases and the output of per unit area increases.
It is little that described " dwarfing " means atypia ground.Described " dwarf plant " means the little plant in atypia ground.Generally speaking, the plant type of such " dwarf plant " or height by the plant type of typical plant or highly reduce about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, more than 60%.Stem, bar or dried length descended when generally speaking, but non-exclusive ground, such dwarf plant was characterised in that and compares with typical plant.
The present invention includes isolating or basic purified polynucleotides molecule or protein composition.The polynucleotide molecule of " separation " or " purifying " or albumen or its biologically-active moiety do not contain usually with the polynucleotide molecule that exists in its natural surroundings basically or in fact or albumen is followed or interactional component.Therefore, when producing by recombinant technology, the polynucleotide molecule of isolated or purified or albumen are substantially free of other cell material or substratum, perhaps are substantially free of precursor or other chemical when chemosynthesis.The polynucleotide molecule of " separation " preferably be not contained in this polynucleotide molecule from biological gene group DNA in the sequence (promptly being positioned at 5 of these polynucleotide ' and 3 ' terminal sequence) (being preferably albumen coded sequence) of natural these polynucleotide of side joint.For example, in different embodiments, isolating polynucleotide molecule can contain the following nucleotide sequence that is less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb or 0.1kb: it is these polynucleotide of natural side joint in the cell genomic dna that these polynucleotide are originated.The protein that is substantially free of cellular material comprises having the protein product that is less than about 30%, 20%, 10%, 5% or 1% (with dry weight basis) contaminating protein.When albumen of the present invention or its biologically-active moiety produce for reorganization, preferably provide the precursor or the non-target protein chemical that are less than about 30%, 20%, 10%, 5% or 1% (with dry weight basis) in the substratum.
The present invention also comprises disclosed polynucleotide molecule and the fragment and the variant of encoded protein thus.Described " fragment " is meant that part polynucleotide molecule or partial amino-acid series reach encoded protein thus.Polynucleotide molecule fragment codified keeps wild-type disclosed herein and the proteic biological activity of MUT1 Zm-D9 and therefore keeping the Plant hormones regulators,gibberellins response type suppresses active protein fragments.Perhaps, as do not encode the usually fragment albumen of retains biological activity of the polynucleotide molecule fragment of hybridization probe.Therefore, the scope of nucleotide sequence fragment can be at least about 20 Nucleotide, about 50 Nucleotide, about 100 Nucleotide, invents proteic total length polynucleotide molecule until code book.
Except as otherwise noted or by apparent in the literary composition, otherwise term " Zm-D9 " is intended to comprise the wild-type that contains the Zm-D9 gene and the polynucleotide molecule of MUT1 allelotrope and fragment and variant.Preferably, the Zm-D9 albumen of allelic these fragments of the wild-type of Zm-D9 gene and MUT1 and variant coding keeps as total length wild-type disclosed herein or the proteic biological activity of MUT1Zm-D9.Term " Zm-D9 " can also be used in reference to by Zm-D9 polynucleotide molecule encoded protein of the present invention at this paper.
Encode at least 15,25,30,50,100,150,200,250,300,350,400,450,500,550 or 600 continuous amino acids of Zm-D9 polynucleotide molecule fragment coding of the proteic biologically-active moiety of Zm-D9 of the present invention or the amino acid sum that in total length wild-type of the present invention or MUT1Zm-D9 albumen, exists (for example SEQ ID NO:2 and 5 is respectively 625 and 622 amino acid).Zm-D9 polynucleotide molecule fragment as hybridization probe or PCR primer does not need the proteic biologically-active moiety of Zm-D9 of encoding usually.
Therefore, Zm-D9 polynucleotide molecule fragment can encoding wild type or the proteic biologically-active moiety of MUT1Zm-D9, and perhaps it can be used as the fragment of hybridization probe or PCR primer for the method for using following discloses.The proteic biologically-active moiety of Zm-D9 can be prepared as follows: separate one of them Zm-D9 polynucleotide molecule of the present invention, the proteic part of Zm-D9 (for example passing through in-vitro recombination expression), and estimate the activity of the proteic Zm-D9 part of Zm-D9.For the polynucleotide molecule of Zm-D9 nucleotide sequence fragment comprises at least 16,20,50,75,100,150,200,250,300,350,400,450,500,550,600,650,700,800,900,1,000,1,100,1,200,1,300,1,4001,500,1,600,1,700,1,800 or 1,850 continuous nucleotides, or the few nucleotide that in total length Zm-D9 polynucleotide disclosed herein, exists (for example being respectively 1878,1875,1869 and 1866 Nucleotide) for SEQ ID NO:1,3,4 and 6.
" variant " is meant similar basically sequence.For polynucleotide molecule, variant is that the one or more inner site in natural polynucleotide molecule comprises the disappearance of one or more Nucleotide and/or the variant of interpolation, and/or the one or more sites in natural polynucleotide comprise the variant of the replacement of one or more Nucleotide." natural " polynucleotide molecule used herein or polypeptide contain natural nucleotide sequence or aminoacid sequence respectively.For polynucleotide molecule, conservative variant comprises that those are owing to genetic code annexs the encode sequence of one of them Zm-D9 amino acid sequence of polypeptide of the present invention of property.Natural allele variant such as these can use well-known Protocols in Molecular Biology to identify, for example adopts the polymerase chain reaction (PCR) and the hybridization technique of following general introduction.The variant polynucleotide molecule also comprises the synthetic polynucleotide of deriving, for example by for example using that site-directed mutagenesis produces but still proteic those polynucleotide of the Zm-D9 of the present invention that encodes.In general, by the sequence alignment program and the parametric measurement of its place's description of this paper, the variant of specific polynucleotide molecule of the present invention and specific polynucleotide molecule have at least about the sequence identity more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
The variant of specific polynucleotide molecule of the present invention (being the reference polynucleotide) can also be estimated by the sequence identity percentage between the polypeptide that compares variant polynucleotide molecule encoded polypeptides and reference polynucleotide encoding.Therefore, for example, disclosing encodes has the polynucleotide molecule that separates of the percentile polypeptide of given sequence identity with the polypeptide of SEQ ID NO:2 and/or 5.Can utilize sequence alignment program that its place of this paper describes and parameter to calculate sequence identity percentage between any two polypeptide.Right for given arbitrarily polynucleotide molecule of the present invention, when relatively the common sequence identity percentage of two polypeptide of their codings is estimated them, the sequence identity percentage between two coded polypeptides is at least about sequence identity more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
" variant " albumen is meant by the disappearance of the one or more inner site in native protein or adds one or more amino acid and/or the one or more sites in native protein have one or more aminoacid replacement and the albumen of deriving and from native protein.Some misfolded proteins that the present invention includes is to have bioactively, that is to say that they still have required native protein biological activity, wild-type promptly as described herein or MUT1Zm-D9 protein-active.These variants can be obtained by for example genetic polymorphism or manual operation.It locates described sequence alignment program and parametric measurement by this paper, and the aminoacid sequence of proteic biological activity variant of natural Zm-D9 of the present invention and native protein has at least about the sequence identity more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.The proteic biological activity variant of the present invention can be less to 1-15 amino-acid residue with this proteic difference, and few, for example 6-10 amino-acid residue individual to 1-10 is few to 5 amino-acid residues, few to 4,3,2 and even 1 amino-acid residue.
Albumen of the present invention can change by comprising aminoacid replacement, disappearance, brachymemma and being inserted in interior several different methods.It generally is known in the art being used for these method of operating.For example, proteic aminoacid sequence variant of Zm-D9 and fragment can prepare by dna mutation.The method that is used for the change of mutagenesis and polynucleotide is well-known in this area.Referring to for example Kunkel (1985) Proc.Natl.Acad.Sci.USA 82:488-492; Kunkel etc., (1987) Methods in Enzymol.154:367-382; United States Patent (USP) the 4th, 873, No. 192; Walker and Gaastra edit, (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York), and the reference of wherein quoting.Be found in Dayhoff etc. about the guidance that does not influence the bioactive suitable aminoacid replacement of target protein, (1978) Atlas of Protein Sequence and Structure (Natl.Biomed.Res.Found., Washington, D.C.) model, the document is attached to herein by reference.The conservative replacement, may be best, and for example an amino acid is replaced by the amino acid that another has similar characteristics.
Therefore, gene of the present invention and polynucleotide molecule had both comprised native sequences, also comprised mutant form.Equally, albumen of the present invention comprises native protein and variant and modified forms.These variants still have required wild-type or MUT1 Zm-D9 activity.Obviously, the sudden change that produces in the DNA of coding variant necessarily can not make sequence be in outside the frame, and preferably can not form the complementation district that can produce secondary mRNA structure.Referring to EP patent application publication number 75,444.
The thorough change that disappearance, insertion and the replacement of the protein sequence that expection this paper is included can not produce protein specificity.But, when the definite effect that is difficult to predict replacement, disappearance in advance or inserts, those of skill in the art will recognize that and to estimate these effects by conventional screening assay.That is to say, can change that for example monitoring is estimated this activity with the stem of Zm-D9 polynucleotide molecule plant transformed of the present invention and/or the change of root elongation by plant or the root morphology of transgenic plant.Referring to for example following embodiment 1 and Fig. 4.
The polynucleotide molecule of variation and albumen also comprise and deriving from such as the sudden change of DNA reorganization and the sequence and the albumen of recombination method.Adopt this method, can operate one or more different Zm-D9 encoding sequences, to obtain having the new Zm-D9 that needs characteristic.Can produce the recombination of polynucleotide library from correlated series polynucleotide group in this way, the sequence area that described polynucleotide group is contained has significant sequence identity, and can be in external or body homologous recombination.For example, use this method, the sequence motifs in coding object construction territory can be reorganized between Zm-D9 gene of the present invention and other known Zm-D9 gene, to obtain the target property that coding the has improvement (K of increase with regard to enzyme for example m) proteic new gene.The strategy of this DNA reorganization is known in this area.Referring to for example Stemmer (1994) Proc.Natl.Acad.Sci.USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etc., (1997) Nature Biotech.15:436-438; Moore etc., (1997) J.Mol.Biol.272:336-347; Zhang etc., (1997) Proc.Natl.Acad.Sci.USA 94:4504-4509; Crameri etc., (1998) Nature 391:288-291; With United States Patent (USP) the 5th, 605,793 and 5,837, No. 458.
Polynucleotide molecule of the present invention can be used for from other biology, particularly other plant, more particularly other monocotyledons separates corresponding sequence.In this way, can use, these sequences be identified based on the sequence homology of itself and the listed sequence of this paper such as methods such as PCR, hybridization.The sequence that the present invention includes the complete Zm-D9 sequence listed based on itself and this paper or its variant and fragments sequence identity and separate.These sequences are included as the sequence of the lineal homologue of disclosed sequence." lineal homologue " is meant the gene that derives from common ancestor's gene, the result that it forms as species and being present in the different plant species.The nucleotide sequence of the gene in being present in different plant species and/or its encoded protein sequence share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, during the sequence identity more than 99%, these genes are considered to lineal homologue.The function of lineal homologue high conservative normally between plant.Therefore, the present invention includes coding Zm-D9 albumen and under stringent condition with the polynucleotide molecule that separates of Zm-D9 sequence disclosed herein or its variant or fragment hybridization.
In PCR method, but the design oligonucleotides primer is used for PCR reaction, with by cDNA or the genomic dna amplification corresponding DNA sequence extracted from any target plant.Design PCR primer and PCR clone's method generally is known in the art, be disclosed in Sambrook etc., and (1989) Molecular Cloning:A Laboratory Manual (the 2nd edition, ColdSpring Harbor Laboratory Press, Plainview, New York).In addition referring to editors such as Innis, (1990) PCR Protocols:A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand edit, (1995) PCRStrategies (Academic Press, New York); And Innis and Gelfand edit (1999) PCR Methods Manual (Academic Press, New York).Known PCR method includes but not limited to use the method for pairing primer, nested primers, monospecific primer, degenerated primer, gene-specific primer, carrier specificity primer, part mispairing primer etc.
In hybridization technique, use all or part of known polynucleotide molecule as probe, itself and other the corresponding polynucleotide selective cross that is present among the cloned genomic dna fragment of selected biology or cDNA fragment (being genome or the cDNA library) group.Hybridization probe can be genomic DNA fragment, cDNA fragment, RNA fragment or other oligonucleotide, and available such as 32But the detection moiety of P or any other detectable label substance markers.Therefore, for example, can prepare hybridization probe based on the synthetic oligonucleotide of Zm-D9 polynucleotide of the present invention by mark.The method for preparing hybridization probe and construction cDNA and genomic library is well known in the art, be disclosed in Sambrook etc., (1989) Molecular Cloning:A LaboratoryManual is (the 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, NewYork).
For example, complete Zm-D9 polynucleotide molecule disclosed herein or its one or more parts can as can with the probe of corresponding Zm-D9 polynucleotide molecule and messenger RNA(mRNA) specific hybrid.In order to realize specific hybrid under multiple condition, these probes are included in the unique sequences in the Zm-D9 polynucleotide sequence, and it is preferably at least about 10 Nucleotide long, most preferably are at least about 20 Nucleotide long.These probes can be used for the corresponding Zm-D9 polynucleotide molecule by the selected plant of pcr amplification.This technology can be used for from the plant of needs separating other encoding sequence, or determines the situation that exists of encoding sequence in the plant as diagnostic assay.Hybridization technique comprises that dull and stereotyped DNA library is (perhaps for plaque or for bacterium colony; Referring to for example Sambrook etc., (1989) Molecular Cloning:ALaboratory Manual (the 2nd edition, Cold Spring Harbor Laboratory Press, Plainview, New York)) screening by hybridization.
The hybridization of these sequences can be carried out under stringent condition.The hybridization degree that described " stringent condition " or " stringent hybridization condition " are meant probe and its target sequence is higher than on detecting and the condition of the hybridization degree of other sequence (for example be at least background 2 times).Stringent condition is a sequence dependent, under varying environment with variant.By control hybridization severity and/or wash conditions, can identify and probe 100% complementary target sequence (homology detection).Perhaps, can adjust stringent condition, to allow in sequence, to exist some mispairing, so that detect similarity (allos detection) than low degree.In general, probe length is less than about 1000 Nucleotide, and length is most preferably less than about 500 Nucleotide.
Typically, stringent condition is meant such condition: wherein salt concn is less than about 1.5M Na ion, typically be about 0.01-1.0M Na ionic concn (or other salt), pH7.0 to 8.3, temperature is at least about 30 ℃ to short probe (for example 10-50 Nucleotide), and long probe (for example greater than 50 Nucleotide) is at least about 60 ℃.Stringent condition can also be realized such as the destabilizing agent of methane amide by adding.Exemplary low stringency condition comprises with the damping fluid that contains 30-35% methane amide, 1M NaCl, 1%SDS (sodium lauryl sulphate) in 37 ℃ of hybridization, and with 1X-2X SSC (20X SSC=3.0M NaCl/0.3M trisodium citrate) in 50-55 ℃ of washing.Exemplary medium stringent condition comprises with 40-45% methane amide, 1M NaCl, 1%SDS in 37 ℃ of hybridization, and with 0.5X-1X SSC in 55-60 ℃ of washing.Exemplary high stringent condition comprises with 50% methane amide, 1M NaCl, 1%SDS in 37 ℃ of hybridization, and with 0.1XSSC in 60-65 ℃ of washing.Randomly, lavation buffer solution can comprise about 0.1% to about 1%SDS.The hybridization time length generally is lower than about 24 hours, about 4 hours to about 12 hours usually.The washing time length is at least and is enough to reach balance time length.
Specificity becomes with post-hybridization washing usually, and key factor is the ionic strength and the temperature of final washing soln.Assorted and the body for DNA-DNA, T mCan use the equation of Meinkoth and Wahl (1984) Anal.Biochem.138:267-284: T m=81.5 ℃+16.6 (logM)+0.41 (%GC)-0.61 (% methane amide)-500/L estimate; Wherein M is the volumetric molar concentration of univalent cation, and %GC is the per-cent of guanine and cytidylic acid(CMP) among the DNA, and the % methane amide is the methane amide per-cent in the hybridization solution, and L is the base pair length of assorted and body.T mThe temperature of the probe hybridization that is meant 50% complementary target sequence and mates fully (under ionic strength of determining and pH).1% mispairing is whenever arranged, T mJust reduce about 1 ℃; Therefore, can adjust T m, hybridization and/or wash conditions, with the sequence hybridization of required identity.For example, if seek the sequence of 〉=90% identity, then can be with T mReduce by 10 ℃.Usually, under ionic strength of determining and pH, select the pyrolysis chain temperature (T of bit sequencing row and its complement m) low about 5 ℃ stringent condition.But very Yan Ge condition can be used specific heat melting temperature(Tm) (T m) low 1,2,3 or 4 ℃ hybridization and/or washing; Medium stringent condition can use specific heat melting temperature(Tm) (T m) low 6,7,8,9 or 10 ℃ hybridization and/or washing; Low stringency condition can use at specific heat melting temperature(Tm) (T m) low 11,12,13,14,15 or 20 ℃ hybridization and/or washing.Those skilled in the art will appreciate that, use this equation, hybridization and washing to form and required T m, the severity of having described hybridization and/or washing soln inherently changes.If required mispairing degree makes T mBe lower than 45 ℃ (aqueous solution) or 32 ℃ (formamide soln), then preferably increase SSC concentration, so that can use higher temperature.The extensive guide of nucleic acid hybridization sees Tijssen (1993) LaboratoryTechniques in Biochem istry and Molecular Biology-Hybridization withNucleic Acid Probes, I portion, the 2nd chapter, (Elsevier, New York) and editor such as Ausubel, (1995) Current Protocols in Molecular Biology, the 2nd chapter, (GreenePublishing and Wiley-Interscience, New York).Referring to Sambrook etc., (1989) Molecular Cloning:A Laboratory Manual (the 2nd edition, Cold Spring HarborLaboratory Press, Plainview, New York).
Following term is used to describe the sequence relation between two or more polynucleotide or the polypeptide: (a) " reference sequences ", (b) " comparison window ", (c) " sequence identity " and (d) " sequence identity percentage ".
(a) " reference sequence " used herein is to be used as sequence comparison basis sequencing row really.Reference sequence can be part or all of particular sequence; For example, as the section of full-length cDNA or gene order, perhaps complete cDNA or gene order.
(b) " comparison window " used herein is meant the continuous and particular section of polynucleotide sequence, wherein the polynucleotide sequence in the comparison window is compared to comprise with reference sequence (it does not comprise interpolation or disappearance) and is added or disappearance (being the room), two polynucleotide are carried out the optimum comparison.Usually, comparison window length is at least 20 continuous nucleotides, randomly can be 30,40,50,100 or longer.It will be understood by those skilled in the art that to avoiding owing in polynucleotide sequence, contain and have vacant position and reference sequence has high similarity, introduce gap penalty usually, and by deducting in the coupling number.
The sequence alignment method that is used for comparison is well-known in this area.Therefore, the sequence identity percentage that can use mathematical algorithm to finish between any two sequences is measured.The limiting examples of these mathematical algorithms is Myers and Miller, the algorithm of (1988) CABIOS 4:11-17; Smith etc., the local alignment algorithm of (1981) Adv.Appl.Math.2:482; Needleman and Wunsch, (1970) the overall comparison algorithm of J.Mol.Biol.48:443-453: Pearson and Lipman, (1988) part of Proc.Natl.Acad.Sci.85:2444-2448 retrieval alignment algorithm: Karlin and Altschul, (1990) algorithm of Proc.Natl.Acad.Sci.USA 872264, by Karlin and Altschul, (1993) Proc.Natl.Acad.Sci.USA90:5873-5877 revises.
The computer realization of these mathematical algorithms can be used for sequence relatively, to determine sequence identity.These realizations include but not limited to: and the CLUSTAL in the PC/Gene program (can be from Intelligenetics, Mountain View, California obtains); GAP, BESTFIT, BLAST, FASTA and TFASTA in ALIGN program (version 2 .0) and the GCG Wisconsin Genetics software package the 10th edition (can be from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA obtains).Use the comparison of these programs can use default parameters to carry out.The CLUSTAL program fully is described in (1988) Gene 73:237-244 (1988) such as Higgins; Higgins etc., (1989) CABIOS 5:151-153; Corpet etc., (1988) NucleicAcids Res.16:10881-90; Huang etc., (1992) CABIOS 8:155-65 and Pearson etc., (1994) Meth.Mol.Biol.24:307-331.The ALIGN program is based on above-mentioned Myers and Miller, the algorithm of (1988).When with ALIGN program comparing amino acid sequence, can use PAM120 weighting residue table, room length point penalty 12 and gap penalty 4.Altschul etc., the blast program of (1990) J.Mol.Biol.215:403 are based on above-mentioned Karlin and Altschul, the algorithm of (1990).BLAST Nucleotide is retrieved available BLASTN program and is carried out, keep the score=100, word length=12 are to obtain inventing proteic nucleotide sequence homologous nucleotide sequence with code book.BLAST albumen is retrieved available BLASTX program and is carried out, keep the score=50, word length=3 are with the aminoacid sequence of acquisition with albumen of the present invention or homologous peptide.In order to obtain to be used for the comparison of having vacant position of comparison purpose, can be as Altschul etc., (1997) NucleicAcidsRes.25:3389 described use Gapped BLAST (in BLAST2.0).Perhaps, can use PSI-BLAST (in BLAST2.0) to carry out iterative searching and come edge relation far away between detection molecules.Referring to above-mentioned Altschul etc., (1997).When using BLAST, Gapped BLAST, PSI-BLAST, can use the default parameters of program (for example BLASTN is used for nucleotide sequence, and BLASTX is used for albumen) separately.Referring to www.ncbi.nlm.nih.gov.Also can compare by estimating manually.
Unless otherwise stated, otherwise sequence identity/similarity provided herein is meant the value of using the 10th edition GAP to use following parameter to obtain: the % identity of nucleotide sequence and % similarity are used room weighting 50, length weighting 3 and nwsgapdna.cmp marking matrix: the % identity of aminoacid sequence and % similarity are used room weighting 8, length weighting 2 and BLOSUM62 marking matrix; Or its any equivalence program.Described " equivalence program " means, when comparing with the corresponding comparison of the 10th edition generation of GAP, for any 2 sequences in question, produce the arbitrary sequence comparison program of comparison with identical Nucleotide or amino-acid residue coupling and same percentage sequence identity.
GAP uses Needleman and Wunsch, and the algorithm of (1970) J.Mol.Biol.48:443-453 is sought and made coupling number maximum and make the room count the comparison of two complete sequences of minimum.GAP considers all possible comparison and null position, and sets up the comparison with the minimum room of maximum match alkali cardinal sum.It allows to provide the room to introduce point penalty in coupling base unit and point penalty is extended in the room.It is favourable to each room of its insertion that GAP must make the coupling room introduce the point penalty number.If point penalty is extended greater than 0 in the room of selecting, then GAP must also make room length multiply by the room to extend point penalty favourable to each room of inserting.In GCG Wisconsin Genetics software package the 10th edition, the point penalty value is introduced in the room of acquiescence and room extension point penalty value is respectively 8 and 2 to protein sequence.For nucleotide sequence, it is 50 that point penalty is introduced in the room of acquiescence, and the room of acquiescence extension point penalty is 3.Introduce in the room and the integer representation that point penalty can be selected from 0-200 is extended in the room.Therefore, for example, introduce in the room and room extension point penalty can be for 0,1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60, more than 65.
GAP has represented a member of best sequence alignment family.This family has a lot of members, but other member does not have better quality.GAP shows the sequence alignment advantage of four aspects: quality, ratio, identity and similarity.Quality is maximized measuring for aligned sequences.Ratio is the base number of quality divided by shorter section.The identity percentage is the percentage of the symbol of actual match.The similarity percentage is the percentage of similarity sign.The symbol relative with the room is left in the basket.When certain to the marking matrix value of symbol during more than or equal to similarity threshold value 0.50, the record similarity.Used marking matrix is BLOSUM62 (referring to Henikoff and Henikoff (1989) Proc.Natl.Acad.Sci.USA 89:10915) in the 10th edition GCG Wisconsin Genetics software package.
(c) " the sequence identity " or " identity " under two polynucleotide or peptide sequence background used herein is meant that comparison obtains maximum to identical residue in seasonable two sequences in specific comparison window.When sequence identity percentage is used in reference to albumen, think that then the common difference in inconsistent residue position is that conserved amino acid replaces, in conserved amino acid replaces, amino-acid residue is replaced by the amino-acid residue that other has similar chemical property (for example electric charge or hydrophobicity), thereby does not change the functional performance of molecule.When sequence difference is conservative the replacement, can raise sequence identity percentage, to proofread and correct the conservative character that replaces.Because of being called as, the different sequence of these conservative replacements has " sequence similarity " or " similarity ".The method of carrying out this adjustment is that those skilled in the art are well-known.Typically, it comprises that replacement is recorded as partly rather than complete mispairing with guarding, thereby has improved sequence identity percentage.Therefore, for example, same amino acid be given keep the score 1 but not conservative the replacement be given under 0 the situation of keeping the score, conservative replacement is given keeping the score between 0 and 1.Conservative keeping the score of replacing can (Intelligenetics, Mountain View California) carry out and calculate by for example using the PC/GENE program.
(d) " sequence identity percentage " used herein is meant definite value by compare two optimum aligned sequences in comparison window, wherein the part of the polynucleotide sequence in the comparison window is compared to comprise with reference sequence (do not contain and add or disappearance) and is added or disappearance (being the room), to optimize the comparison of two sequences.Following calculating percentage: measure in two sequences and have the identical nucleic acid base or the positional number of amino-acid residue, obtain the matched position number, divided by the total positional number in the comparison window, and the result be multiply by 100, acquisition sequence identity percentage with this matched position number.
It is the polynucleotide that contain DNA that the application of term " polynucleotide " is not limited to the present invention.Persons skilled in the art will appreciate that polynucleotide can comprise the combination of ribonucleotide and ribonucleotide and deoxyribonucleotide.These deoxyribonucleotides and ribonucleotide comprise natural molecule and synthetic analogues these two.Polynucleotide of the present invention also comprise all sequences form, include but not limited to single stranded form, double chain form, hair clip, loop-stem structure etc.
Zm-D9 polynucleotide molecule of the present invention can provide in being used for the expression cassette that target plant Zm-D9 expresses.This expression cassette comprises 5 ' and the 3 ' regulating and controlling sequence that effectively is connected with Zm-D9 polynucleotide molecule of the present invention." effectively connect " the functional connection that is meant two or more elements.For example, the herbicide-tolerant polynucleotide molecule is a kind of functional connection with effective connection between the regulating and controlling sequence (being promotor), and it can make the herbicide-tolerant polynucleotide developed by molecule.Effectively the element that connects can be continuous or discontinuous.When being used in reference to the connection of two protein-coding regions, described effective connection is meant that the coding region is in the identical frame.Expression cassette can comprise at least one in addition and want cotransformation other gene in the biology.Perhaps, other gene can provide on a plurality of expression cassettes.Such expression cassette can provide a plurality of restriction sites and/or recombination site, in order to insert the Zm-D9 polynucleotide molecule, makes under its transcriptional control that is in control region.Expression cassette can contain marker gene selectively in addition.
Expression cassette is included in transcribing with translation initiation district (being promotor), Zm-D9 polynucleotide molecule of the present invention and transcribing with the translation termination district (being the terminator) of working in the plant by 5 '-3 ' direction of transcribing.Control region (being promotor, transcription regulatory region and translation termination district) and/or Zm-D9 polynucleotide molecule of the present invention to host cell or can be each other natural/similarly.Perhaps, control region and/or Zm-D9 polynucleotide molecule of the present invention are to host cell or can be allogenic each other.This paper is used for indicator sequence " allogenic " and is meant the sequence that derives from alien species, perhaps, if from same species, is then taken place significantly to change by its natural form aspect composition and/or genomic gene seat by the artificial interference of having a mind to.For example, the promotor that effectively is connected with heterologous polynucleotide molecules is from the species that are different from polynucleotide molecule source species, perhaps, if from identical/similar species, then one of them or the two take place significantly to change by its original form and/or genomic gene seat, perhaps promotor is not the natural promoter of the polynucleotide that effectively connect.Mosaic gene used herein comprises the encoding sequence that effectively is connected with transcription initiation region, and described transcription initiation region is allogenic for encoding sequence.
May be best although use allogeneic promoter to express described sequence, also can use the natural promoter sequence.These constructs can change the Zm-D9 expression level in plant or the vegetable cell.Therefore, can change the phenotype of plant or vegetable cell.
The terminator can be natural to transcription initiation region, target Zm-D9 polynucleotide molecule to effective connection can be natural, to plant host can be natural, perhaps can derive from another source (be external source or allogenic) with respect to promotor, target Zm-D9 polynucleotide molecule, plant host or its any combination.The terminator can obtain from the Ti-plasmids of Agrobacterium tumefaciems (A.tumefaciens) easily, for example octopine synthase and nopaline synthase terminator.In addition referring to Guerineau etc., (1991) Mol.Gen.Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon etc., (1991) Genes Dev.5:141-149; Mogen etc., (1990) Plant Cell 2:1261-1272; Munroe etc., (1990) Gene91:151-158; Ballas etc., (1989) Nucleic Acids Res.17:7891-7903 and Joshi etc., (1987) Nucleic Acids Res.15:9627-9639.
Under suitable situation, express in order in transforming plant, to increase, can optimize polynucleotide.That is to say that the codon of available plant optimization comes synthetic polyribonucleotides, express to improve.The argumentation of selecting about the preferred codon of host referring to for example Campbell and Gowri (1990) Plant Physiol.92:1-11.These methods can obtain from the field of synthetic plant optimization gene.Referring to for example United States Patent (USP) the 5th, 380,831 and 5,436, No. 391, and Murray etc., (1989) Nucleic Acids Res.17:477-498, described document is attached to herein by reference.
Known in cell host other sequence modification of reinforcing gene expression.These modifications comprise sequence, exon-intron splice site signal, swivel base increment tumor-necrosis factor glycoproteins of removing the false poly-adenosine signal of coding and the sequence that may fully characterize disadvantageous other this class of genetic expression.The known of expressing in host cell by reference calculates, and the G-C content of sequence can be adjusted to the mean level (ML) of given cell host.When possible, sequence is modified, with the hair clip secondary mRNA structure of avoiding predicting.
Expression cassette can contain 5 ' leader sequence in addition.This class leader sequence can be used for strengthening translation.The translation leader sequence is known in the art, comprising: picornavirus leader sequence, for example EMCV leader sequence (encephalomyocarditis 5 ' non-coding region) (Elroy-Stein etc., (1989) Proc.Natl.Acad.Sci.USA 86:6126-6130); The marmor upsilon leader sequence, TEV leader sequence (marmor erodens) (Gallie etc. for example, (1995) Gene 165 (2): 233-238), MDMV leader sequence (maize dwarf mosaic virus) (Virology 154:9-20), and human immunoglobulin heavy chain conjugated protein (BiP) (Macejak etc., (1991) Nature 353:90-94); The untranslated leader of alfalfa mosaic virus glutelin mRNA (AMV RNA 4) (Jobling etc., (1987) Nature 325:622-625); Tobacco mosaic virus (TMV) leader sequence (TMV) (Gallie etc., (1989) are stated from Molecular Biology of RNA, and Cech edits, (Liss, NewYork), the 237-256 page or leaf); And corn chlorotic mottle poison leader sequence (MCMV) (Lommel etc., (1991) Virology 81:382-385).In addition referring to Della-Cioppa etc., (1987) PlantPhysiol.84:965-968.
When the preparation expression cassette, can operate multiple dna fragmentation, thereby dna sequence dna in the right direction and that frame is fit to when suitable is provided.For this reason, can use aptamers or connector to connect dna fragmentation, perhaps can comprise other operation, with the restriction site that facilitates, remove redundant DNA, remove restriction site etc.For this purpose, can comprise vitro mutagenesis, primer reparation, restriction, annealing, replacement again (for example conversion and transversion).
In the present invention's practice, can use multiple promotor, comprise the natural promoter of herbicide-tolerant polynucleotide sequence.Can select promotor based on required result.Can be with nucleic acid of the present invention and constitutive promoter, organize the combination of preferred promoter or other promotor, be used for expressing plant.
Such constitutive promoter for example comprises core promoter and other constitutive promoter of disclosed Rsyn7 promotor in No. the 6th, 072,050, WO 99/43838 and United States Patent (USP); Core CaMV 35S promoter (Odell etc., (1985) Nature 313:810-812); Rice actin (McElroy etc., (1990) Plant Cell 2:163-171); Ubiquitin (Christensen etc., (1989) Plant Mol.Biol.12:619-632 and Christensen etc., (1992) PlantMol.Biol.18:675-689); PEMU (Last etc., (1991) Theor.Appl.Genet.81:581-588); MAS (Velten etc., (1984) EMBO are J.3:2723-2730); ALS promotor (United States Patent (USP) the 5th, 659, No. 026) etc.Other constitutive promoter comprises for example United States Patent (USP) the 5th, 608,149,5,608,144,5,604,121,5,569,597,5,466,785,5,399,680,5,268,463,5,608,142 and 6,177, No. 611.
The Chemical Regulation promotor can be used for regulating and control the genetic expression of plant by application external source Chemical Regulation thing.Based on this purpose, promotor can be chemical inducible promoter, uses the chemical substance inducible gene expression in the case, or chemical repressible promoter, in the case by using the chemical substance inhibition of gene expression.Chemical inducible promoter is known in the art, including but not limited to can be by benzenesulfonamide herbicide safener activated corn In2-2 promotor, can be used as before the hydrophobic electrophilic compound activated corn GST promotor of emergent weedicide, and by Whitfield's ointment activated tobacco PR-1a promotor.The Chemical Regulation promotor that other merits attention comprises that the steroid responsive promoter is (referring to for example Schena etc., (1991) Proc.Natl.Acad.Sci.USA 88:10421-10425 and MeNellis etc., (1998) Plant J.14 (2): the glucocorticoid inducible type promotor among the 247-257) and tsiklomitsin induction type and tsiklomitsin inhibition type promotor (referring to for example Gatz etc., (1991) Mol.Gen.Genet.227:229-237 and United States Patent (USP) the 5th, 814,618 and 5,789, No. 156), described document is attached to herein by reference.
But the using-system preferred promoter is expressed enhanced Zm-D9 and is oriented to the specified plant tissue.Organize preferred promoter to comprise Yamamoto etc., (1997) Plant is (2): 255-265 J.12; Kawamata etc., (1997) Plant Cell Physiol.38 (7): 792-803; Hansen etc., (1997) Mol.Gen Genet.254 (3): 337-343; Russell etc., (1997) Transgenic Res.6 (2): 157-168; Rinehart etc., (1996) Plant Physiol.112 (3): 1331-1341; VanCamp etc., (1996) Plant Physiol.112 (2): 525-535; Canevascini etc., (1996) Plant Physiol.112 (2): 513-524; Yamamoto etc., (1994) Plant Cell Physiol.35 (5): 773-778; Lam (1994) Results Probl.Cell Differ.20:181-196; Orozco etc., (1993) Plant Mol Biol.23 (6): 1129-1138; Matsuoka etc., (1993) Proc Natl.Acad.Sci.USA90 (20): 9586-9590 and Guevara-Garcia etc., (1993) Plant is (3): 495-505 J.4.If needed, can modify such promotor, to weaken expression.
Certain embodiments of the present invention utilization to be to organize the preferred promoter plant transformed, stem preferred promoter especially, and it effectively is connected with the proteic nucleotide sequence of coding Zm-D9.In one embodiment of the invention, MS-S2A promotor (Abrahams etc., (1995) PlantMol Biol 27:513-28) effectively is connected with encoding wild type or the proteic polynucleotide sequence of MUT1Zm-D9.The selection of promotor, and inherent tissue specificity should influence degree or intensity that the form of expressing wild-type of the present invention or the proteic transgenic plant of MUT1Zm-D9 changes.With regard to the MS-S2A promotor, stem means that preferably existing file records the expression relevant with tieing up tube elements (not display data).The MS-S2A promotor seems to expression the best of MUT1 Zm-D9, and Actin muscle-a kind of composition type expression promoter that in leaf texture, has than high expression level, when being used for MUT1 ZM-D9 protein expression, form changes very appropriateness or slight.
The leaf preferred promoter is known in the art.Referring to for example Yamamoto etc., (1997) Plant is (2): 255-265 J.12; Kwon etc., (1994) Plant Physiol.105:357-67; Yamamoto etc., (1994) Plant Cell Physiol.35 (5): 773-778; Gotor etc., (1993) Plant is J.3:509-18; Orozco etc., (1993) Plant Mol.Biol.23 (6): 1129-1138; With Matsuoka etc., (1993) Proc.Natl.Acad.Sci.USA 90 (20): 9586-9590.(referring to above).
The root preferred promoter is known, can be selected from numerous promotors that can be obtained by document, or from the beginning separate from multiple suitable mutually species.Referring to for example Hire etc., (1992) Plant Mol.Biol.20 (2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3 (10): 1051-1061 (the root-specific controlling elements of French bean GRP1.8 gene); Sanger etc., (1990) Plant Mol.Biol.14 (3): 433-443 (root-specific promoter of Agrobacterium tumefaciems mannopine synthase (MAS) gene); And Miao etc., (1991) Plant Cell 3 (1): 11-22 (full length cDNA clone of coding kytoplasm glutamine synthetase (GS), it can be expressed in soybean root and root nodule).In addition referring to Bogusz etc., (1990) Plant Cell 2 (7): 633-641, wherein described from isolating two root-specific promoters of hemoglobin gene of the non-pulse family Ulmaceae of fixed nitrogen mountain jute (Parasponia andersonii) and the relevant non-pulse family of non-fixed nitrogen mountain jute (Trematomentosa).The promotor of these genes is connected with beta-glucuronidase enzyme reporter gene, and importing to non-pulse family tobacco (Nicotiana tabacum) and pulse family Root or stem of Littleleaf Indianmulberry (Lotus corniculatus) in these two, the root-specific promoter activity is all kept under these two kinds of situations.Leach and Aoyagi (1991) described they to the analysis of the promotor of the rolC of high expression level in the rhizobiaceae (Agrobacterium rhizogenes) and rolD root induction gene (referring to Plant Science (Limerick) 79 (1): 69-76).They infer enhanser and organize preferred terminator dna is dissociated in these promotors.Teeri etc., (1989) use the gene that merges with lacZ to show, edaphic bacillus (Agrobacterium) the T-DNA gene of coding octopine synthase especially has activity in tip of a root epidermis, TR2 ' gene is a root-specific in complete plant, and stimulated by the wound in the leaf texture, using with sterilant or larvacide gene is that especially desirable feature combination is (referring to EMBO J.8 (2): 343-350).TR1 ' the gene that merges with nptII (neomycin phosphotransferase II) shows similar feature.Other root preferred promoter comprises VfENOD-GRP3 gene promoter (Kuster etc., (1995) Plant Mol.Biol.29 (4): 759-772) with rolB promotor (Capana etc., (1994) Plant Mol.Biol.25 (4): 681-691).In addition referring to United States Patent (USP) the 5th, 837,876,5,750,386,5,633,363,5,459,252,5,401,836,5,110,732 and 5,023, No. 179.
Needing to use weak promoter under the situation of low expression level.Generally speaking, described " weak promoter " means with low-level driving encoding sequence expression promoter.The level of the described low-level level that means about 1/1000 transcript to the level of about 1/100,000 transcript to about 1/500,000 transcript.Perhaps, recognize that weak promoter only also comprises at some cell and expression promoter in other cell not, to produce overall low expression level.Under the situation of promotor, can lack or modify the part promoter sequence, to reduce expression level with unacceptable high level expression.
Weak constitutive promoter like this comprises for example core promoter of Rsyn7 promotor (No. the 6th, 072,050, WO99/43838 and United States Patent (USP)), core CaMV 35S promoter etc.Other constitutive promoter comprises for example United States Patent (USP) the 5th, 608,149,5,608,144,5,604,121,5,569,597,5,466,785,5,399,680,5,268,463 and 5,608, No. 142.Referring to United States Patent (USP) the 6th, 177, No. 611, this patent is hereby incorporated by in addition.
Expression cassette can also comprise the selected marker who is used to select transformant.The selected marker is used for the selection of transformant or tissue.Marker gene comprises the gene of the antibiotics resistance of encoding, for example encode those of neomycin phosphotransferase II (NEO) and hygromix phosphotransferase (HPT), and give herbicidal compounds (for example Glufosinate, bromoxynil, imidazolone and 2, the gene of 4-dichlorophenoxyacetic acid ester (2,4-D)) resistance.Other selected marker comprises phenotypic markers, for example beta-galactosidase enzymes and fluorescin, green fluorescent protein (GFP) (Su etc. for example, (2004) Biotechnol Bioeng 85:610-9 and Fetter etc., (2004) Plant Cell 16:215-28), cyan fluorescent protein (CYP) (Bolte etc., (2004) J.CellScience 117:943-54 and Kato etc., (2002) Plant Physiol 129:913-42) and yellow fluorescence protein (derive from the PhiYFPTM of Evrogen, referring to Bolte etc., (2004) J.CellScience 117:943-54).About other selected marker, usually referring to Yarranton (1992) Curr.Opin.Biotech.3:506-511; Christopherson etc., (1992) Proc.Natl.Acad.Sci.USA 89:6314-6318; Yao etc., (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol.6:2419-2422; Barkley etc., (1980) are stated from The Operon, the 177-220 page or leaf; Hu etc., (1987) Cell 48:555-566; Brown etc., (1987) Cell49:603-612; Figge etc., (1988) Cell 52:713-722; Deuschle etc., (1989) Proc.Natl.Acad.Aci.USA 86:5400-5404; Fuerst etc., (1989) Proc.Natl.Acad.Sci.USA 86:2549-2553; Deuschle etc., (1990) Science 248:480-483; Gossen (1993) Ph D dissertation, Ruprecht-Karls-Universitat Heidelberg; Reines etc., (1993) Proc.Natl.Acad.Sci.USA 90:1917-1921; Labow etc., (1990) Mol.Cell.Biol.10:3343-3356; Zambretti etc., (1992) Proc.Natl.Acad.Sci.USA 89:3952-3956; Baim etc., (1991) Proc.Natl.Acad.Sci.USA 88:5072-5076; Wyborski etc., (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol.Struc.Biol.10:143-162; Degenkolb etc., (1991) Antimicrob.AgentsChemother.35:1591-1595; Kleinschnidt etc., (1988) Biochemistry27:1094-1104; Bonin (1993) Ph D dissertation, Ruprecht-Karls-Universitat Heidelberg; Gossen etc., (1992) Proc.Natl.Acad.Sci.USA 89:5547-5551; Oliva etc., (1992) Antimicrob.Agents Chemother.36:913-919; Hlavka etc., (1985) Handbook ofExperimental Pharmacology, the 78th volume (Springer-Verlag, Berlin); Gill etc., (1988) Nature 334:721-724.These disclosures are attached to herein by reference.
Selectable marker gene listed above does not have restricted.Can use any selected marker in the present invention.
In one embodiment, with the molecular targeted chloroplast(id) that is used to express of herbicide-tolerant polynucleotide.In this way, do not have directly to insert under the situation of chloroplast(id) at herbicide-tolerant polynucleotide, expression cassette will comprise the nucleic acid of the transit peptides of encoding in addition, so that the target gene product is imported in the chloroplast(id).Such transit peptides is known in the art.Referring to for example Von Heijne etc., (1991) PlantMol.Biol.Rep.9:104-126; Clark etc., (1989) J.Biol.Chem.264:17544-17550; Della-Cioppa etc., (1987) Plant Physiol.84:965-968; Romer etc., (1993) Biochem.Biophys.Res.Commun.196:1414-1421; With Shah etc., (1986) Science 233:478-481.
Chloroplast targeted sequence is known in the art, comprises ribulose-1,5-bisphosphate, the chloroplast(id) small subunit of 5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho etc., (1996) Plant Mol.Biol.30:769-780; Schnell etc., (1991) J.Biol.Chem.266 (5): 3335-3342); 5-(enol pyruvoyl) shikimic acid-3-phosphate synthase (EPSPS) (Archer etc., (1990) J.Bioenerg.Biomemb.22 (6): 789-810); Tryptophan synthetase (Zhao etc., (1995) J.Biol.Chem.270 (11): 6081-6087); Plastocyanin (Lawrence etc., (1997) J.Biol.Chem.272 (33): 20357-20363); Chorismate synthase (Schmidt etc., (1993) J.Biol.Chem.268 (36): 27447-27457); And light harvesting chlorophyll a/b conjugated protein (LHBP) (Lamppa etc., (1988) J.Biol.Chem.263:14996-14999).In addition referring to Von Heijne etc., (1991) Plant Mol.Biol.Rep.9:104-126; Clark etc., (1989) J.Biol.Chem.264:17544-17550; Della-Cioppa etc., (1987) Plant Physiol.84:965-968; Romer etc., (1993) Biochem.Biophys.Res.Commun.196:1414-1421; With Shah etc., (1986) Science 233:478-481.
The method that is used to transform chloroplast(id) is known in the art.Referring to for example Svab etc., (1990) Proc.Natl.Acad.Sci.USA 87:8526-8530; Svab and Maliga (1993) Proc.Natl.Acad.Sci.USA 90:913-917; Svab and Maliga (1993) EMBO J.12:601-606.This method dependence particle gun transmission contains the DNA of selected marker, and this DNA is passed through homologous recombination target plastom.In addition, the tissue of RNA polymerase that can be by nuclear coding and plastid guiding is preferably expressed reticent genetically modified that the trans-activation plastid carries, and realizes the plastid conversion thus.Such system at McBride etc., reports among (1994) Proc.Natl.Acad.Sci.USA 91:7301-7305.
Can optimize by the herbicide-tolerant polynucleotide of target chloroplast(id), in order in chloroplast(id), to express, with the codon usage variance between explanation plant nuclear and this organoid.In this way, can use the preferred codon of chloroplast(id) to synthesize the herbicide-tolerant polynucleotide molecule.Referring to for example U.S. Patent number 5,380,831, this patent is attached to herein by reference.
Method of the present invention comprises polypeptide or polynucleotide molecule is imported in the plant." importing " is to point to plant polynucleotide molecule or polypeptide are provided, and presentation mode can make sequence enter vegetable cell inside.Method of the present invention does not rely on the concrete grammar that imports sequence in plant, as long as polynucleotide molecule or polypeptide enter at least one cell interior of plant.With the method in polynucleotide molecule or the polypeptide importing plant is known in the art, includes but not limited to stable conversion method, instantaneous conversion method and virus-mediated method.
" stable conversion " means the target constructs that imports in the plant and is incorporated in the Plant Genome, and can be gone down by its filial generation heredity." instantaneous conversion " means polynucleotide molecule and is directed in the plant, and unconformability perhaps imports to polypeptide in the plant in Plant Genome.
Transformation Program and the program of polypeptide or polynucleotide sequence of importing in plant can be according to changing as the type (promptly being unifacial leaf or dicotyledonous) of plant that transforms target or vegetable cell.The appropriate methodology that imports polypeptide and polynucleotide to vegetable cell comprises microinjection (Crossway etc., (1986) electroporation (Riggs etc. Biotechniques 4:320-334),, (1986) agrobacterium-mediated conversion (United States Patent (USP) the 5th Proc.Natl.Acad.Sci.USA 83:5602-5606),, 563, No. 055 and United States Patent (USP) the 5th, 981, No. 840), directed gene shifts (Paszkowski etc., (1984) EMBO is J.3:2717-2722) and trajectory particle accelerated process (referring to for example United States Patent (USP) the 4th, 945, No. 050; United States Patent (USP) the 5th, 879, No. 918; United States Patent (USP) the 5th, 886,244 and 5,932, No. 782; Tomes etc., (1995) are stated from Plant Cell, Tissue, and OrganCulture:Fundamental Methods, Gamborg and Phillips edit, (Springer-Verlag, Berlin); McCabe etc., (1988) Biotechnology 6:923-926); Transform (WO 00/28058) with Lec1.In addition referring to Weissinger etc., (1988) Ann.Rev.Genet.22:421-477; Sanford etc., (1987) Particulate Science andTechnology 5:27-37 (onion); Christou etc., (1988) Plant Physiol.87:671-674 (soybean); McCabe etc., (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.Biol.27P:175-182 (soybean); Singh etc., (1998) Theor.Appl.Genet.96:319-324 (soybean); Datta etc., (1990) Biotechnology 8:736-740 (paddy rice); Klein etc., (1988) Proc.Natl.Acad.Sci.USA 85:4305-4309 (corn); Klein etc., (1988) Biotechnology 6:559-563 (corn); United States Patent (USP) the 5th, 240,855,5,322,783 and 5,324, No. 646; Klein etc., (1988) Plant Physiol.91:440-444 (corn); Fromm etc., (1990) Biotechnology8:833-839 (corn); Hooykaas-Van Slogteren etc., (1984) Nature (London) 311:763-764; United States Patent (USP) the 5th, 736, No. 369 (cereal); Bytebier etc., (1987) Proc.Natl.Acad.Sci.USA 84:5345-5349 (lily); De Wet etc., (1985) are stated from TheExperimental Manipulation of Ovule Tissues, editors such as Chapman, (Longman, New York), 197-209 page or leaf (pollen); Kaeppler etc., (1990) Plant CellReports 9:415-418 and Kaeppler etc., (1992) Theor.Appl.Genet.84:560-566 (conversion of whisker mediation); D ' Halluin etc., (1992) Plant Cell 4:1495-1505 (electroporation); Li etc., (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (paddy rice); Osjoda etc., (1996) NatureBiotechnology 14:745-750 (corn transforms by Agrobacterium tumefaciems); All these documents all are attached to herein by reference.
In specific embodiment, can use multiple instantaneous conversion method that Zm-D9 sequence of the present invention is offered plant.Such instantaneous conversion method includes but not limited to Zm-D9 albumen or its variant and fragment are directly imported in the plant, perhaps the Zm-D9 transcript is imported in the plant.Such method comprises for example microinjection or partickle bombardment.Referring to for example Crossway etc., (1986) Mol Gen.Genet.202:179-185; Nomura etc., (1986) Plant Sci.44:53-58; Hepler etc., (1994) Proc.Natl.Acad.Sci.91:2176-2180 and Hush etc., (1994) The Journal of Cell Science 107:775-784, all documents all are attached to herein by reference.Perhaps, the Zm-D9 polynucleotide molecule can use technology instantaneous conversion known in the art in plant.These technology comprise virus carrier system and precipitate polynucleotide molecule in the mode of getting rid of follow-up DNA release.Therefore, can be by transcribing with particle bonded DNA, but its frequency that is released to be incorporated in the genome is greatly reduced.These methods comprise uses bag by polyethylenepolyamine (polyethylimine) (PEI; Sigma#P3143) particle.
In other embodiments, can polynucleotide molecule of the present invention be imported in the plant by plant is contacted with virus or viral nucleic acid.Usually, such method comprises constructs of the present invention is mixed in viral DNA or the RNA molecule.Will be appreciated that Zm-D9 aminoacid sequence of the present invention can be used as the part of viral polyprotein at first and synthesizes, this part after a while can be in vivo or is external by proteolysis processing, produces required recombinant protein.In addition, will be appreciated that promotor of the present invention also comprises the promotor of transcribing use by viral rna polymerase.It is known in the art relating to method viral DNA or RNA molecule, that be used for polynucleotide are imported plant and express proteins encoded therein.Referring to for example United States Patent (USP) the 5th, 889,191,5,889,190,5,866,785,5,589,367,5,316, No. 931 and Porta etc., (1996) Molecular Biotechnology 5:209-221; Described document is attached to herein by reference.
The method that specific position target in Plant Genome inserts polynucleotide molecule is known in the art.In one embodiment, use the site-specific recombination system to realize the insertion of polynucleotide molecule in required genome position.Referring to for example WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all documents all are attached to herein by reference.In brief, polynucleotide molecule of the present invention can be included in and shift in the expression cassette two recombination sites that do not cause reorganization of side joint.Import in the stable plant of mixing in its genome of target site two recombination sites that do not cause reorganization of described target site side joint with shifting expression cassette corresponding to the site of shifting expression cassette.Suitable recombinase is provided, makes the transfer expression cassette be incorporated into target site.Therefore, the herbicide-tolerant polynucleotide molecule is incorporated into the specific chromosome position in the Plant Genome.
Cell transformed can be cultivated into plant according to traditional method.Referring to for example McCormick etc., (1986) Plant Cell Reports 5:81-84.Can cultivate these plant then, with the plant or the different plants pollination of same conversion, and the filial generation that obtains of evaluation constitutive expression desired phenotype feature.Can cultivate two generations or many generations, keep and heredity with being stabilized, gather in the crops seed then, to guarantee to realize the expression of desired phenotype feature with the expression of guaranteeing the desired phenotype feature.In this way, the invention provides with the stable transformed the seed (being also referred to as " transgenic seed ") that mixes in its genome of polynucleotide molecule of the present invention (expression cassette for example of the present invention).
Pedigree breeding is since two genotypic hybridization, for example target original seed system has one or more with another and the target original seed is the inbred lines of the required feature of complementary (the promptly stable polynucleotide molecule of the present invention, the activity of the polypeptide of the present invention of regulation and control and/or level etc. of mixing).If two original parents do not provide all to need feature, then in propagating population, can comprise other source.In the pedigree method, make good plant selfing, and selecting in the filial generation continuously.In filial generation subsequently, heterozygous state is replaced by the pure lines that produced by self-pollination and selection.Typically, in the pedigree breeding method, carry out the selfing and the selection of 5 or more a plurality of continuous filial generations: F1 → F2; F2 → F3; F3 → F4; F4 → F5 etc.After the capacity inbreeding, the successive filial generation will be used to increase the seed of growing inbred lines.In specific embodiment, inbred lines comprises the allelotrope that isozygotys at its about locus more than 95%.
Backcross except be used for producing backcross transform, also can be used for and the pedigree breeding combination, with the hybrid that changes target original seed system and use the original seed system that changes to produce.As previously mentioned, backcross can be used for from one be the one or more specific required feature transfer of (donor parents) in the inbred lines that is called recurrent parent, this inbred lines possesses good on the whole agronomy feature, but does not have one or more required characteristics.But, this identical program can be used for making the genotype of filial generation tendency recurrent parent, but simultaneously by stopping in early days backcrossing and proceeding selfing and select to keep many components of nonrecurrent parent.For example, the F1 of generation such as commercialization hybrid.This commercialization hybrid can be backcrossed with one of its parent system, produces BC1 or BC2.Filial generation is carried out selfing and is selected, so that new inbred lines of growing has the various features of recurrent parent, but has the several required feature of nonrecurrent parent.This method balance the value and the intensity of used recurrent parent in novel hybrid and the breeding.
Therefore, one embodiment of the invention are the methods that make target corn inbred lines backcross and transform, this method may further comprise the steps: make target corn inbred lines plant and the mutator gene of giving required feature (promptly reducing plant height or plant type) or the hybridization of genetically modified donor plant, the mutator gene or the genetically modified F1 progeny plant of required feature given in selection, and selected F1 progeny plant and target corn inbred lines plant are backcrossed.This method can also may further comprise the steps: obtain the molecule marker spectrum of target corn inbred lines, and select to have the progeny plant of required feature and target inbred lines molecule marker spectrum with this molecule marker spectrum.By same mode, this method can be used for producing the F1 hybrid seed by increasing last following step: the required feature of target corn inbred lines is transformed and the hybridization of different milpas, give the mutator gene or the genetically modified F1 hybrid corn seed of required feature with generation.
Recurrent selection is the method that is used to improve plant population in the plant breeding program.This method need make individual plant hybridization pollination each other, to form filial generation.Cultivate filial generation, and screen good filial generation by numerous systems of selection, it comprises individual plant, half sibs filial generation, full sibs filial generation, self-bred progeny and topcross filial generation.Selected filial generation is hybridization pollination each other, to form another group filial generation.Plant this population, and select good plant once more, carry out hybridization pollination each other.Recurrent selection is a working cycle, therefore can repeat repeatedly as required.The purpose of recurrent selection is the quality of improvement population.The population of improvement can be used as the source of breeding material then, with the inbred lines that obtains to be used for hybrid or be used as the parent of synthetic cultivated variety.Synthetic cultivated variety is by the formed offspring of the hybridization of several selected inbred lines.
Mix to be chosen in and strengthen with molecule marker that to select to unite when using be a kind of useful technology.In mix selecting, select seed from individuality according to phenotype and/or genotype.Then, the seed that these are selected mixes, and is used for cultivating of future generation.Mix and select to cultivate plants in batch the group, make the plant self-pollination, gather in the crops seed in batch, use the sample plantation of the seed of gathering in the crops in batch of future generation then.Directly pollination can replace self-pollination to be used as the part of the procedure of breeding.
Mutation breeding is to can be used for new proterties is introduced a kind of in numerous methods of original seed system.For the plant breeder, spontaneous generation or artificial induction's sudden change can be the useful source of variability.The target of induced mutations is to improve the mutation rate of required proterties.Can improve mutation rate by multiple different methods, comprise temperature, long-term seed stores, conditions of tissue culture, radiation (X ray for example, gamma-rays (for example cobalt 60 or caesium 137), neutron (fission products of uranium 235 in the reactor), β radiation (from radio isotope such as phosphorus 32 or carbon 14 emissions) or uviolizing (preferably from 2500nm to 2900nm)), or chemical mutagen (base analogue (5-bromouracil) for example, related compound (8-Ethoxycaffeine), microbiotic (Streptonigrin), alkylating agent (sulfur mustard, mustargen, epoxide, vinyl-amine, vitriol, sulfonate, sulfone, lactone), trinitride, azanol, nitrous acid or acridine.In case observe required proterties by mutagenesis, then can as backcrossing this proterties be mixed in the existing germplasm by traditional breeding technology.The details of mutation breeding is found in " Principals of Cultivar Development, " Fehr, 1993Macmillan Publishing Company, and its disclosure is attached to herein by reference.In addition, the sudden change that produces in other is can be used for producing the conversion of backcrossing of the original seed system that contains these sudden changes.
Term plant used herein comprises vegetable cell, plant protoplast, plant cell tissue's culture, plant callus, plant piece and complete vegetable cell in plant or plant part (for example embryo, pollen, ovule, seed, leaf, flower, branch, fruit, nuclear/benevolence, ear fringe (ears), cob, shell, bar, root, the tip of a root, flower pesticide etc.) that can aftergrowth.Seed means commercial grower in order to grow or to breed the purpose beyond the species and the mature seed produced.The offspring of regenerated plant, variant and mutant are also included within the scope of the invention, as long as these parts comprise the polynucleotide of importing.
The present invention can be used for transforming any plant species, includes but not limited to monocotyledons and dicotyledons.The example of target plant species includes but not limited to corn (Zea mays); Rape class (Brassica sp.) (for example Brassica napus (B.napus), turnip type rape (B.rapa), mustard type rape (B.juncea)), especially those can be used as the rape class in seed oil source; Clover (Medicago sativa), paddy rice (Oryza sativa), rye (Secale cereale), Chinese sorghum (Sorghum bicolor, Sorghum vulgare), broomcorn millet (pearl millet (Pennisetumglaucum) for example, millet (Panicum miliaceum), grain (Setaria italica), ragimillet (Eleusinecoracandj)), Sunflower Receptacle (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Man ihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananascomosus), Citrus tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), Fructus Fici (Ficuscasica), piscidia (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew nut (Anacardium occidentale), Queensland nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Betavulgaris), sugarcane (Saccharum spp.), oat, barley, vegetables, ornamental plant and softwood tree.
Vegetables comprise that tomato (Lycopersicon esculentum), lettuce (for example Lactucasativd), French beans (Phaseolus vulgaris), lima bean (Phaseolus limensis), pea (Lathyrus spp.) and cucumber (Cucumis) belong to member, for example cucumber (C.sativus), hami melon (C.cantalupensis) and muskmelon (C.melo).Ornamental plant comprises rhododendron (Rhododendronspp.), Flower of Largeleaf Hydrangea (Macrophylla hydrangea), lotus (Hibiscus rosasanensis), rose (Rosa spp.), turmeric (Tulipa spp.), flower of Chinese Narcissus (Narcissus spp.), petunia (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrimd) and chrysanthemum.
Can be used for implementing softwood tree of the present invention and comprise for example pine tree, for example torch pine (Pinustaeda), slash pine (Pinus elliotii), yellow oregon pine (Pinus ponderosa), black pine (Pinuscontorta) and pine (Pinus radiata); Pseudotsuga menziesii (Mirbel) Franco (Pseudotsuga menziesii); California hemlock spruce (Tsuga canadensis); Sitka spruce (Picea glauca); Chinese larch (Sequoiasempervirens); Fir, for example silver fir (Abies amabilis) and glue fir (Abies balsamea); And cdear, for example Pacific red cedar (Thuja plicata) and yellow cedar (Chamaecyparisnootkatensis).In specific embodiment, plant of the present invention is cultivated plant (for example corn, clover, Sunflower Receptacle, rape, soybean, cotton, safflower, peanut, Chinese sorghum, wheat, broomcorn millet, a tobacco etc.).In other embodiments, corn and soybean plants are best, and in further other embodiment, maize plant is best.
Other target plant comprises cereal grass, oils spermatophyte and the leguminous plants that the target seed is provided.The target seed comprises cereal seed, for example corn, wheat, barley, paddy rice, Chinese sorghum, rye etc.The oils spermatophyte comprises cotton, soybean, safflower, Sunflower Receptacle, rape, corn, clover, palm, coconut etc.Leguminous plants comprises Kidney bean and pea.Kidney bean comprises guar-bean, locust bean, Semen Trigonellae, soybean, French bean, cowpea, mung bean, lima bean, broad bean, root of Szemao crotalaria, garbanzo etc.
Be provided at the concentration and/or the active method of regulation and control polypeptide of the present invention in the plant.Generally speaking, concentration and/or active natural control plant with respect to the sequence of the present invention that does not contain importing, plant part or cell increase or reduce at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.Regulation and control among the present invention can occur in plant-growth to the process of required etap and/or afterwards.In specific embodiment, especially corn is regulated and control polypeptide of the present invention in monocotyledons.
Zm-D9 polypeptide expression level can directly detect by the Zm-D9 polypeptide level in the plant for example, perhaps Zm-D9 activity (for example measuring the height of whole plant height or stem or the bar) indirect detection by the Zm-D9 polypeptide in the plant for example.Measure the active method of Zm-D9 in its place's description of this paper.
In specific embodiment, polypeptide of the present invention or polynucleotide molecule are imported in the vegetable cell.Subsequently, use method known to those skilled in the art,, select to have imported the vegetable cell of sequence of the present invention such as but not limited to southern blotting technique analysis, dna sequencing, pcr analysis or phenotype analytical.Grow for some time under the plant formation condition by plant or plant part that previous embodiments is changed or revises, this section period is enough to regulate and control concentration and/or the activity of polypeptide of the present invention in plant.The condition that plant forms is well known in the art, and at its place's brief discussion of this paper.
What also will recognize is, can use can not be in transforming plant the level and/or the activity of polynucleotide molecule regulation and control polypeptide of pilot protein or rna expression.For example, polynucleotide of the present invention can be used for designing polynucleotide constructs, this construct can be used for changing or mutation biology in the method for genome nucleotide sequence in.Such polynucleotide constructs includes but not limited to RNA:DNA carrier, RNA:DNA mutational vector, RNA:DNA repair vector, mixes the duplex oligonucleotide, self complementary RNA:DNA oligonucleotide and cause the oligonucleotidase of reorganization.These constructs and using method are known in the art.Referring to United States Patent (USP) the 5th, 565,350,5,731,181,5,756,325,5,760,012,5,795,972 and 5,871, No. 984; All patents all are attached to herein by reference.In addition referring to WO 98/49350, WO99/07865, WO 99/25821 and Beetham etc., (1999) Proc.Natl.Acad.Sci.USA 96:8774-8778; Described document is attached to herein by reference.
Therefore, recognize that method of the present invention does not rely on mixes whole polynucleotide in the genome, as long as plant or its cell are owing to changing in the polynucleotide molecule transfered cell.In one embodiment of the invention, genome can be changed in the polynucleotide molecule transfered cell afterwards.For example, polynucleotide or its any part all can be mixed in the Plant Genome.Genome of the present invention changes increase, disappearance and the replacement that includes but not limited to the Nucleotide in the genome.Although method of the present invention does not rely on interpolation, disappearance and the replacement of any given number Nucleotide, generally acknowledge that this class is added, lacked or replacement comprises at least 1 Nucleotide.
In one embodiment, increase the activity and/or the level of Zm-D9 polypeptide of the present invention.Can realize Zm-D9 polypeptide level of the present invention and/or active increase by the Zm-D9 polypeptide is provided to plant.As discussing in its place of this paper, known in the artly provide many methods of polypeptide to plant, include but not limited to polypeptide directly imported in the plant and the polynucleotide constructs that coding has an active polypeptide of Zm-D9 is imported in the plant on (instantaneous ground or stably).To recognize that also method of the present invention can be used the polynucleotide molecule that can not instruct albumen or rna expression in transforming plant.Therefore, the level of Zm-D9 polypeptide and/or active can increasing by gene or its promotor that changes coding Zm-D9 polypeptide.Referring to for example Kmiec, United States Patent (USP) 5,565,350; Zarling etc., PCT/US93/03868.Therefore, provide the mutagenesis of carrying sudden change in Zm-D9 gene plant, wherein sudden change increases the Zm-D9 expression of gene, or increases the Zm-D9 activity of coded Zm-D9 polypeptide.
In other embodiments, import activity and/or the level that reduces or remove Zm-D9 polypeptide of the present invention in the plant by the level or the active polynucleotide that will suppress Zm-D9 polypeptide of the present invention.Described polynucleotide can directly suppress Zm-D9 expresses by the translation that stops the Zm-D9 messenger RNA(mRNA), or suppresses Zm-D9 indirectly by the coded polypeptide that suppresses coding proteic Zm-D9 gene transcription of Zm-D9 or translation and express.The method of inhibition or removal genetic expression is well-known in this area in plant, and any such method all can be used for suppressing the expression of Zm-D9 plant in the present invention.In other embodiments of the present invention, reduce or remove the activity of Zm-D9 polypeptide with the encoding sequence transformed plant cells of the polypeptide that suppresses the Zm-D9 polypeptide active.In other embodiments, can reduce or remove the activity of Zm-D9 polypeptide by the gene that destroys coding Zm-D9 polypeptide.Present invention resides in the mutagenesis plant of carrying sudden change in the Zm-D9 gene, wherein sudden change reduces the Zm-D9 expression of gene, or suppresses the Zm-D9 activity of the Zm-D9 polypeptide of coding.
Some aspects of genetically engineered plant need reduce the activity (being also referred to as gene silencing or gene inhibition) of specific gene.Many gene silent technologies are well-known in this area, include but not limited to that antisense technology is (referring to for example Sheehy etc., (1988) Proc.Natl.Acad.Sci.USA85:8805-8809; With U.S. Patent number 5,107,065; 5,453,566; With 5,759,829); Suppress altogether (Taylor (1997) Plant Cell 9:1245 for example; Jorgensen (1990) TrendsBiotech.8 (12): 340-344; Flavell (1994) Proc.Natl.Acad.Sci.USA91:3490-3496; Finnegan etc., (1994) Bio/Technology 12:883-888; With Neuhuber etc., (1994) Mol.Gen.Genet.244:230-241); RNA disturbs (Napoli etc., (1990) Plant Cell 2:279-289; U.S. Patent number 5,034,323; Sharp (1999) Genes Dev.13:139-141; Zamore etc., (2000) Cell 101:25-33; With Montgomery etc., (1998) Proc.Natl.Acad.Sci.USA 95:15502-15507); The gene silencing of virus induction (Burton etc., (2000) Plant Cell 12:691-705; And Baulcombe (1999) Curr.Op.Plant Bio.2:109-113); Target-RNA-specific ribozyme (Haseloff etc., (1988) Nature 334:585-591); Hairpin structure (Smith etc., (2000) Nature 407:319-320; WO 99/53050; WO 02/00904; WO 98/53083; Chuang and Meyerowitz (2000) Proc.Natl.Acad.Sci.USA 97:4985-4990; Stoutjesdijk etc., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat.Rev.Genet.4:29-38; Pandolfini etc., BMCBiotechnology 3:7, U.S. Patent Publication numbers 20030175965; Panstruga etc., (2003) Mol.Biol.Rep.30:135-140; Wesley etc., (2001) Plant is J.27:581-590; Wang and Waterhouse (2001) Curr.Opin.Plant Biol.5:146-150; U.S. Patent Publication numbers 20030180945; And WO 02/00904, all these documents all are attached to herein by reference); Ribozyme (Steinecke etc., (1992) EMBO is J.11:1525; With Perriman etc., (1993) Antisense Res.Dev.3:253); Oligonucleotide mediated target is revised (for example WO 03/076574 and WO 99/25853); Zinc refers to targeted molecular, and (for example WO 01/52620; WO 03/048345; With WO 00/42219); Transposon tagging (Maes etc., (1999) TrendsPlant Sci.4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol.Lett.179:53-59; Meissner etc., (2000) Plant is J.22:265-274; Phogat etc., (2000) J.Biosci.25:57-63; Walbot (2000) Curr.Opin.Plant Biol.2:103-107; Gai etc., (2000) Nucleic Acids Res.28:94-96; Fitzmaurice etc., (1999) Genetics153:1919-1928; Bensen etc., (1995) Plant Cell 7:75-84; Mena etc., (1996) Science 274:1537-1540; With United States Patent (USP) the 5th, 962, No. 764); Each of described document all is attached to herein by reference; And the combination of other method and above method well known by persons skilled in the art.
What recognize is, can make up at least a portion complementary antisense constructs with the messenger RNA(mRNA) (mRNA) of Zm-D9 sequence with polynucleotide of the present invention.Antisense nucleotide that makes up and corresponding mRNA hybridization.Can modify antisense sequences, as long as this sequence is hybridized with corresponding mRNA and disturbed its expression.In this way, can use with corresponding antisense sequences have 70%, the antisense constructs of preferred 80%, more preferably 85% sequence identity.In addition, the part of antisense nucleotide can be used for destroying target gene expression.Generally speaking, can use at least 50 Nucleotide, 100 Nucleotide, 200 Nucleotide, 300,400,450,500, the sequence of the Nucleotide more than 550.
Polynucleotide of the present invention can also be used for suppressing native gene plant with sense orientation expresses.It is known in the art using polynucleotide method of inhibition of gene expression in plant of sense orientation.In general, these class methods relate to containing in plant the DNA construct that drives expression promoter and transform plant, described promotor and effectively being connected to the small part polynucleotide corresponding to the native gene transcript.Typically, the sequence of this nucleotide sequence and native gene transcript has significant sequence identity, preferred about sequence identity more than 65%, more preferably from about the sequence identity more than 85%, most preferably from about the sequence identity more than 95%.Referring to United States Patent (USP) the 5th, 283,184 and 5,034, No. 323, described patent is attached to herein by reference.Therefore, many methods can be used for reducing or removing the activity of Zm-D9 polypeptide.Can use more than one method to reduce the activity of single Zm-D9 polypeptide.In addition, can also use combined method to reduce or remove the activity of Zm-D9 polypeptide.
In certain embodiments, the expression cassette of the polynucleotide of expressing with expression inhibiting Zm-D9 transforms the activity that the Zm-D9 vegetable cell reduces or remove Zm-D9.These polynucleotide can directly suppress one or more Zm-D9 protein expressions by the translation that stops the Zm-D9 messenger RNA(mRNA), or suppress one or more Zm-D9 protein expressions indirectly by the polypeptide of transcribing or translating of the proteic corn gene of coding inhibition coding Zm-D9.The method of inhibition or removal genetic expression is well-known in this area in plant, and any such method all can be used to suppress the proteic expression of one or more Zm-D9 in the present invention.
According to the present invention, if being lower than, the protein level of Zm-D9 do not carrying out hereditary change or mutagenesis protein level with the identical Zm-D9 in the plant that suppresses this Zm-D9 protein expression on statistics, then the expression of Zm-D9 is suppressed.In specific embodiments of the present invention, the proteic protein level of Zm-D9 in change of the present invention plant be lower than be not mutant or also not by hereditary change with the plant that suppresses this Zm-D9 protein expression in the proteic protein level of identical Zm-D9 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.The proteic expression level of Zm-D9 for example can directly be measured by measuring the Zm-D9 protein level of expressing in corn plant cell or the plant, perhaps for example by detecting the active indirect measurement of the proteic Zm-D9 of Zm-D9 in corn plant cell or the plant.Measure the active method of the proteic Zm-D9 of Zm-D9 in its place's description of this paper.
In other embodiments of the present invention, reduce or remove the proteic activity of one or more Zm-D9 with expression cassette maize transformation vegetable cell, described expression cassette contains the polynucleotide that coding suppresses the polypeptide of one or more Zm-D9 protein-actives.According to the present invention,, the proteic Zm-D9 activity of Zm-D9 do not carrying out hereditary change on statistics to suppress the proteic Zm-D9 activity of identical Zm-D9 in the active plant of the proteic Zm-D9 of this Zm-D9, the then proteic Zm-D9 activity inhibited of Zm-D9 if being lower than.In specific embodiments of the present invention, the proteic Zm-D9 activity of Zm-D9 in change of the present invention plant be lower than also do not carrying out hereditary change with the plant that suppresses this Zm-D9 protein expression in the proteic Zm-D9 of identical Zm-D9 active 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or 5%.According to the present invention, when the detection method of describing by its place of this paper can not detect the proteic Zm-D9 of Zm-D9 when active, then should activity by " removal/elimination ".Measure the active method of the proteic Zm-D9 of Zm-D9 in its place's description of this paper.
In other embodiments, can reduce or the proteic activity of removal Zm-D9 by destroying the proteic gene of coding Zm-D9.Present invention resides in the mutagenesis maize plant that carries sudden change in the Zm-D9 gene, wherein said sudden change reduces the Zm-D9 expression of gene, or suppresses the proteic Zm-D9 activity of Zm-D9 of coding.
Therefore, can use several different methods reduction or the proteic activity of removal Zm-D9.Can use more than one method to reduce the proteic activity of single Zm-D9.In addition, also can use combined method to reduce or remove the proteic activity of two or more different Zm-D9.
The limiting examples of the method for reduction or removal Zm-D9 protein expression provides hereinafter.
A. Method based on polynucleotide
In certain embodiments of the invention, with the expression cassette maize transformation vegetable cell of polynucleotide that can expression inhibiting Zm-D9 protein expression.Term used herein " expression " is meant the biosynthesizing of gene product, comprises transcribing and/or translating of described gene product.For example, for target of the present invention, can in maize plant, the expression cassette of the polynucleotide of at least 1 Zm-D9 protein expression of expression inhibiting be in maize plant, to produce the expression cassette that suppresses at least 1 proteic RNA molecule of transcribing and/or translating of Zm-D9.Be meant transcribing of encoding sequence and translate from dna molecular " expression " or " generation " albumen or polypeptide to produce albumen or polypeptide, and be meant the translation generation albumen or the polypeptide of RNA encoding sequence from RNA molecule " expressions " or " generation " albumen or polypeptide.
The example that suppresses the polynucleotide of Zm-D9 protein expression in maize plant provides hereinafter.
1. there is justice to suppress/suppress altogether
In certain embodiments of the invention, can be by having justice to suppress or suppressing the inhibition of acquisition altogether to the Zm-D9 protein expression.For inhibition altogether, expression cassette is designed for all or part of RNA molecule of " justice is arranged " direction expression corresponding to the proteic messenger RNA(mRNA) of coding Zm-D9.Expressing excessively of RNA molecule can cause natural gene to express reduction.Therefore, screening shows the department of botany that maximum Zm-D9 protein expression suppresses with suppressing a plurality of departments of botany that expression cassette transforms altogether to identify those.
The polynucleotide that are used for common inhibition can be corresponding to all or part of of the proteic sequence of Zm-D9 of encoding, 5 of Zm-D9 albumen transcript ' and/or 3 ' non-translational region is all or part of, these two all or part of of the encoding sequence of the proteic transcript of Zm-D9 of perhaps encoding and non-translational region.In certain embodiments, when polynucleotide comprised Zm-D9 protein-coding region all or part of, expression cassette was designed to remove the initiator codon of polynucleotide, makes protein product not transcribed.
Suppress to can be used for suppressing gene expression in plants altogether, to produce the undetectable plant of proteic protein level by these genes encodings.Referring to for example Broin etc., (2002) Plant Cell14:1417-1432.Inhibition can also be used for suppressing a plurality of proteic expression of same plant altogether.Referring to No. the 5th, 942,657, United States Patent (USP) for example.The method of expressing with the native gene that suppresses altogether in the plant is described in the following document: Flavell etc., (1994) Proc.Natl.Acad.Sci.USA 91:3490-3496; Jorgensen etc., (1996) Plant Mol.Biol.31:957-973; Johansen and Carrington (2001) Plant Physiol.126:930-938; Broin etc., (2002) Plant Cell 14:1417-1432; Stoutjesdijk etc., (2002) Plant Physiol.129:1723-1731; Yu etc., (2003) Phytochemistry 63:753-763; With United States Patent (USP) the 5th, 034,323,5,283,184 and 5,942, No. 657; Each of described document all is attached to herein by reference.Can comprise the efficient that common inhibition is improved in the poly-dT district by 5 of 3 of adopted sequence ' position and the poly-adenosine signal ' position that has at expression cassette.Referring to U.S. Patent Publication No. 20020048814, this patent is attached to herein by reference.Typically, the sequence of this nucleotide sequence and native gene transcript has significant sequence identity, preferred about sequence identity 65% or more, the sequence identity more than 85% more preferably from about, the about sequence identity more than 95% of optimum.Referring to United States Patent (USP) the 5th, 283,184 and 5,034, No. 323, described patent is attached to herein by reference.
2. Antisense Suppression
In certain embodiments of the invention, can be by the inhibition of Antisense Suppression acquisition to the Zm-D9 protein expression.For Antisense Suppression, expression cassette is designed to express and all or part of complementary RNA molecule of the proteic messenger RNA(mRNA) of Zm-D9 of encoding.The expression reduction that can cause natural gene is expressed in crossing of antisense rna molecule.Therefore, a plurality of departments of botany that screening transforms with the Antisense Suppression expression cassette show those departments of botany that maximum Zm-D9 protein expression suppresses with evaluation.
What be used for that the polynucleotide of Antisense Suppression can be corresponding to the complement of coding Zm-D9 proteic sequence is all or part of, Zm-D9 albumen transcript 5 ' and/or the complement of 3 ' non-translational region is all or part of, these two complement all or part of of the encoding sequence of the proteic transcript of Zm-D9 of perhaps encoding and non-translational region.In addition, antisense polynucleotides can be fully complementary with target sequence complement 100% identity of target sequence (promptly with) or part complementary (promptly with the identity of the complement of target sequence less than 100%).Antisense Suppression can be used for suppressing the expression of multiple protein in the same plant.Referring to No. the 5th, 942,657, United States Patent (USP) for example.In addition, the part of antisense nucleotide can be used for destroying target gene expression.Generally speaking, can use at least 50 Nucleotide, 100 Nucleotide, 200 Nucleotide, 300,400,450,500, the sequence of the Nucleotide more than 550.Suppress method that the native gene in the plant expresses with Antisense Suppression and for example be described in Liu etc., (2002) Plant Physiol.129:1732-1743 and United States Patent (USP) the 5th, 759,829 and 5, in 942, No. 657, each all is attached to described document herein by reference.Can comprise the efficient that Antisense Suppression is improved in the poly-dT district by 5 of 3 of the antisense sequences in expression cassette ' position and poly-adenosine signal ' position.Referring to U.S. Patent Publication No. 20020048814, this patent is attached to herein by reference.
3. double-stranded RNA disturbs
In certain embodiments of the invention, can pass through double-stranded RNA (dsRNA) and disturb the inhibition that obtains the Zm-D9 protein expression.Disturb for dsRNA, in same cell, express with above to suppress altogether described similarly have adopted RNA molecule and with adopted RNA molecule complementary antisense rna molecule is wholly or in part arranged, produce the inhibition that corresponding endogenous messenger RNA(mRNA) is expressed.
Design the expression cassette that contains adopted sequence and antisense sequences and can realize the expression of justice and antisense molecule.Perhaps, can use independently expression cassette to justice and antisense sequences.Screening shows the department of botany that maximum Zm-D9 protein expression suppresses with the various plants system that one or more dsRNA disturb expression cassette to transform with evaluation then.Disturb the method that suppresses endogenous plant genetic expression to be described in Waterhouse etc., (1998) Proc.Natl.Acad.Sci.USA95:13959-13964 with dsRNA; Liu etc. are among (2002) Plant Physiol.129:1732-1743 and WO99/49029, WO 99/53050, WO 99/61631 and the WO 00/49035; Each all is attached to described document herein by reference.
4. hairpin RNA disturbs and contains the hairpin RNA interference of intron
In certain embodiments of the invention, can disturb or contain the inhibition of hairpin RNA (ihpRNA) the interference acquisition of intron by hairpin RNA (hpRNA) to the Zm-D9 protein expression.These methods efficiently suppress the expression of native gene.Referring to Waterhouse and Helliwell (2003) Nat.Rev.Genet.4:29-38 and the reference wherein mentioned.
Disturb for hpRNA, expression cassette is designed to express the RNA molecule that forms hairpin structure with self hybridization, and described hairpin structure contains single-stranded loop district and base pairing stem.All or part of adopted sequence being arranged and with this adopted sequence complementary antisense sequences is wholly or in part arranged corresponding to endogenous messenger RNA(mRNA) contained in base pairing stem district, and the expression of described endogenous messenger RNA(mRNA) encoding gene is suppressed.Therefore, the base pairing stem district of molecule determines RNA interferential specificity usually.The hpRNA molecule efficiently suppresses native gene expresses, and its inductive RNA disturbs and can be inherited by the plant offspring.Referring to for example Chuang and Meyerowitz (2000) Proc.Natl.Acad.Sci.USA 97:4985-4990; Stoutjesdijk etc., (2002) Plant Physiol.129:1723-1731; With Waterhouse and Helliwell (2003) Nat.Rev.Genet.4:29-38.The method of using hpRNA to disturb inhibition or silencer to express is described in for example Chuang and Meyerowitz (2000) Proc.Natl.Acad.Sci.USA 97:4985-4990; Stoutjesdijk etc., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell (2003) Nat.Rev.Genet.4:29-38; Pandolfini etc. are in BMCBiotechnology 3:7 and the U.S. Patent Publication No. 20030175965; Each all is attached to described document herein by reference.At Panstruga etc., the hpRNA construct instantaneous measurement of the efficient expressed of silencer has in vivo been described among (2003) Mol.Biol.Rep.30:135-140, the document is attached to herein by reference.
For ihpRNA, disturbing molecule has the universal architecture identical with hpRNA, but the RNA molecule contain in addition can be by the intron of montage in expressing the cell of ihpRNA.Use intron that the size of encircling after the montage in the hairpin RNA molecule is minimized, this has increased jamming effectiveness.Referring to for example Smith etc., (2000) Nature 407:319-320.In fact, the interference table of use ihpRNA such as Smith mediation reveals 100% native gene expression inhibiting.The method that disturb to suppress endogenous plant genetic expression with ihpRNA for example is described in Smith etc., (2000) Nature407:319-320; Wesley etc., (2001) Plant is J.27:581-590; Wang and Waterhouse (2001) Curr.Opin.Plant Biol.5:146-150; Waterhouse and Helliwell (2003) Nat.Rev.Genet.4:29-38; In Helliwell and Waterhouse (2003) Methods 30:289-295 and the U.S. Patent Publication No. 20030180945, each of described document all is attached to herein by reference.
Be used for that hpRNA interferential expression cassette can also be designed to adopted sequence and antisense sequences does not correspond to endogenous RNA.In this embodiment, have adopted sequence and antisense sequences to be positioned at ring sequence flank, the nucleotide sequence that described ring sequence contains is all or part of corresponding to the endogenous messenger RNA(mRNA) of target gene.Therefore, be ring district decision RNA interferential specificity.Referring to for example WO
02/00904, the document is attached to herein by reference.
Can pass through to use the hpRNA construct to realize transcriptional gene silencing (TGS), wherein the inverted repeats of hair clip and the gene promoter area consensus sequence identity of wanting silence.With hpRNA be processed as can with the interactional short rna in homologous promoter district can trigger the degraded or methylate, thereby cause silence (Aufsatz etc., (2002) PNAS 99 (4): 16499-16506; Mette etc., (2000) EMBO J 19 (19): 5194-5201).
V. the interference of amplicon mediation
The amplicon expression cassette contains the sequence in plant virus source, and this sequence contains all or part of target gene, but does not contain all genes of natural viral usually.The virus sequence that exists in the transcription product of expression cassette can make transcription product instruct and himself duplicate.That the transcript that is produced by amplicon can be justice with respect to target sequence (being the proteic messenger RNA(mRNA) of Zm-D9) or antisense.J.16:3675-3684 the method that suppresses endogenous plant genetic expression with amplicon for example is described in Angell and Baulcombe (1997) EMBO; Angell and Baulcombe (1999) Plant J.20:357-362 with United States Patent (USP) the 6th, 646, in No. 805, each of described document all is attached to herein by reference.
6. ribozyme
In certain embodiments, the polynucleotide of being expressed by expression cassette of the present invention are catalytic RNAs, perhaps the proteic messenger RNA(mRNA) of Zm-D9 are had specific ribozyme activity.Therefore, polynucleotide can make endogenous messenger RNA(mRNA) degraded, cause the proteic expression of Zm-D9 to reduce.This method for example is described in United States Patent (USP) the 4th, 987, No. 071, and this patent is attached to herein by reference.
7. siRNA or microRNA
In certain embodiments of the invention, the RNA that can pass through the gene of expression coding microRNA (miRNA) disturbs the inhibition that obtains the Zm-D9 protein expression.MiRNA is by about 22 regulatory factors that ribonucleotide is formed.MiRNA efficiently suppresses native gene to express.Referring to for example Javier etc., (2003) Nature 425:257-263, the document is attached to herein by reference.
Disturb for miRNA, expression cassette is designed to express the RNA molecule of miRNAs gene in the simulation.The RNA of miRNA genes encoding forms hairpin structure, this structure and another native gene (target sequence) complementation of the sequence that contains 22 Nucleotide.Be to suppress the Zm-D9 protein expression, the sequence of 22 Nucleotide is selected from Zm-D9 albumen transcript sequence, and contains 22 Nucleotide of described Zm-D9 protein sequence at sense orientation, and with 21 Nucleotide that the corresponding antisense sequences of adopted sequence complementary is arranged.The miRNA molecule efficiently suppresses native gene expresses, and their inductive RNA interference are inherited by the plant offspring.
B. Genetic expression based on polypeptide suppresses
In one embodiment, the zinc finger protein of polynucleotide encoding combines with the proteic gene of coding Zm-D9 in maize plant or cell, causes genetic expression to reduce.In specific embodiment, zinc finger protein combines with the control region of Zm-D9 protein gene.In other embodiments, zinc finger protein combines with the proteic messenger RNA(mRNA) of coding Zm-D9, and stops its translation.Select the method for zinc finger protein target site for example to be described in United States Patent (USP) the 6th, 453, No. 242, the method for using zinc finger protein to suppress the genetic expression in the plant is described in for example U.S. Patent Publication No. 20030037355; Each of described document all is attached to herein by reference.
C. Protein-active based on polypeptide suppresses:
In certain embodiments of the invention, polynucleotide encoding and the protein bound antibody of at least a Zm-D9, and reduce the proteic Zm-D9 activity of Zm-D9.In another embodiment, the combination of antibody causes the conversion of antibody-Zm-D9 albumen composition to increase by cell Quality Control mechanism.Antibody in vegetable cell expression and by expressing antibodies in vegetable cell and make antibody and protein binding to suppress molecular pathways well-known in this area.Referring to for example Conrad and Sonnewald (2003) Nature Biotech.21:35-36, the document is attached to herein by reference.
D. Gene disruption:
In certain embodiments of the invention, reduce or the proteic activity of removal Zm-D9 by destroying the proteic gene of coding Zm-D9.Can destroy the proteic gene of coding Zm-D9 by any means known in the art.For example, in one embodiment, destroy gene by transposon tagging.In another embodiment, following destruction gene: use at random or directed mutagenesis mutagenesis maize plant, and the plant of selecting the Zm-D9 protein-active to reduce or change.
1. transposon tagging
In one embodiment of the invention, use transposon tagging reduction or the proteic Zm-D9 activity of removal Zm-D9.Transposon tagging comprises transposon is inserted in the endogenous Zm-D9 gene, to reduce or to remove the Zm-D9 protein expression." Zm-D9 gene " is meant the proteic gene of coding Zm-D9 of the present invention.
In this embodiment, transposon is inserted reduce in the control region of the proteic gene of coding Zm-D9 or the coding region or remove the proteic expression of Zm-D9.Can use the exon that is arranged in the Zm-D9 gene, intron, 5 ' or the transposon of 3 ' non-translated sequence, promotor or any other regulating and controlling sequence reduce or remove coding proteic expression of Zm-D9 and/or activity.
The method of transposon tagging that is used for the plant specific gene is well-known in this area.Referring to for example Maes etc., (1999) TrendsPlant Sci.4:90-96; Dharmapuri and Sonti (1999) FEMS Microbiol.Lett.179:53-59; Meissner etc., (2000) Plant is J.22:265-274; Phogat etc., (2000) J.Biosci.25:57-63; Walbot (2000) Curr.Opin.Plant Biol.2:103-107; Gai etc., (2000) Nucleic Acids Res.28:94-96; Fitzmaurice etc., (1999) Genetics 153:1919-1928).In addition, at Bensen etc., (1995) Plant Cell 7:75-84; Mena etc., (1996) Science 274:1537-1540; With United States Patent (USP) the 5th, 962, the TUSC method of selecting Mu to insert in selected genes has been described in No. 764, each of described document all is attached to herein by reference.
2. the active mutant plant that reduces
Reducing or removing other method that native gene is expressed in the plant also is known in this area, and can be applied to the present invention similarly.These methods comprise the mutagenesis of other form, for example (adopt PCR) aspect reverse genetics and use the mutagenesis of ethyl methane sulfonate inductive, deletion mutagenesis and fast neutron deletion mutagenesis, with the department of botany that identifies that native gene has lacked.The example of these methods is referring to Ohshima etc., (1998) Virology 243:472-481; Okubara etc., (1994) Genetics 137:867-874; With Quesada etc., (2000) Genetics 154:421-436; Each of described document all is attached to herein by reference.In addition, use the rapid automatized method TILLING (the local sudden change of directional induction genome) of sex change HPLC or selected PCR product selectivity endonuclease digestion screening chemical induction sudden change also to can be applicable to the present invention.Referring to McCallum etc., (2000) Nat.Biotechnol.18:455-457, the document is attached to herein by reference.
Influence genetic expression or disturb the sudden change of proteins encoded function (Zm-D9 activity) well-known in this area.Insertion sudden change in gene extron produces null mutant usually.The conserved residues sudden change is effective especially to the Zm-D9 activity that suppresses proteins encoded.Described that to be suitable for to remove or to suppress Plant hormones regulators,gibberellins signal transduction activity in the plant DELLA albumen be the conserved residues of the mutagenesis of target.Referring to for example Itoh, H., M.Ueguchi-Tanaka etc., (2002) Plant Cell14:57-70.This class mutant can separate according to well-known method, and the sudden change in the different Zm-D9 locus can be piled up by genetic cross.Referring to for example Gruis etc., (2002) Plant Cell14:2863-2882.
In another embodiment of the invention, because gene is reverse and the reorganization of duplicate loci, dominant mutant can be used for triggering the RNA silence.Referring to for example Kusaba etc., (2003) Plant Cell 15:1455-1467.
The present invention includes other method that reduces or remove the Zm-D9 protein-active.Change or mutant plant in the example of other method of genome nucleotide sequence be known in the art, include but not limited to use RNA:DNA carrier, RNA:DNA mutational vector, RNA:DNA repair vector, mix double chain oligonucleotide, self complementary RNA:DNA oligonucleotide and cause the oligonucleotidase of reorganization.These carriers and using method are known in the art.Referring to for example United States Patent (USP) the 5th, 565,350,5,731,181,5,756,325,5,760,012,5,795,972 and 5,871, No. 984, described each patent all is attached to herein by reference.In addition referring to WO98/49350, WO 99/07865, WO 99/25821 and Beetham etc., (1999) Proc.Natl.Acad.Sci.USA 96:8774-8778; Described each document all is attached to herein by reference.
In certain embodiments, polynucleotide molecule of the present invention can be piled up mutually with the arbitrary combination of herbicide-tolerant polynucleotide sequence, have the plant of required proterties with generation.Proterties used herein is meant the phenotype that derives from particular sequence or sequence set.For example, polynucleotide molecule of the present invention can pile up mutually with any other polynucleotide molecule that coding has a polypeptide of sterilant and/or pesticide activity: for example other bacillus thuringiensis (Bacillus thuringiensis) toxalbumin (is described in U.S. Patent number 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; With Geiser etc., (1986) Gene48:109), lectin (Van Damme etc., (1994) Plant Mol.Biol.24:825), pentin (being described in U.S. Patent number 5,981,722) etc.The combination that produces can also comprise a plurality of copies of any herbicide-tolerant polynucleotide molecule.Polynucleotide molecule of the present invention can also pile up with any other gene or the assortment of genes, the plant that has multiple required proterties combination with generation, include but not limited at the required proterties of animal-feed: for example high oil base is because of (for example U.S. Patent number 6,232,529); Equilibrated amino acid (hordothionins (U.S. Patent number 5,990,389 for example; 5,885,801; 5,885,802; With 5,703,409); Barley high-lysine (Williamson etc., (1987) Eur.J.Biochem.165:99-106; With WO 98/20122) and homomethionine albumen (Pedersen etc., (1986) J.Biol.Chem.261:6279; Kirihara etc., (1988) Gene 71:359; With Musumura etc., (1989) Plant Mol.Biol.12:123)); The digestibility that the increases (storage protein of modification (the U. S. application series number 10/053,410 that submit to November 7 calendar year 2001) for example; And Trx (the U. S. application series number 10/005,429 that submit to December 3 calendar year 2001)); The disclosure of described document is attached to herein by reference.
Polynucleotide molecule of the present invention can also with pile up mutually at following required proterties: disease or Herbicid resistant (fumonisin detoxification gene (U.S. Patent number 5,792,931) for example; Avirulence and disease resistance gene (Jones etc., (1994) Science 266:789; Martin etc., (1993) Science 262:1432; Mindrinos etc., (1994) Cell 78:1089); Produce acetolactate synthestase (ALS) mutant of Herbicid resistant, for example S4 and/or Hra sudden change; Glutamine synthase inhibitor, for example Glufosinate (phosphinothricin) or Glufosinate (basta) (for example bar gene); And glyphosate resistance (EPSPS gene)); And processing or the required proterties of converted products, for example high oil (for example U.S. Patent number 6,232,529); Modified oil (fatty acid desaturase gene (U.S. Patent number 5,952,544 for example; WO 94/11516)); Treated starch (for example ADPG pyrophosphorylase (AGP enzyme), starch synthase (SS), Q-enzyme (SBE) and starch-debranching enzyme (SDBE)); (for example U.S. Patent number 5.602,321 with promoting polymkeric substance that polyhydroxyalkanoatefrom (PHA) is expressed or biological plastics; β-Tong Liuxiemei, polyhydroxybutyrate synthase and Acetoacetyl-CoA reductase (Schubert etc., (1988) J.Bacteriol.170:5837-5847)); Its disclosure is attached to herein by reference.People can also make up polynucleotide molecule of the present invention and the polynucleotide molecule of agronomic traits is provided, the for example male sterile of described agronomic traits is (for example referring to U.S. Patent number 5,583,210), bar intensity is (referring to U.S. Patent number 6,803,498), flowering time is (referring to U.S. Patent number 6,573,430) or transformation technology proterties, for example Cycle Regulation or gene targeting (for example WO 99/61619, WO 00/17364 and WO 99/25821); The disclosure of described document is attached to herein by reference.
These stacked combination can generate by any means, and described method comprises and be not limited to, by any conventional or TopCross method or genetic transformation cross-breeding plant.If pile up sequence by genetic transformation plant, then can be at any time and with any order composite object polynucleotide sequence.For example, can will comprise the transgenic plant of one or more required proterties as the target that imports other proterties by conversion subsequently.In the cotransformation scheme, can import proterties simultaneously with the herbicide-tolerant polynucleotide molecule that the arbitrary combination that transforms box provides.For example, if will import 2 sequences, then 2 sequences can be included in the conversion box separately (trans) or be included in (cis) in the same conversion box.The expression of sequence can be driven by identical promoters or different promoters.In some cases, may need to import the conversion box that will suppress the herbicide-tolerant polynucleotide expression.This can be combined with other arbitrary combination that suppresses box or overexpression box, to generate required proterties combination in plant.To recognize that also polynucleotide sequence can use site-specific recombination system to pile up in the genome position of needs.Referring to for example WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, they all are attached to herein by reference.
The lipid acid that changes plant that comprises interesting in the multiple change of phenotype is formed, is changed the aminoacids content of plant, changes the pathogenic agent defense mechanism of plant etc.These results can realize by expression that the allos product is provided or the expression that increases endogenous product in the plant.Perhaps, described result can realize by the reductions that provide one or more endogenous products to express, especially enzyme in the plant or cofactor.These changes cause transforming the phenotypic alternation of plant.
Target gene has reflected commodity market, and relates to those people's of this crop exploitation target.Crop and target market change, and along with developing country opens the world market, new crop and technology also will be emerged in large numbers.In addition, along with our increase to the understanding of agronomic traits and feature (for example output and hybrid vigour), the selection of the gene that is used to transform is also with corresponding change.The general classification of target gene comprises those genes that for example relate to information, and for example zinc refers to, relates to those genes of communication, kinases for example, and relate to those special genes, for example heat shock protein.Transgenosis classification more specifically for example comprises the gene of the important agronomic traits of coding, insect-resistant, disease resistance, Herbicid resistant, sterile, seed feature and commercialization product.Target gene generally comprises those genes that relate to oil, starch, carbohydrate or Nutrition and Metabolism, and those genes that influence grain size, sucrose carrying capacity etc.
Important proterties on agricultural, for example oil, starch and protein content can also change through genetic engineering except that using traditional breeding way.Modification comprises the content, the level that increases Methionin and sulphur that increase oleic acid, saturated and unsaturated oil, indispensable amino acid and starch conversion is provided.The protein modified U.S. Patent number 5,703 that is described in of Hordothionin, 049,5,885,801,5,885,802 and 5,990,389, these patents are attached to herein by reference.Another example is the seed albumen that is rich in Methionin and/or sulphur by soybean 2S albumin coding, it is described in U.S. Patent number 5,850,016, with the chymotrypsin inhibitor that derives from barley, it is described in Williamson etc., (1987) Eur.J.Biochem.165:99-106, and the disclosure of these documents is attached to herein by reference.
The derivative that can prepare encoding sequence by site-directed mutagenesis is to increase previously selected amino acid whose level in the coded polypeptide.For example, the gene source of coding barley high-lysine polypeptide (BHL) is in the barley chymotrypsin inhibitor, U. S. application sequence number 08/740,682 and WO 98/20133 that on November 1st, 1996 submitted to, the disclosure of described document is attached to herein by reference.Other albumen comprises the vegetable-protein that is rich in methionine(Met), for example from sunflower seeds (Lilley etc., (1989) Proceedings of the World Congress on VegetableProtein Utilization in Human Foods and Animal Feedstuffs, Applewhite edits, (American Oil Chemists Society, Champaign, Illinois), the 497-502 page or leaf; Described document is attached to herein by reference); Corn (Pedersen etc., (1986) J.Biol.Chem.261:6279; Kirihara etc., (1988) Gene 71:359; These two documents all are attached to herein by reference); And paddy rice (document is attached to herein by reference for Musumura etc., (1989) Plant Mol.Biol.12:123).Other is important function of gene coding latex, Floury 2, somatomedin, the seed storage factor and transcription factor on agricultural.
The insect-resistant gene can be encoded to the resistance of the insect that causes a large amount of underproduction, as rootworm, caterpillar, corn stalk borer etc.These genes comprise Tribactur toxoprotein gene (U.S. Patent number 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; With Geiser etc., (1986) Gene 48:109).
The gene of coding disease resistance proterties comprises the detoxification gene, for example the gene of anti-fumonisin (U.S. Patent number 5,792,931); Avirulence (avr) and disease resistance (R) gene (Jones etc., (1994) Science266:789; Martin etc., (1993) Science 262:1432; With Mindrinos etc., (1994) Cell 78:1089); Or the like.
The Herbicid resistant proterties can comprise the gene of the Herbicid resistant of encoding, described weedicide is used to suppress the effect of acetolactate synthase (ALS), especially sulfonylurea herbicide (acetolactate synthase (ALS) gene that for example contains sudden change, produce such resistance, particularly S4 and/or Hra sudden change); Described weedicide is used to suppress the active effect of glutamine synthase, for example Glufosinate ammonium (phosphinothricin) or Glufosinate ammonium (basta) (for example bar gene); Or glyphosate (for example EPSPS gene and GAT gene; Referring to for example US publication 20040082770 and WO03/092360); Other such gene perhaps known in the art.The bar genes encoding is to the resistance of weedicide Glufosinate ammonium (basta), and the nptII genes encoding is to the resistance of microbiotic kantlex and Geneticin, and the als gene mutant code is to the grand resistance of weedicide chlorine sulphur.
Sterile gene also can be encoded in expression cassette, for physics castrate provide one alternative.The example of the gene of Shi Yonging comprises preferred gene of male tissue and the gene with male sterile phenotype in this way, and as QM, it is described in U.S. Patent number 5,583,2100.Other gene comprises kinase gene and encodes male and female gamete are grown all those genes of virose compound.
The quality of seed reflects proterties, for example the quality and quantity and the Mierocrystalline cellulose level of You level and type, saturated and unsaturated, indispensable amino acid.In corn, the hordothionin albumen of modification is described in U.S. Patent number 5,703, and 049,5,885,801,5,885,802 and 5,990,389.
The commercialization characteristic also can be by one or more starch that for example is used for alcohol production or genes encodings that protein expression is provided of increasing.Another important commercial purposes that transforms plant for example is described in U.S. Patent number 5,602,321 for generating polymer and protoplastis.Promote the expression of polyhydroxyalkanoatefrom (PHA) such as the gene of β-Tong Liuxiemei, PHB enzyme (polyhydroxybutyrate synthase) and Acetoacetyl-CoA reductase (referring to Schubert etc., (1988) J.Bacteriol.170:5837-5847).
Exogenous genes products comprises plant enzyme and product and from other source those products of (comprising prokaryotic organism and other eukaryote).These products comprise enzyme, cofactor, hormone etc.Can increase protein level, especially have the level of the modified protein that the amino acid of improvement distributes, to improve the using value of plant.This realizes by expressing described albumen with enhanced aminoacids content.
" plant of being tried or vegetable cell " is meant wherein and realized gene alteration such as plant transformed or vegetable cell with respect to target gene, perhaps serve as reasons plant or cytogenetics that so changes and plant or the vegetable cell that contains this change." contrast " or " control plant " or " control plant cell " provides the reference point of measuring the phenotype variation that is tried plant or vegetable cell.
Control plant or vegetable cell for example can comprise: (a) wild-type plant or cell, and promptly itself and parent material have the homologous genes type, are used for gene alteration, produce to be tried plant or cell; (b) have the homologous genes type with parent material but used invalid construct (construct that promptly objective trait is not had known effect for example contains the construct of marker gene) plant transformed or vegetable cell; (c) be the plant or the vegetable cell of the segregant of non-conversion in the offspring who is tried plant or vegetable cell; (d) with tried the identical but plant or the vegetable cell that stimulate of the contact conditioned disjunction that can induce target gene to express not of plant or vegetable cell heredity, or (e) under the condition of not expressing target gene, tried plant or vegetable cell itself.
It is in order to set forth that following embodiment is provided, rather than in order to limit.
Embodiment 1
The separation of corn D9 gene
Two kinds of DELLA albumen of corn coding (dwarf plant 8, D8, and dwarf plant 9, D9), its several dominant mutants separated (Winkler and Freeling (1994) Planta193:341-348).Although on genetics, described Zm-D9 (Winkler and Freeling (1994) Planta 193:341-348), also on molecular level, do not characterize this gene in the past.In the process of this work, separated and analyzed two Zm-D9 allelotrope.Wild-type Zm-D9 allelotrope is the RNA separation of B73 by corn by RT PCR.Zm-D9MUT1 allelotrope separates (Fig. 1) by PCR by the call oneself genomic dna of seedling of the reactionless system of GA of D9xB73 of separation.Prediction Zm-D9 MUT1 does not have intron, and the DELLA gene of other report does not have yet.For guaranteeing to obtain the correct coding sequence of Zm-D9 MUT1, by RT-PCR check cDNA.
Zm-D8 is arranged in BIN1.09, and Zm-D9 is positioned at karyomit(e) 5, (Helentjaris etc., (1988) genetics 118:353-363 in the colinearity of BIN 5.00 (syntenous) district; Winkler and Freeling (1994) Planta 193:341-348; Lawrence etc., (2005) PlantPhysiol.138:55-58).For checking isolating allelotrope is the Zm-D9 form, starts a plurality of projects, comprises the BAC library screening and carries out the phenotype recurrence by transgenosis.Zm-D9 mapped to can not with heredity and the B73 BACbacb.pk425.i4 that links together of physical map, although the sign of its certain and karyomit(e) 1 and 5 on these two is chain.Different for showing the Zm-D9 that infers with Zm-D8, and determine its chromosomal localization, carry out pcr analysis (Fig. 2 of oat addition line; Ananiev etc., (1997) Proc.Natl.Sci.USA94:3524-3529), and by the confirmation of sequencing reaction product.This analyzes confirmation, and the Zm-D9 position of inferring is identical with karyomit(e) 5, and is different with the Zm-D8 locus on the karyomit(e) 1.
Determine two proteic Subcellular Localization of corn DELLA (Fig. 3) by the fluorescin syzygy.The Zm-D9 location of inferring is similar with the location of Zm-D8, and consistent with the location in nuclear.Write down hereof numerous DELLA albumen in other botanical system appraise and decide the position (Silverstone etc., (2001) Plant Cell 10:155-169; Ogawa etc., (2000) gene 245:21-29; Fleck and Harberd (2002) Plant J.32:935-947; Gubler etc., (2002) Plant Physiol.129:191-200; Wen and Chang (2002) Plant Cell14:87-100).Use following Multis ite Gateway (Invitrogen, USA) construct (Hiei etc., 1994 in the middle of the Japan Tobacco of improvement; Ishida etc., 1996) tungsten particle of bag quilt: PHP23800, attB4:UBI PRO:attB1:ZM-D8:attB2:AC-GFP1:NOSTERM:attB3 or PHP25355, attB4:UBIPRO:attB1:ZM-D9:attB2:AcGFP1:NOS TERM:attB3.In dark, germinate back 3 days (DAG) in room temperature, use Biolistic PDS-1000/He particle transfer system (BioRad, USA) and the 650psi sliver with above construct particle bombardment HG11 etiolated seedling.When DAG is 6 days, with CARV rotating disk confocal microscope (Fryer Company, USA) etiolated seedling that bombarded of visual inspection.
Produced the Arabidopis thaliana T2 plant of carrying by the corn DELLA cDNA of clover vascular system sclerenchyma cell preferred L S-S2a promoters driven.Dwarfing effect (by the most serious to least serious) shows as following Zm-D8 MUT〉Zm-D8 MPL 〉=Zm-D9MUT1〉Zm-D8=Zm-D9 (referring to Fig. 4).In addition, transfer-gen plant seems to have the floral shape of change as seen in fig. 5.As if in Zm-D8 MUT, Zm-D8 MPL, Zm-D9 and Zm-D9 MUT1, filigree preferentially is shortened, make pollen sac shorter than column cap.Zm-D8 MUT transfer-gen plant seems to be subjected to maximum effect, and Zm-D9 and Zm-D8MPL transfer-gen plant are influenced hardly.As if the filigree of GUS and Zm-D8 transfer-gen plant unaffected.Shown specific Z m-D8MUT transgenic lines also is male sterile, the pollen because it does not come off.
Disclosed result's conclusive evidence in Fig. 2-5, the isolating Zm-D9 of inferring allelotrope is the true allelotrope of Zm-D9.Isolating wild-type Zm-D9 is encoded on maize chromosome 5, with the same (Winkler and Freeling, 1994) before Zm-D9 measured.In the time will being merged by Zm-D9 encoded protein and fluorescent mark protein translation, it is present in the subcellular location consistent with nuclear, and other DELLA albumen also is like this.The most meaningfully, carry the allelic Arabidopis thaliana transfer-gen plant of Zm-D9MUT1 and downgrade, and carry wild-type those be normal plant type.These results confirm, in fact are responsible for corn by the isolating sudden change of the D9 sudden change maize seedling DELLA albumen allelotrope of downgrading and downgrade mechanism, and the identity that confirms this gene is Zm-D9.
Also in T2 Arabidopis thaliana plant, studied expressing the effect of the proteic root architecture of corn DELLA.All plants all grew on vertical square culture dish in the vehicle-mounted lighting system of ArabiSun under the length 18 hour daytime.Measure average root length and the average tip of a root quantity of every strain plant in the time of back 10 days in germination, the result is summarized in table 1.Comparison is compared according to plant (GUS), and the average root length of plant of carrying Zm-D8MPL, Zm-D8MUT and MUT1 Zm-D9 construct is obviously shorter, and the tip of a root of every strain plant obviously still less.Therefore, the same with the Zm-D8 gene, the Zm-D9 gene participates in control, especially root length and the root branch (the average tip of a root number with every strain plant is a card) of root architecture.
Table 1. is expressed the effect (Boyes etc., (2001) Plant Cell 13:1499-1510) of corn DELLA albumen to the root architecture in transgenosis (T2) Arabidopis thaliana plant main vegetative period 1.03.
Construct Average root length (cm) Average tip of a root quantity
MS-S2a PRO::GUS 6.34 a 9.73 d
MS-S2a PRO::Zm-D8 5.97 a 9.10 d
MS-S2a PRO::Zm-D8MPL 4.82 c 4.30 e
MS-S2a PRO::Zm-D8MUT 4.69 c 5.00 e
MS-S2a PRO::Zm-D9 5.77 ab 9.66 d
MS-S2a PRO::MUT1Zm-D9 5.15 bc 6.00 e
It is not remarkable different group each other that the indication of subscript letter is analyzed in 95% confidence level according to LSD.Data are collected in 4-15 replicate(determination) by 4 independent transformation events.
Embodiment 2
The regeneration of Zm-D9 maize transformation plant and transgenic plant
With containing Zm-D9 and the selected marker PAT (Wohlleben etc. that effectively are connected with MS-S2A promotor (MS-S2a PRO), (1988) plasmid bombardment Gene 70:25-37) derives from the prematurity maize of greenhouse supply plant, and described selected marker PAT gives the resistance to the weedicide bialaphos.Perhaps, the selected marker is independently providing on the plasmid.The following conversion.Culture medium prescription is as follows.
The preparation of target tissue
Remove the shell of fringe, and surface sterilization 20 minutes in adding the 30%Clorox sodium hypochlorite solution of 0.5%Micro denaturing agent, with aseptic water washing 2 times.Downcut immature embryo, and (on the cotyledon dish side direction) under the plumular axis side direction be placed on reach 4 hours on the 560Y substratum, each flat board is put 25 embryos, aligns in the 2.5cm target area then, prepares bombardment.
Preparation contains the plasmid vector of the Zm-D9 that effectively is connected with effable promotor in plant.Following use CaCl 2The precipitator method are deposited in this plasmid DNA and the plasmid DNA that contains the PAT selected marker on 1.1 μ m (mean diameter) tungsten particles: the aqueous solution of the tungsten particle of 100 μ l preparation; The Tris edta buffer liquid (the total DNA of 1 μ g) of 10 μ l (1 μ g) DNA; 100 μ l 2.5M CaCl 2With 10 μ l 0.1M spermidines.
When on the multitube vortice, keeping every kind of reagent is added in the tungsten particle suspension successively.To the of short duration supersound process of final mixture, and make its incubation 10 minutes under constant vortex.At the precipitation after date, each is managed of short duration centrifugal, remove liquid, with 500ml 100% washing with alcohol, and centrifugal 30 seconds.Remove liquid once more, add 105 μ l, 100% ethanol to final tungsten particle precipitation.For particle gun bombardment,, 10 μ l point samples in each huge carrier center, and are dried about 2 minutes before bombardment to tungsten/of short duration supersound process of DNA particle.
Sample plate is bombarded with horizontal #4 in particle gun.All samples is all accepted the single bombardment of 650 PSI, and the particle/DNA that is equipped with by every control takes out 10 sample aliquot altogether.
After bombardment, embryo remained on reach 2 days on the 560Y substratum, be transferred to then and contain the 560R that 3mg/ rises bialaphos and select substratum, and per 2 weeks carry out the cultivation of going down to posterity for 1 time.After selecting in about 10 weeks, the resistant calli clone who selects is transferred to the 288J substratum, to start plant regeneration.After somatic embryo maturation (2-4 week), the somatic embryo of well growing is transferred to germination medium, and is transferred to the illumination cultivation chamber.After about 7-10 days, the 272V that the plantlet of growing is transferred in pipe does not have hormone culture-medium cultivation 7-10 days, grows fine until plantlet.Then, plant is transferred in flat board (the being equivalent to 2.5 ＂ basins) liner that is covered with potting soil, is placed on 1 week of growth in the culturing room, be placed on regrowth 1-2 week in the greenhouse subsequently, then be transferred in classical 600 basins (1.6 gallons), and grow to maturation.Monitoring plant, and record plant height.MUT1 Zm-D9 plant height reduces about 60% in this stage.
Bombardment substratum (560Y) contains 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml/l ErikssonShi vitamine mixture (1000X SIGMA-1511), 0.5mg/l vitamin, 120.0g/l sucrose, 1.0mg/l 2, and 4-D and 2.88g/l L-proline(Pro) (are adjusted to pH 5.8 back D-I H with KOH 2The O constant volume); (use D-I H 2.0g/l take off acetyl gellan gum (Gelrite) 2Add behind the O constant volume); With 8.5mg/l Silver Nitrate (adding) at medium sterilization and after being cooled to room temperature.Select substratum (560R) to contain 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml/lErikssonShi vitamine mixture (1000X SIGMA-1511), 0.5mg/l vitamin, 30.0g/l sucrose and 2.0mg/l 2,4-D (is adjusted to pH 5.8 back D-I H with KOH 2The O constant volume); (use D-I H 3.0g/l take off the acetyl gellan gum 2Add behind the O constant volume); And 0.85mg/l Silver Nitrate and 3.0mg/l bialaphos (the two all adds at medium sterilization and after being cooled to room temperature).
Plant regeneration substratum (288J) contains 4.3g/l MS salt (GIBCO 11117-074), (0.100g nicotinic acid, 0.02g/l vitamin, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine are with purified D-I H for 5.0ml/l MS VITAMIN mother liquor 2The O constant volume) (Murashige and Skoog (1962) Physiol.Plant.15:473), 100mg/l inositol, 0.5mg/l zeatin, 60g/l sucrose and 1.0ml/l 0.1mM dormin (are used purified D-I H after being adjusted to pH5.6 2The O constant volume); (use D-I H 3.0g/l take off the acetyl gellan gum 2Add behind the O constant volume); With 1.0mg/l indolylacetic acid and 3.0mg/l bialaphos (adding) at medium sterilization and after being cooled to 60 ℃.No hormone culture-medium (272V) contains 4.3g/l MS salt (GIBCO 11117-074), (0.100g/l nicotinic acid, 0.02g/l vitamin, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine are with purified D-I H for 5.0ml/l MS VITAMIN mother liquor 2The O constant volume), 0.1g/l inositol and 40.0g/l sucrose (are used purified D-I H after being adjusted to pH5.6 2The O constant volume); (using purified D-I H with the 6g/l bacteria Agr 2Add behind the O constant volume), sterilize and be cooled to 60 ℃.
Bombardment and substratum
Bombardment substratum (560Y) contains 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml/l ErikssonShi vitamine mixture (1000X SIGMA-1511), 0.5mg/l vitamin, 120.0g/l sucrose, 1.0mg/l 2, and 4-D and 2.88g/l L-proline(Pro) (are adjusted to pH 5.8 back D-I H with KOH 2The O constant volume); (use D-I H 2.0g/l take off the acetyl gellan gum 2Add behind the O constant volume); With 8.5mg/l Silver Nitrate (adding) at medium sterilization and after being cooled to room temperature.Select substratum (560R) to contain 4.0g/l N6 basis salt (SIGMA C-1416), 1.0ml/l ErikssonShi vitamine mixture (1000X SIGMA-1511), 0.5mg/l vitamin, 30.0g/l sucrose and 2.0mg/l 2,4-D (is adjusted to pH 5.8 back D-I H with KOH 2The O constant volume); (use D-I H 3.0g/l take off the acetyl gellan gum 2Add behind the O constant volume); With 0.85mg/l Silver Nitrate and 3.0mg/l bialaphos (the two all adds at sterilising medium and after being cooled to room temperature).
Plant regeneration substratum (288J) contains 4.3g/l MS salt (GIBCO 11117-074), (0.100g nicotinic acid, 0.02g/l vitamin, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine are with purified D-I H for 5.0ml/l MS VITAMIN mother liquor 2The O constant volume) the 0.1mM dormin of (Murashige and Skoog (1962) Physiol.Plant.15:473), 100mg/l inositol, 0.5mg/l zeatin, 60g/l sucrose and 1.0ml/l (is being adjusted to pH 5.6 back D-I H 2The O constant volume); (use D-I H 3.0g/l take off the acetyl gellan gum 2Add behind the O constant volume); With 1.0mg/l indolylacetic acid and 3.0mg/l bialaphos (adding) at medium sterilization and after being cooled to 60 ℃.No hormone culture-medium (272V) contains 4.3g/l MS salt (GIBCO 11117-074), (0.100g/l nicotinic acid, 0.02g/l vitamin, 0.10g/l pyridoxine hydrochloride and 0.40g/l glycine are with purified D-I H for 5.0ml/l MS VITAMIN mother liquor 2The O constant volume), 0.1g/l inositol and 40.0g/l sucrose (are being regulated pH to 5.6 back purified D-I H 2The O constant volume); (using purified D-I H with 6g/l bacterium-agar 2Add behind the O constant volume), sterilize and be cooled to 60 ℃.
Embodiment 3
The agrobacterium-mediated corn that contains Zm-D9 transforms and transforms the regeneration of plant
Corn for the mediation of the edaphic bacillus (Agrobacterium) that carries out with one or more Zm-D9 Nucleotide polynucleotide molecules of the present invention transforms, and uses method (U.S. Patent number 5,981,840 and PCT patent publication No. WO98/32326 of Zhao; Its content is hereby incorporated by).In brief, from immature embryo, described embryo is contacted by corn dividing with edaphic bacillus suspension, wherein this bacterium can be transferred to target Zm-D9 polynucleotide at least 1 cell (step 1: infect step) of at least 1 immature embryo.In this step, with immature embryo immerse begin in the edaphic bacillus suspension inoculation.Embryo and edaphic bacillus are cultivated for some time altogether (step 2: be total to culturing step).After infecting step, immature embryo is cultivated on solid medium.After this common cultivation stage, imagined optional " dormancy " step.In this sleep step, incubation embryo in the presence of the microbiotic of at least a kind of known inhibition edaphic bacillus growth, and do not add the selective agent (step 3: sleep step) of vegetable transformant.Immature embryo is cultivated on the solid medium of selective agent having microbiotic but do not have, be used for the elimination of edaphic bacillus and the resting stage of cells infected.Then, the embryo of inoculation is cultivated containing on the substratum of selective agent, and reclaimed the transformed calli (step 4: select step) of growth.Immature embryo is cultivated having on the solid medium of selective agent, made transformant produce selective growth.Callus regeneration is a plant (step 5: regeneration step), and will cultivate on solid medium at the callus of growing on the selective medium, with regeneration plant then.
Embodiment 4
Regeneration with Zm-D9 soybean transformation embryo and conversion plant
Culture condition
Soybean embryo generation suspension culture (cultivated variety Jack) is remained among the 35ml liquid nutrient medium SB196 (prescription vide infra) on 150rpm, the 26 ℃ of rotary shakers, according to 16:8 hour the daytime/the cold white fluorescent lighting of noctilucence cycle, light intensity is 60-85 μ E/m2/s.The inoculation of about 35mg tissue is gone among the 35ml fresh liquid SB196, to 2 weeks culture was gone down to posterity in per 7 days and to cultivate (preferably go down to posterity to cultivate and be spaced apart per 7 days 1 time) 1 time.
Be used in the plasmid described in following examples and dna fragmentation by particle gun bombardment method soybean transformation embryo generation suspension culture (Klein etc., (1987) Nature, 327:70).
Start soybean embryo generation suspension culture
Starting soybean 2 times every month and cultivate, is 5-7 days between each the startup.
Had the beanpod of immature seed by available soybean plant strain selection in 45-55 days after plantation, the shell of removing them is placed in the magenta sterile cupboard.Jolting sterilization in 15 minutes soybean seeds in containing the 5%Clorox solution of 1 ivory soap (distilled water of 95ml high pressure steam sterilization adds 5ml Clorox and 1 soap).Thorough mixing.Use 2 bottles 1 liter sterile distilled water flushing seed, will place on the independent microslide less than those seeds of 4mm.Downcut the little end of seed, cotyledon is extruded kind of a skin.Cotyledon is transferred to the flat board (each flat board is put 25-30 cotyledon) that contains the SB1 substratum.Dull and stereotyped with the fiber band parcel, and stored for 8 weeks.Then, downcut secondary embryo, and place the SB196 liquid nutrient medium to reach 7 days.
Prepare bombardment DNA
Complete plasmid or DNA plasmid fragment that use contains target gene and selected marker are bombarded.The plasmid DNA that conventional preparation is used to bombard, and use at Promega TMThe method purifying of describing among the Protocols and Applications Guide, second edition (the 106th page).Plasmid by the gel separation double digested obtains the plasmid fragment of carrying the Zm-D9 polynucleotide.In each case, digestion 100 μ g plasmid DNA in being suitable for the 0.5ml certain enzyme mixture of target plasmid.The dna fragmentation that obtains carries out gel electrophoresis on 1% SeaPlaque GTG agarose (BioWhitaker Molecular Applications) separates, by downcutting the dna fragmentation that contains the Zm-D9 polynucleotide on the sepharose.Use the GELase digestive ferment according to manufacturer's scheme by the agarose purify DNA.
The sterile distilled water that will contain 50 μ l equal portions of 3mg gold grain (3mg gold) adds 1 μ g/ μ l dna solution (Zhi Bei complete plasmid or dna fragmentation as mentioned above), the 50 μ l2.5M CaCl of 5 μ l 2In 20 μ l 0.1M spermidines.With mixture with level 3 joltings of vortex vibrator 3 minutes, and in desk centrifuge centrifugal 10 seconds.After with 400 μ l, 100% washing with alcohol, supersound process is suspended in particulate matter in 40 μ l, 100% ethanol.5 μ l DNA suspension are assigned to each flight dish of Biolistic PDS1000/HE plant tray.Per 5 μ l sample aliquot contain the about 0.375mg gold of each bombardment (being every dish).
Tissue preparation and bombard with DNA
The embryo suspension culture in 7 day age of about 150-200mg is placed empty aseptic 60 * 15mm culture dish, and culture dish covers with plastic wire.Bombard tissue with each dull and stereotyped 1 or 2 shooting, film rupture pressure is set to 1100PSI, plays the chamber and is evacuated to 27-28 inch of mercury vacuum tightness.Tissue is placed apart from keeping/stop 3.5 inches places of agreement that contracts a film or TV play to an actor or actress.
Transform the selection of embryo
Use Totomycin (when using the hygromix phosphotransferase HPT gene) or chlorine sulphur grand (when using the acetolactate synthase als gene) to select to transform embryo as selected marker as selected marker.
Totomycin (HPT) is selected
After bombardment, tissue is placed fresh SB196 substratum, and cultivate as mentioned above.In bombardment back 6 days, change SB196 with the fresh SB196 that contains 30mg/L Totomycin selective agent.Upgrade weekly and select substratum.In 4-6 week after selection, can be observed is grown thickly by unconverted downright bad embryo grows green transforming tissue.Take out isolating chlorenchyma, and inoculation is gone in the porous flat plate, to produce conversion embryo generation suspension culture new, clonal propagation.
Chlorine sulphur grand (ALS) is selected
After bombardment, tissue is distributed in 2 flasks that contain fresh SB196 substratum, and cultivates as mentioned above.After bombardment 6-7 days, change SB196 with the fresh SB196 that contains the grand selective agent of 100ng/ml chlorine sulphur.Upgrade weekly and select substratum.In 4-6 week after selection, can be observed is grown thickly by unconverted downright bad embryo grows green transforming tissue.Take out isolating chlorenchyma, and inoculate to go into to contain in the porous flat plate of SB196, to produce conversion embryo generation suspension culture new, clonal propagation.
Somatic embryos of soybean is regenerated as plant
In order to obtain complete stool by embryo generation suspension culture, must regenerating tissues.
The embryo maturation
Under cold white fluorescence (the cold white Econowatt F40/CW/RS/EW of Phillips) and Agro (Phillips F40Agro) bulb (40 watts),, embryo is cultivated 4-6 week in 26 ℃ in SB196 with 16:8 hour photoperiod and 90-120uE/m2s light intensity.After this, the embryo clump is moved to solid nutrient agar SB166 and reach 1-2 week.Then each clump gone down to posterity to cultivate in substratum SB103 and reached for 3 weeks.
Embryo dehydration and germination
Sophisticated single embryo is placed empty little culture dish (35 * 10mm) about its dehydrations of 4-7 angel.With fiber band seal plate (producing little moistening chamber).The semina of dehydration is implanted in the SB71-4 substratum, in this substratum, they is germinateed under above-mentioned identical culture condition.Take out the plantlet that germinates by the germination substratum, and the water cleaning down, be planted in then among the Redi-Earth of 24-hole tray (24-cell pack tray), cover with transparent plastic lousing.After 2 weeks, remove cover, plant is through 1 week of cold resistant training.If it is strong that plantlet seems, then they are transplanted to 10 " Redi-Earth of basin, each basin is 3 plantlets at the most.After week, gather in the crops mature seed, cracked and analyzing proteins at 10-16.
Culture medium prescription
SB196-FN less salt liquid proliferated culture medium (every liter)-
MS FeEDTA-100 * stoste 1 10ml
MS vitriol-100 * stoste, 2 10ml
FN less salt halogenide-100 * stoste, 3 10ml
FN less salt P, B, Mo-100 * stoste 4 10ml
B5 VITAMIN (1ml/L) 1.0ml
2,4-D (10mg/L final concentration) 1.0ml
KNO 3 2.83gm
(NH 4) 2SO 4 0.463gm
L-asparagine 1.0gm
Sucrose (1%) 10gm
pH 5.8
FN less salt stoste
Mother liquor # 1000ml 500ml
1MS Fe EDTA 100 * stoste
Na 2EDTA * 3.724g 1.862g
FeSO 4-7H 2O 2.784g 1.392g
*At first add, when stirring, be dissolved in the black bottle
2MS vitriol 100 * stoste
MgSO 4-7H 2O 37.0g 18.5g
MnSO 4-H 2O 1.69g 0.845g
ZnSO 4-7H 2O 0.86g 0.43g
CuSO 4-5H 2O 0.0025g 0.00125g
3FN less salt halogenide 100 * stoste
CaCl 2-2H 2O 30.0g 15.0g
KI 0.083g 0.0715g
CoCl 2-6H 2O 0.0025g 0.00125g
4FN less salt P, B, Mo 100 * stoste
KH 2PO 4 18.5g 9.25g
H 3BO 3 0.62g 0.31g
Na 2MoO 4-2H 2O 0.025g 0.0125g
SB1 solid medium (every liter) contains: 1 bag MS salt (Gibco/BRL-catalog number (Cat.No.) 11117-066); 1ml B5 VITAMIN 1000X mother liquor; 31.5g sucrose; 2ml 2,4-D (20mg/L final concentration); PH 5.7; With 8g TC agar.
SB 166 solid mediums (every liter) contain: 1 bag MS salt (Gibco/BRL-catalog number (Cat.No.) 11117-066); 1ml B5 VITAMIN 1000X mother liquor; 60g maltose; 750mg MgCl 2Hexahydrate; The 5g gac; PH 5.7; Take off the acetyl gellan gum with 2g.
SB 103 solid mediums (every liter) contain: 1 bag MS salt (Gibco/BRL-catalog number (Cat.No.) 11117-066); 1ml B5 VITAMIN 1000X mother liquor; 60g maltose; 750mg MgCl 2Hexahydrate; PH 5.7; Take off the acetyl gellan gum with 2g.
SB 71-4 solid medium (every liter) contains: 1 bottle of GamborgShi B5 salt w/ sucrose (Gibco/BRL-catalog number (Cat.No.) 21153-036); PH 5.7; With 5g TC agar.
2,4-D stoste is the Phytotech catalog number (Cat.No.) D295 pre-preparation acquisition of 1mg/ml by concentration.
The B5 VITAMIN stoste (every 100ml) that is stored in-20 ℃ with sample aliquot contains: the 10g inositol; 100mg nicotinic acid; The 100mg pyridoxine hydrochloride; With 1g VitB1.If this solution dissolves soon inadequately, then apply low-level heat through heating stirrer.The grand mother liquor of chlorine sulphur contains the 0.01N solution of ammonium hydroxide of 1mg/ml.
Embodiment 5
Carry out the regeneration that the Sunflower Receptacle meristematic tissue transformed and transformed plant with Zm-D9
(other is referring to european patent number EP0486233 with the following conversion of the expression cassette Sunflower Receptacle meristematic tissue that contains the Zm-D9 polynucleotide molecule of the present invention that effectively is connected with the MS-S2A promotor, this patent is hereby incorporated by, with Malone-Schoneberg etc., (1994) Plant Science 103:199-207).Use 1 wheat head thresing machine to make sophisticated sunflower seeds (Helianthus annuus L.) shelling.The 20%Clorox liquid lime chloride that adds 2 polysorbas20s with every 50ml solution was with seed-coat sterilization 30 minutes.Wash seed twice with sterile distilled water.
By the Schrammeijer that revises etc., the plumular axis explant that (Schrammeijer etc., (1990) Plant CellRep.9:55-60) described program preparation is split.After the surface sterilization program, seed immersed and reach 60 minutes in the distilled water.Fracture the then cotyledon of every seed obtains clean section on the plumular axis plane.After cutting off root tips, with explant between prophyll vertically to dividing.The cut surface of two hemisection faces upwards is placed on the GBA substratum, the GBA substratum is by Murashige and Skoog mineral element (Murashige etc., (1962) Physiol.Plant.15:473-497), (Shepard (1980) is stated from the ShepardShi vitamin addn: EmergentTechniques for the Genetic Improvement of Crops (University ofMinnesota Press, St.Paul, Minnesota), the 40mg/l adenine sulfate, 30g/l sucrose, 0.5mg/l6-benzyl-aminopurine (BAP), 0.25mg/l indole-3-acetic acid (IAA), 0.1mg/l gibberic acid (GA3), pH5.6 and 8g/l plant agar are formed.
Before edaphic bacillus is handled, explant is carried out micropellet bombardment (Bidney etc., (1992) Plant Mol.Biol.18:301-313).30-40 explant is placed on the dull and stereotyped center of 60 * 20mm with ring-type carries out this processing.The little bullet of 1.8mm tungsten of about 4.7mg is resuspended in the aseptic TE damping fluid of 25ml (pH 8.0 for 10mM Tris HCl, 1mM EDTA), and the 1.5ml sample aliquot is used in each bombardment.Every flat board is at PDS
Figure A200780022452D0074145105QIETU
150mm nytex screen bombardment through placing 2cm on the sample in the particle accelerator 2 times.
In all transformation experiments, all use and unload nail root knurl edaphic bacillus bacterial strain EHA105.According to Holsters etc., (1978) the described freeze-thaw method of Mol.Gen.Genet.163:181-187 will contain among the binary plasmid carrier importing edaphic bacillus bacterial strain EHA105 of expression cassette, and described expression cassette comprises the Zm-D9 polynucleotide molecule that effectively is connected with described promotor.This plasmid also comprises kantlex selected marker (being nptII).The bacterium that is used for the Plant Transformation experiment is at liquid YEP substratum (10g/l yeast extract, 10g/l bacto peptone and 5g/l NaCl, pH 7.0) middle incubated overnight (28 ℃ and 100RPM continuously stirring), described liquid YEP substratum contains keeps bacterial isolates and the required suitable microbiotic of binary plasmid.Suspension is at OD 600Use when reaching about 0.4-0.8.Precipitation edaphic bacillus cell, and with containing 12.5mM MES pH 5.7,1g/l NH 4Cl and 0.3g/l MgSO 4The inoculation medium resuspension to final OD 600Be 0.5.
The fresh explant that bombarded is placed edaphic bacillus suspension, mix, and left standstill 30 minutes.Then explant is transferred to the GBA substratum, and tangent plane downwards in 26 ℃ to cultivate altogether 18 hour daytime.After cultivating 3 days altogether, explant is transferred among the 374B (not having growth regulator and sucrose level to be reduced to 1% GBA substratum) that adds 250mg/l cefotaxime and 50mg/l sulphuric acid kanamycin.Explant is selected to cultivate 2-5 week, transfer to the fresh 374B substratum relaying supervention that does not contain kantlex then and educated for 1 to 2 week.To have explant differentiation, antibiotics resistant, that do not produce the vitellarium of the branch be suitable for excising as yet and transfer in the GBA substratum that contains the 250mg/l cefotaxime, handle in order to the plant hormone that carried out 3 days for the second time.There is situation in the NPTII that measures from the leaf sample of the branch of green, anti-kantlex by ELISA, and by measuring the situation that exists of Zm-D9 determination of activity transgene expression.
The positive branch grafting of NPTII is arrived Hybrid 6440 growth in vitro Sunflower Receptacle seedling rootstocks.The seed that makes surface sterilization is sprouted in 48-0 substratum (half dense Murashige and Skoog salt, 0.5% sucrose, 0.3% take off the acetyl gellan gum, and pH 5.6), and grows cultivating under the described condition at explant.Remove seedling top, on hypocotyl, make the 1cm terrace cut slice, then the branch that transforms is inserted otch.Whole zone is with Parafilm (parafilm) parcel, with the protection branch.Can after 1 all vitro culture, the plant of grafting be transferred in the soil.Transplant in soil is remained under the high humidity, then greenhouse is slowly adapted to.Sophisticated T in the greenhouse 0The transform portion of plant (parent's generation) is identified by the NPTIIELISA of leaf extract.
Embodiment 6
The expression of Zm-D9 and sign
Express spectra shows, sees that by growing viewpoint d9 demonstrates the preference to sophisticated noble cells, especially those and bar bonded noble cells, and d8 more relevant with somatoblast or meristematic cell (Figure 10).For example, the expression of d8 in branch split plot of meristematic tissue, bar (internode) and zone of transition is obviously higher, and d8 and d9 roughly express in the maturation zone of internode coequally.The high expression level of d8 and d9 is respectively in fringe and vascular bundle.Generally speaking, two kinds of expression of gene having of dimension pipe organ-/ tissue all are higher than the expression of non-dimension pipe organ-/ tissue.This is with among the dicotyledons vascular system and DELLA mRNA on every side and proteic situation consistent (Haywood etc., (2005) the Plant Journal 42:49-68 of existing; Israelsson etc., (2005) Plant Journal 44:494-504), prompting is located between dicotyledons and monocotyledons and is guarded.
For further analyzing the allelic character of D9, set up structural domain exchange construct, and it is transformed in the Arabidopis thaliana.5 heredity districts of exchange (Figure 11) between d9 and the ABC of clone of D9, the chimeric the ABC of clone that use obtains sets up S2A PRO::DELLA intermediate and common integrative vector, is used for transforming as described in to natural corn allelotrope.Carry out the morphometric Analysis (Figure 14) of structural domain exchange transfer-gen plant in T1 generation.The E600K sudden change that derives from D9 is necessary and enough for dwarfing and the variation of more precocious phenotype.D9 E600K influences different with their main chain allelotrope (Figure 14) with the form that D9K597E produces.The most noticeable difference is plant height, silique length, to the fate of blooming and the quantity of the lotus throne leaf when blooming.For whole 4 kinds of situations, d9 (E600K) plant all demonstrates the feature that is similar to D9.It seems that on average stem and the silique of the plant that d9 (E600K) mosaic produces are the shortest, the lotus throne leaf when any in 10 constructs bloomed is all minimum.On the contrary, the silique length of D9 K597E, to the fate of blooming and the lotus throne number of sheets amount when blooming come second high or the 3rd height.There is not other polymorphism to show height or flowering time change pattern clearly.This sudden change and other sudden change therefore in the corn plant type to having specific end use aspect the change of the high production capacity configuration of seed type.
Corn DELLA dwarfing allelotrope is found the acceleration Arabidopis thaliana and blooms.D8 MPL and D8 MUT will be bloomed and be moved precontract 6 days (Figure 12).Noticeable is that D9 quickens to bloom to reach 11 days (26.5%).As if this effect is relevant with Plant hormones regulators,gibberellins-insensitivity, because the flowering time of d8 and d9 transfer-gen plant is significantly not different with the GUS contrast.The D9 gene is bloomed T0GS3xGaspe Flint and is delayed (Figure 13), and d9 causes blooming in advance and (uses the basis of ground all nodes as the maturation transformation; Figure 12).Also not observing flowering time in d8 or D8 MPL transgenic corns changes.
Select the MS-S2A promotor, to drive the expression of 5 corn DELLA allelotrope in transgenic arabidopsis.Rice actin 1 promotor is used for driving these allelotrope and expresses in the transgenic arabidopsis group of doing sth. in advance.The T1 plant of these transformant does not show any visible phenotype, and prompting rice actin 1 promotor is not expressed DELLA albumen in suitable tissue.People's such as the corn express spectra (Figure 10) of known this result, d8 and d9 and Haywood works ((2005) Plant Journal 42:49-68) shows that DELLA albumen is related with the vascular system of mRNA in 3 species, select the MS-S2A promotor, be used for carrying out corn DELLA expression at Arabidopis thaliana and corn.Select transgenic method, so that can directly carry out corn allelotrope relatively, this should not can be activated sub-dependency effect and twists.These data have been proved conclusively the purposes of tissue-specific promoter as the strategy that changes plant architecture.
Embodiment 7
The Zm-D9 variant
A. do not change the variant nucleotide sequence (SEQ IDNO:1,3,4 or 6) of the Zm-D9 of amino acid sequence coded
Be used to produce the variant nucleotide sequence at the Zm-D9 nucleotide sequence shown in the SEQ ID NO:1,3,4 or 6, when comparing with SEQ ID NO:1,3,4 or 6 initial unaltered ORF nucleotide sequence, the nucleotide sequence of the open reading-frame (ORF) that described variant nucleotide sequence has has about nucleotide sequence homology of 70%, 76%, 81%, 86%, 92% and 97%.These functional variants use the standard cipher sublist to produce.Although the nucleotide sequence of variant is changed, do not change by the open reading-frame (ORF) amino acid sequence coded.
The variant aminoacid sequence of B.Zm-D9
Produce the variant aminoacid sequence of Zm-D9.In this embodiment, change an amino acid.Specifically, investigate the aminoacid sequence of statement in SEQ ID NO:2 or 5, to determine suitable amino acid change.By the amino acid that will change being selected with reference to (with another lineal homologues of a plurality of species and other gene family member) albumen comparison.Referring to Fig. 7.Selected amino acid is considered to not be in (non-high conservative) under the high selective pressure, but is had the aminoacid replacement of similar chemical feature (being the similar functionality side chain) easily.Use can change suitable amino acid in the comparison of the albumen shown in Fig. 7.In case identify target amino acid, just be implemented in the method for summarizing among the embodiment 6A.Use this method to produce the variant that has about 70%, 75%, 81%, 86%, 92% and 97% nucleotide sequence identity with SEQ ID NO:1,3,4 or 6.
Other variant aminoacid sequence of C.Zm-D9
In the present embodiment, produce the artificial sequence protein that has 82%, 87%, 92% and 97% identity with respect to the reference protein sequence.After this a need of work is identified conserved regions and variable region by the comparison shown in Fig. 7, uses the aminoacid replacement table then advisably.This part will be discussed hereinafter in more detail.
The aminoacid sequence that will change to a great extent based on the conserved regions in Zm-D9 albumen or other DELLA albumen decision.Referring to Fig. 7.Based on sequence alignment, the Zm-D9 of Gai Bianing represents with lowercase in proteic a plurality of districts possibly, and conserved regions is represented with capitalization.It is generally acknowledged and to guard replacement in following conserved regions, and do not change function.In addition, the technician should be understood that the functional variant of Zm-D9 aminoacid sequence of the present invention can have the nonconservative amino acid change of minority in conserved regions.
Conserved regions is present between the about amino acid 37-109,224-504,528-625 of SEQ ID NO:2.Non-conserved regions is about amino acid/11-36,110-223, the 505-527 of SEQ ID NO:2.Set up the artificial sequence protein that is different from original series with the interval of 80-85%, 85-90%, 90-95% and 95-100% identity then.With these interval mid points is target, and degree of freedom for example adds or deduct 1%.Aminoacid replacement can be realized by the customization perl script.The replacement table provides in following table 2.
Table 2. replaces table
Amino acid The best replacement of height phase Sihe What change puts in order Note
I L、V 1 50:50 replaces
L I、V 2 50:50 replaces
V I、L 3 50:50 replaces
A G 4
G A 5
D E 6
E D 7
W Y 8
Y W 9
S T 10
T S 11
K R 12
R K 13
N Q 14
Q N 15
F Y 16
M L 17 First methionine(Met) can not change
H Na There is not good replacement
C Na There is not good replacement
P Na There is not good replacement
At first identify and do not answer reformed any conserved amino acid in the albumen, and " being marked as " isolates with replacement.Initial methionine undoubtedly will be added this tabulation automatically.Change then.
H, C and P are changed in no instance.At first Isoleucine is changed, scan C-terminal from N-terminal.Be leucine then, and analogize downwards, up to reaching suitable target spot along tabulation.Can implement mediant and replace, so that do not cause reverse change.Tabulation is 1-17 by the mark preface, so change beginning with many Isoleucines as required, is leucine then, and by that analogy, up to methionine(Met).Obviously, many in this way amino acid do not need to be changed.L, I and V relate to two selectable best 50:50 that replace and replace.
The variant aminoacid sequence is registered as output.Calculate the identity percentage with perl script.Use this method, produce the Zm-D9 protein variant that has about 82%, 87%, 92% and 97% amino acid identity with SEQ ID NO:1,3,4 or 6 initial unaltered ORF nucleotide sequence.
Article " one " and " a kind of " are used in reference to the grammatical object of (promptly at least 1) this article more than 1 or 1 at this paper.As an example, " a kind of factor " means the factor more than 1 or 1.
All publications and the patent application mentioned in this manual all show those skilled in the art in the invention's level.All publications and patent application all are attached to herein by reference, and its degree is pointed out to be attached to herein by reference as each independent publication or patent application particularly and individually.
Although described in detail aforementioned invention for the clear purpose of understanding by elaboration and embodiment, obviously, can in the scope of claim of enclosing, implement some change and modification.
Embodiment 8
Expression and the sign of Zm-D9 in corn
Test structure territory exchange construct (referring to embodiment 6) d9 E600k and D9 K597E in corn (T0 generation) are so that determine that they are to plant height, number of sheets amount, the fate that comes off to the fate that goes out fringe, to pollen first with to the influence of the fate of pollinating.The phenotypic data of d9 E600k and D9 K597E is contrasted with using the allelic data of MUT1 Zm-D9 allelotrope and Zm-D8 MPL.In all cases, the S2a promotor all is used to drive expression of structural gene separately.Vector construction is specified in embodiment 6.Than the unconverted milpa of the homologous genes type of growing simultaneously in the greenhouse, the plant height of D9 K597E is not changed.Equally, the number of sheets amount of D9 K597E, the fate that comes off to the fate that goes out fringe, to pollen first and similar to the fate and the unconverted plant of pollination.MUT1 D9 and d9 E600K transfer-gen plant on average have 1 extra leaf than D9 K597E.D9 E600 K and MUT1 D9 transfer-gen plant reduce (reducing by 50% and 30% respectively) than D9 K597E highly significant.On the other hand, d9 E600k and MUT1 D9 go out fringe, pollen comes off and the date of pollinating aspect postpone than D9 K597E.Pollen comes off and postpones nearly 14 days (for MUT1 D9) and 10 days (for d9 E600k).Pollination postpones to plant similar with these transgenosiss.In addition, these transfer-gen plants show about 1 extra joint than D9K597E.These data demonstrate aspect height and the similar pattern of Arabidopis thaliana (short stem), change phenotype, the wherein delay of maturation of MUT1D9 and d9 E600k but demonstrate opposite maturation.These data acknowledgements in the meaning of Arabidopis thaliana and the corn polymorphism that this plant type and flowering time change in these two.MUT1 D9 and d9E600k sudden change can have specific end use to plant type, joint number or the ripening degree that changes alternative plant configuration in the high production capacity configuration of seed type of corn.
Sequence table
<110>Pioneer Hi-Bred International，Inc.
＜120〉corresponding to the isolating polynucleotide molecule and the use of the mutation allele and the wild-type allele of corn D9 gene
Method
<130>2075-PCT
<140>PCT/US2007/066826
<141>2007-04-18
<150>60/793,048
<151>2006-04-19
<150>60/834,024
<151>2006-07-28
<160>8
<170>FastSEQ for Windows Version 4.0
<210>1
<211>1878
<212>DNA
＜213〉corn (Zea mays)
<400>1
Figure A200780022452D00821
<210>2
<211>625
<212>PRT
＜213〉corn
<400>2
Figure A200780022452D00832
Figure A200780022452D00841
Figure A200780022452D00851
<210>3
<211>1875
<212>DNA
＜213〉corn
<400>3
Figure A200780022452D00861
Figure A200780022452D00871
<210>4
<211>1869
<212>DNA
＜213〉corn
<400>4
Figure A200780022452D00872
Figure A200780022452D00881
<210>5
<211>622
<212>PRT
＜213〉corn
<400>5
<210>6
<211>1866
<212>DNA
＜213〉corn
<400>6
Figure A200780022452D00902
Figure A200780022452D00911
<210>7
<211>15
<212>PRT
＜213〉corn
<220>
＜223〉insertion/deletion sequence (indel sequence)
<400>7
Figure A200780022452D00912
<210>8
<211>11
<212>PRT
＜213〉corn
<220>
＜223〉insertion/deletion sequence
<400>8
Figure A200780022452D00921

Claims (32)

1. isolated polypeptide, it comprises and is selected from following aminoacid sequence:
(a) comprise the aminoacid sequence of SEQ ID NO:2 or 5;
(b) aminoacid sequence that has at least 93% sequence identity with SEQ ID NO:2, wherein said polypeptide has the Zm-D9 activity;
(c) aminoacid sequence that has at least 95% sequence identity with SEQ ID NO:5, wherein said polypeptide has MUT1 Zm-D9 activity;
(d) by the nucleotide sequence coded aminoacid sequence of under stringent condition, hybridizing with the complement of SEQ ID NO:1 or 3, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(e) by the nucleotide sequence coded aminoacid sequence of under stringent condition, hybridizing with the complement of SEQ ID NO:4 or 6, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(f) contain the aminoacid sequence of at least 124 continuous amino acids of SEQ ID NO:2, wherein said polypeptide keeps the Zm-D9 activity; With
(g) comprise the aminoacid sequence of at least 124 continuous amino acids of SEQ ID NO:5, wherein said polypeptide keeps MUT1 Zm-D9 activity.
2. isolating polynucleotide molecule, it comprises and is selected from following nucleotide sequence:
(a) comprise SEQ ID NO:1,3,4 or 6 nucleotide sequence;
(b) coding comprises the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 5;
(c) under stringent condition with the nucleotide sequence of the complement hybridization of (a) nucleotide sequence, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(d) nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 or 3, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9;
(e) contain at least 698 continuous nucleotides of SEQ ID NO:1 or 3 or the nucleotide sequence of its complement;
(f) amino acid sequence coded and SEQ ID NO:2 nucleotide sequence with at least 93% sequence identity, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9; With
(g) nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:4 or 6, wherein said polynucleotide molecule coding has the active polypeptide of MUT1 Zm-D9;
(h) contain at least 395 continuous nucleotides of SEQ ID NO:4 or 6 or the nucleotide sequence of its complement; With
(i) nucleotide sequence with at least 95% sequence identity of amino acid sequence coded and SEQ ID NO:5, wherein said polynucleotide molecule coding has the active polypeptide of MUT1Zm-D9.
3. expression cassette that comprises the polynucleotide molecule of claim 2.
4. the expression cassette of claim 3, wherein said polynucleotide molecule with in plant, drive expression promoter and effectively be connected.
5. non-human host cell of containing the expression cassette of claim 3 or 4.
6. the host cell of claim 5, wherein said host cell is vegetable cell, bacterial cell or fungal cell.
7. a kind of plant, it contains and drive the polynucleotide molecule that expression promoter effectively is connected in plant, and wherein said polynucleotide molecule contains and is selected from following nucleotide sequence:
(a) contain SEQ ID NO:1,3,4 or 6 nucleotide sequence;
(b) coding contains the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 5;
(c) under stringent condition with the nucleotide sequence of the complement hybridization of (a) nucleotide sequence, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(d) nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 or 3, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9;
(e) contain at least 698 continuous nucleotides of SEQ ID NO:1 or 3 or the nucleotide sequence of its complement;
(f) amino acid sequence coded and SEQ ID NO:2 nucleotide sequence with at least 93% sequence identity, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9; With
(g) nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:4 or 6, wherein said polynucleotide molecule coding has the active polypeptide of MUT1 Zm-D9;
(h) contain at least 395 continuous nucleotides of SEQ ID NO:4 or 6 or the nucleotide sequence of its complement; With
(i) nucleotide sequence with at least 95% sequence identity of amino acid sequence coded and SEQ ID NO:5, wherein said polynucleotide molecule coding has the active polypeptide of MUT1Zm-D9.
8. the plant of claim 7, wherein said plant is a cell.
9. the plant of claim 7, wherein said plant is a monocotyledons.
10. the plant of claim 9, wherein said monocotyledons is selected from corn, wheat, paddy rice, Chinese sorghum, rye, broomcorn millet and barley.
11. the plant of claim 7, wherein said plant are dicotyledons.
12. the plant of claim 11, wherein said dicotyledons is selected from Arabidopis thaliana, soybean, Sunflower Receptacle, safflower, clover, rape, cotton and peanut.
13. each plant among the claim 7-12, wherein said polynucleotide molecule mixes in the genome of plant with being stabilized.
14. the plant of claim 13, wherein said plant are seed.
15. comprising, a method that increases the polypeptide level in the plant, this method import in the described plant containing the polynucleotide molecule that is selected from following nucleotide sequence:
(a) comprise SEQ ID NO:1,3,4 or 6 nucleotide sequence;
(b) coding comprises the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 5;
(c) under stringent condition with the nucleotide sequence of the complement hybridization of (a) nucleotide sequence, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(d) nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 or 3, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9;
(e) contain at least 698 continuous nucleotides of SEQ ID NO:1 or 3 or the nucleotide sequence of its complement;
(f) amino acid sequence coded and SEQ ID NO:2 nucleotide sequence with at least 93% sequence identity, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9; With
(g) nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:4 or 6, wherein said polynucleotide molecule coding has the active polypeptide of MUT1 Zm-D9;
(h) contain at least 395 continuous nucleotides of SEQ ID NO:4 or 6 or the nucleotide sequence of its complement; With
(i) nucleotide sequence with at least 95% sequence identity of amino acid sequence coded and SEQ ID NO:5, wherein said polynucleotide molecule coding has the active polypeptide of MUT1Zm-D9.
16. comprising, a method that is used for regulating the polypeptide level of plant, this method import in the described plant containing the polynucleotide molecule that is selected from following nucleotide sequence:
(a) comprise SEQ ID NO:1,3,4 or 6 nucleotide sequence;
(b) coding comprises the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 5;
(c) under stringent condition with the nucleotide sequence of the complement hybridization of (a) nucleotide sequence, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(d) nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 or 3, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9;
(e) contain at least 698 continuous nucleotides of SEQ ID NO:1 or 3 or the nucleotide sequence of its complement;
(f) amino acid sequence coded and SEQ ID NO:2 nucleotide sequence with at least 93% sequence identity, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9; With
(g) nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:4 or 6, wherein said polynucleotide molecule coding has the active polypeptide of MUT1 Zm-D9;
(h) contain at least 395 continuous nucleotides of SEQ ID NO:4 or 6 or the nucleotide sequence of its complement; With
(i) nucleotide sequence with at least 95% sequence identity of amino acid sequence coded and SEQ ID NO:5, wherein said polynucleotide molecule coding has the active polypeptide of MUT1Zm-D9.
17. the method for claim 15 or 16, wherein said polynucleotide molecule stably mixes in the genome of plant.
18. each method among the claim 15-17, wherein said plant are vegetable cell.
19. each method among the claim 15-17, wherein said plant are dicotyledons.
20. the method for claim 19, wherein said dicotyledons is selected from Arabidopis thaliana, soybean, Sunflower Receptacle, safflower, clover, rape, cotton and peanut.
21. each method among the claim 15-17, wherein said plant are monocotyledons.
22. the method for claim 21, wherein said monocotyledons is selected from corn, wheat, paddy rice, Chinese sorghum, rye, broomcorn millet and barley.
23. the method for each of claim 15, wherein said plant are seed.
24. method that is used to change plant-growth, described method comprises uses the polynucleotide constructs inverting biological, described polynucleotide constructs contains and can drive the nucleotide sequence that described nucleotide sequence expression promoter effectively is connected in described biology, and wherein said nucleotide sequence is selected from:
(a) comprise SEQ ID NO:1,3,4 or 6 nucleotide sequence;
(b) coding comprises the nucleotide sequence of the aminoacid sequence of SEQ ID NO:2 or 5;
(c) under stringent condition with the nucleotide sequence of the complement hybridization of (a) nucleotide sequence, wherein said stringent condition is included among 50% methane amide, 1M NaCl, 1% SDS in 37 ℃ of hybridization, and in 0.1X SSC in 60 ℃ to 65 ℃ washings;
(d) nucleotide sequence that has at least 92% sequence identity with SEQ ID NO:1 or 3, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9;
(e) contain at least 698 continuous nucleotides of SEQ ID NO:1 or 3 or the nucleotide sequence of its complement;
(f) amino acid sequence coded and SEQ ID NO:2 nucleotide sequence with at least 93% sequence identity, wherein said polynucleotide molecule coding has the active polypeptide of Zm-D9; With
(g) nucleotide sequence that has at least 96% sequence identity with SEQ ID NO:4 or 6, wherein said polynucleotide molecule coding has the active polypeptide of MUT1 Zm-D9;
(h) contain at least 395 continuous nucleotides of SEQ ID NO:4 or 6 or the nucleotide sequence of its complement; With
(i) nucleotide sequence with at least 95% sequence identity of amino acid sequence coded and SEQ ID NO:5, wherein said polynucleotide molecule coding has the active polypeptide of MUT1Zm-D9.
25. the method for claim 24, wherein said nucleotide sequence effectively is connected to produce the antisense transcript with described promotor.
26. the method for claim 24, wherein with respect to unconverted plant, the height of described plant reduces.
27. the method for claim 24, wherein with respect to unconverted plant, the height of described plant increases.
28. the method for claim 27, wherein with respect to unconverted plant, the root architecture of described plant changes.
29. the method for claim 24, wherein said plant are monocotyledons.
30. the method for claim 29, wherein said monocotyledons is selected from corn, wheat, paddy rice, Chinese sorghum, rye, broomcorn millet and barley.
31. the method for claim 24, wherein said plant are dicotyledons.
32. the method for claim 31, wherein said dicotyledons is selected from Arabidopis thaliana, soybean, Sunflower Receptacle, safflower, clover, rape, cotton and peanut.
CNA2007800224528A 2006-04-19 2007-04-18 Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use Pending CN101479294A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
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CN113481315A (en) * 2021-07-06 2021-10-08 齐鲁师范学院 Molecular marker related to dwarfing of maize plants
CN115786567A (en) * 2022-10-10 2023-03-14 安徽农业大学 Semi-dominant maize dwarfing related molecular marker and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481315A (en) * 2021-07-06 2021-10-08 齐鲁师范学院 Molecular marker related to dwarfing of maize plants
CN113481315B (en) * 2021-07-06 2022-05-17 齐鲁师范学院 Molecular marker related to dwarfing of maize plants
CN115786567A (en) * 2022-10-10 2023-03-14 安徽农业大学 Semi-dominant maize dwarfing related molecular marker and application thereof
CN115786567B (en) * 2022-10-10 2023-05-16 安徽农业大学 Semi-dominant corn dwarf related molecular marker and application thereof

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