CN102964439A - Transcription factor for regulating and controlling aging of leaf blades and improving multiple stress tolerance and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant gene engineering and specifically relates to a transcription factor for regulating and controlling aging of leaf blades and improving multiple stress tolerance and an application thereof. A plant WRKY58 protein provided by the invention is derived from arabidopsis, chili and other species, but is not limited to the species, and the plant WRKY58 protein comprises an analogous protein, wherein the homeology of the analogous protein with AtWRKY58 (the amino acid sequence is as shown in SEQ ID NO: 2) in the arabidopsis is higher than 30%. An encoding gene of the arabidopsis WRKY58 protein is one of the following nucleotide sequences: (1) SEQ ID NO: 1 in a sequence table; and (2) polynucleotide with the amino acid sequence as shown in the SEQ ID NO: 2 in a coding sequence table. The invention can provide a protein resource and a method in the aspects of deferring the aging of the leaf blades and improving the yield, quality and multiple stress tolerance of crops.

Description

A kind of regulation and control leaf senile, the transcription factor and the application thereof that improve multiple resistance

Technical field

The invention belongs to the plant gene engineering technology field, be specifically related to a kind of regulation and control leaf senile, improve transcription factor and the application of multiple resistance.

Background technology

Plant senescence is the gradually capable property decline of the inner a series of sequencing of plant individual, final process deathward.Leaf senile is the integral part of growth and development of plants, consisted of the last stage that plant leaf is grown, be considered to a kind of of apoptosis (PCD), carry out in the mode that relies on leaf age, and induced by many environmental factorss, the interaction of the inside and outside factor of the complexity in period of being grown affects.Therefore leaf senile is not passive process, but the process of an active control.In the during Leaf Senescence, cellularstructure, physiological metabolism, genetic expression aspect all experience a series of orderly variations.In addition, owing to relate to transhipment and the recycling of nutritive substance in the during Leaf Senescence, concerning whole plant, leaf senile is the plant health growth, breeds key (Buchanan-Wollaston V etc., the Plant Biotechnol J of superior progeny, 1 (1): 3-12,2003; Lim PO and Nam HG etc., Curr Top Dev Biol, 67:49-83,2005).

Plant hormone has extremely important effect to development of plants and response environment.In numerous plant hormones, phytokinin (CK) to the plant leaf aging delay and ethene (ET) is very clear and definite to the promoter action of leaf senile, and in different plant species, all obtained relatively consistent result.Other associated hormone, such as dormin (ABA), Plant hormones regulators,gibberellins (GA), methyl jasmonate (MeJA) and Whitfield's ointment (SA) etc. all play in various degree promoter action to the aging of blade, but for their mechanism of action, particularly the network regulation system of their complexity still knows little about it.

Jasmonic (JA) and Whitfield's ointment (SA) mainly are the hormones of involved in plant defensive raction, but it also has certain effect in during Leaf Senescence.Excised leaf is executed JA outward can accelerate its senescence process, but affecting the synthetic mutant of JA Coi1In do not observe the phenomenon that leaf senile delays, do not change (Xie DX etc., Science, 280:1091-1094,1998 of 12% leaf senile correlation gene wherein; Buchanan-Wollaston V etc., Plant Journal, 42:567-585,2005).During Leaf Senescence, the rapid rising of SA content in the plant materials, but in not old and feeble blade, do not have such phenomenon.After this, Morris etc. is by right NahGTransgenic line, and related mutants Pad4With Npr1Analysis, find old and feeble marker gene in these mutant SAGsExpress and change to some extent (Morris K etc., Plant Journal, 23:677-685,2000).SA may be to have affected the process that changes from aging to necrocytosis in the Leaf senescence development on the effect of aging.

The WRKY transcription factor is gained the name because of the aminoacid sequence that its N end contains the high conservative that is comprised of WRKYGQK.In Arabidopis thaliana, find 74 WRKY members, then contained 105 complete WRKY members in the paddy rice.Research finds that (T) of WRKY structural domain and single-minded combination thereof be TGAC(C/T (T)) sequence (claiming again W-box) is present in the upstream regulatory region territory with disease-resistant, damage, senescence-associated gene and Induced by Salicylic Acid gene more.Existing a large amount of evidences show stress reaction (Eulgem T etc., EMBO J, 18:4689-4699,1999 in close relations that WRKY and above-mentioned several biology and abiotic stress excite; Chen C etc., Plant Physiol, 129:706-716,2002).In addition, expression (Johnson CS etc., Plant Cell, 14:1359-1375,2002 of WRKY gene in the processes such as fruit maturation, seed trichome development, invertase signal conduction and the conduction of Plant hormones regulators,gibberellins signal, have all been found; Sun C etc., Plant Cell, 15:2076-2092,2003).

Dong etc. have studied the effect of WRKY transcription factor aspect plant disease-resistant of 72 Arabidopis thalianas, find that wherein 49 are subject to pathogenic bacteria Pseudomonas syringae pv tomato( Pst) and inducing of SA and producing express to change, and then the expression of regulating genes involved participates in the disease-resistant process of Arabidopis thaliana.Key gene as regulator control system acquired resistance SAR NPR1(Cao WH etc., Plant Physiol, 143:707-719,2007) and PR1Deng, all contain W-box (Rushton PJ etc., Plant Mol Biol, 29:691-702,1995) in its promotor.Usually the expression level of NPR1 in plant is very low, but with pathogenic infection or after executing SA outward, its expression amount increases 2-3 doubly (Cao WH etc., Plant Physiol, 143:707-719,2007), and the ability of anti-multiple pathogens significantly increases simultaneously.Dna sequence analysis shows, NPR1The sequence of 28 Nucleotide contains 3 W-box in the promotor.These W-box are that the WRKY protein binding is necessary, if certain Nucleotide wherein is replaced, and the just no longer with it combination of WRKY albumen, the expression that its SA induces is namely eliminated fully, and at this moment, plant becomes very responsive to the reaction of pathogenic agent.Other has research to find, WRKY18, WRKY59, WRKY70Are direct targets of NPR1 Deng 8 genes, participated in the SAR process of plant, shown that WRKY participates in complicacy (Wang D etc., Science, 308:1036-1040,2005 of the mechanism of SAR; Wang D etc., Plos Pathogens, 2:1042-1050,2006). PR1Gene promoter drives GUSGene expression analysis and Northern engram analysis show that all it is subject to the activation of WRKY6, and this activation faster, stronger (Robatzek S etc., Genes Dev, 16:1139-1149,2002) under the defense response inductive condition.WRKY also plays an important role in signal cross and dialogue.The discoveries such as Li, in Arabidopis thaliana, AtWRKY70 is subjected to inducing of SA, and JA and ethene then do not have inducing action; On the contrary, behind JA processing blade, AtWRKY70 does not express AtCOR1 and is then induced in a large number; Equally, exist NahGIn the transfer-gen plant, SA the synthetic and inducing action of WRKY disappeared; Excessively expressing in the plant of AtWRKY70 the gene relevant with SAR PR-2With PR-5High level expression, the disease resistance of plant improves, and JA induces AtCOR1Gene is then suppressed, but the content of body interior SA, JA and ethene does not significantly change (Li J etc., Plant Cell, 16:319-331,2004; Abuqamar S etc., Plant Journal, 48:28-44,2006).In Arabidopis thaliana, except AtWRKY70, AtWRKY25 and AtWRKY33 may also be important factor of determination (Li J etc., Plant Cell, 16:319-331,2004 of balance plant SA, JA defence signal pathway; Zheng XL etc., Plant Cell Reports, 26:1195-1203,2007).This external leaf senile aspect, the part member of the WRKY factor that involved in plant is disease-resistant has also participated in this physiological process, illustrates that the function of WRKY family has the feature of diversification.Murray etc. (Murray SL etc., MPMI, 20:1431-1438,2007) studies show that AtWRKY53 is the relevant important regulating and controlling factor of a disease resistance, and its afunction mutant is more vulnerable to PstInfection, on the contrary Ralstonia solanacearum is shown stronger tolerance (Hu J etc., PloS ONE, 3:e2589,2008), but evidence suggests that also AtWRKY53 is transcription factor (the Hinderhofer K etc. that a leaf senile commitment is expressed, Planta, 213:469-473,2001; Miao Y etc., Plant Mol Biol, 55:853-867,2004).

At present less to the research of AtWRKY58, mainly rest on the phenomenon observation stage; Research for WRKY family regulating plant leaf senile and multiple resistance is also few in number, particularly heredity and the molecular mechanism of its regulation and control is also almost known nothing.By the Arabidopis thaliana mode system, the important gene resource of regulating plant leaf senile and multiple resistance has great importance on the genetic improvement for crop in the excavation WRKY family.

Summary of the invention

The purpose of this invention is to provide a kind of transcription factor and the application that can regulate and control leaf senile, improve multiple resistance.

The transcription factor that can regulate and control leaf senile, improve multiple resistance provided by the invention for plant transcription factor WRKY58 albumen, derives from the species such as Arabidopis thaliana, capsicum, as shown in Figure 2, but is not limited to these species, comprise with Arabidopis thaliana ( Arabidopsis thaliana) in AtWRKY58(NCBI gene accession number be AT3G01080, aminoacid sequence is shown in SEQ ID NO:2) homology be higher than 30% albuminoid.

Arabidopis thaliana transcription factor WRKY58 albumen provided by the present invention, the protein of amino acid residue sequence shown in the SEQ ID NO:2, or with replacement, disappearance or the interpolation through one or several amino-acid residue of the amino acid residue sequence of SEQ ID NO:2, and has protein with the identical activity of amino acid residue sequence of SEQ ID NO:2.

The encoding gene of Arabidopis thaliana transcription factor WRKY58 is one of following Nucleotide:

(1) dna sequence dna shown in the SEQ ID NO:1;

(2) polynucleotide of aminoacid sequence shown in the coding SEQ ID NO:2.

Dna sequence dna shown in the SEQ ID NO:1 is by 1272 based compositions, and the reading frame of this gene is to hold the 1st to 1272 bit bases from 5 '; Aminoacid sequence is comprised of 423 amino-acid residues shown in the SEQ ID NO:2.

The present invention also comprises the expression vector of the encoding gene of described plant transcription factor WRKY58 albumen.

The present invention also comprises the clone that contains described plant transcription factor WRKY58 expression vector.

The present invention also comprises the application of described plant transcription factor WRKY58 albumen, the expression vector that comprises the encoding gene that will contain described WRKY albumen changes paddy rice, wheat, cotton or seeding corn and other crops over to, thereby expresses this albumen promotion growth and strengthen multiple resistance to cross.Also comprise expression by suppressing the WRKY58 encoding gene with the increase yield potential that delays senility, or strengthen the multiple resistance that it expresses to improve crop.

The expression vector and the clone that turn the encoding gene that albumen of the present invention is arranged all belong to protection scope of the present invention.Albumen of the present invention is playing an important role aspect Delaying Leaf-Senescence, raising crop yield and the multiple resistance.

Description of drawings

Fig. 1 is AtWRKY58 and other Arabidopis thaliana WRKY family transcription factor phylogenetic tree.

Fig. 2 is the systematic evolution tree of plant WRKY58.

Fig. 3 is 30 days synthetic key genes of wild-type Arabidopis thaliana, AtWRKY58 mutant material and Whitfield's ointment of long day growth ICS1Mutant Sid2-2Phenotype after SA processes.

Embodiment

The acquisition of embodiment 1, Arabidopis thaliana transcription factor AtWRKY58 insertion mutation body material.

1.1 the Arabidopis thaliana material is prepared.

Be taken at 16h L/8h D illumination, cultivate the blade of 30 days Arabidopis thaliana Col-0 under 23 ℃ of conditions.

1.2 RNA extracts.

Get the about 0.1g of Arabidopis thaliana material.After liquid nitrogen fully grinds, transfer to the 1.5ml centrifuge tube, add 1ml TRIzol (invitrogen company), behind the mixing, room temperature was placed 15 minutes, added the 0.2ml chloroform: primary isoamyl alcohol (24:1), acutely shake after 15 seconds room temperature and placed 5 minutes, 13000rpm, 4 ℃ are centrifugal 15 minutes.Get supernatant liquor and add the equal-volume Virahol, careful mixing, room temperature was placed 15 minutes, 13000rpm, 4 ℃ are centrifugal 15 minutes.70% washing with alcohol precipitation, drying at room temperature 15 minutes.Be dissolved in an amount of ddH that processed through 0.1% DEPC ₂In the O water, be stored in-80 ℃ for subsequent use.

1.3 cDNA the first chain is synthetic and reverse transcription PCR.

Adopt the cDNA first chain synthetic agent box of Shen, Shanghai energy lottery industry biotech company (SHBC), according to operational guidance total RNA reverse transcription is become cDNA.Reaction system and reaction conditions are respectively: total RNA of 2 μ g preparation, and 0.5 μ l Rnase inhibitor adds deionized water to the 8.5 μ l that DEPC processed, 65 ℃ of the Oligo of 2 μ l (dT), 18 primer., 5min, room temperature is placed 10min, the brief centrifugal 5s of 13000rpm.Add successively again 4 μ l, 5 * First-Strand buffer, 0.5 μ l RNase Inhibitor, 2 μ l 100mM DTT, 2 μ l dNTP, 1 μ l MMLV Reverse Transcriptase.Careful mixing; 37 ℃ of reverse transcriptions 1 hour, 90 ℃ 5 minutes; Cooled on ice; 13000rpm of short duration centrifugal 5 seconds, deposit in-20 ℃ stand-by.

1.4 the evaluation of AtWRKY58 insertion mutation body material.

According to TAIR(http: //the AtWRKY58 total length CDS sequence and the insertion point that provide on www.arabidopsis.org/), designed three primer AtWRKY58-LB(5'TGGTTCACGTAGTGGGCCATCG 3'), AtWRKY58-LP:(5'AAGCCATGATATTTCATTAAGACC 3') and AtWRKY58-RP:(5'GACTCGTGACATCAACTCGAAC 3') as the primer of PCR reaction.The PCR reaction system is 50 μ l, and reaction conditions is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 40s, 55 ℃ of renaturation 40s, 72 ℃ are extended 70s, circulate 35 times, and 72 ℃ are fully extended 5min.The PCR product of gained obtains homozygous mutation body material through 1% agarose gel electrophoresis isolation identification.

Embodiment 2: Arabidopis thaliana transcription factor AtWRKY58 functional analysis.

2.1 sequence comparing analysis.

With the Genedoc software analysis homology of AtWRKY58 and other Arabidopis thalianas WRKY family protein.Analytical results shows that AtWRKY58 has the conservative WRKY domain of typical case, as shown in Figure 1, use MEGA software to set up the phylogenetic tree of AtWRKY58 with other Arabidopis thalianas WRKY family protein, the result shows that AtWRKY58 belongs to the WRKY-Ia subfamily, and is nearest with AtWRKY3, AtWRKY4 and AtWRKY32 sibship.In the plants such as paddy rice, capsicum, clover, also there is the albumen higher with the AtWRKY58 homology.Fig. 2 has showed the Phylogenetic of WRKY58 albumen in the plant.

2.2. mutant leaf senile index analysis.

2.2.1 Arabidopis thaliana matrix is cultivated:

Matrix components: vermiculite: black earth: perlite 9: 3: 0.5

Nutrient solution prescription:

After matrix is soaked into nutritive medium, seed is sowed in the earthen bowl, covers with preservative film, place under 4 ℃ of dark conditions, change (16h L/8h D) illumination after 2 days over to, cultivate under 23 ℃ of conditions.The Arabidopis thaliana growth was tested in 30 days.

Mutant Atwrky58After processing, Whitfield's ointment do not show obvious leaf presenility phenotype, with the synthetic key gene of Whitfield's ointment ICS1Mutant Sid2-2Phenotype similar, significantly be different from wild-type material (Fig. 3).This shows that WRKY58 regulation and control Leaf senescence development relies on the SA signal pathway.

Embodiment 3: the application of transcription factor WRKY58

Of the present invention WRKY58Gene can be building up in the existing plant expression vector with existing method, can add any promotor that comprises constitutive promoter, strengthens promotor, inducible promoter, tissue-specific promoter, etap specificity promoter before its transcription initiation Nucleotide.For the ease of turning WRKY58Gene plant cell or plant are identified and screen, can process employed carrier, as add the alternative mark of plant ( BARGene, GUSGene, luciferase gene etc.) or antibiotic marker thing (kantlex, Streptomycin sulphate, Totomycin etc.) with resistance.The plant host that is converted both can be monocotyledons, also can be dicotyledons, such as paddy rice, corn, soybean, willow, turfgrass etc.Carry of the present invention WRKY58The expression vector of gene can Ti-plasmids, Ri plasmid, plant viral vector, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant that transforms bred plant through tissue culture, obtain the plant that corresponding regulation and control, crop yield and quality trait obtain improveing to obtain the crop leaf Senescence.

＜110〉Fudan University

＜120〉a kind of regulation and control leaf senile, the transcription factor and the application thereof that improve multiple resistance

<130> 001

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 1272

<212> DNA

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400> 1

atggcggttg aagacgatgt atctttgata agaacgacga cgttagtggc accaacaaga 60

cccacgatta cagttcctca tagacctccg gcgatcgaaa cggcggcgta tttctttggc 120

ggtggagatg ggcttagtct aagcccaggg ccactttctt ttgtctcttc tttgtttgtt 180

gataacttcc ctgacgtctt gacgccggat aaccaacgga cgacgtcgtt tactcatctt 240

ctcacctcgc ccatgttctt tcccccgcag tcgtcagctc ataccggctt tattcaacca 300

cggcagcagt cacaaccgca accacaacga ccagacacgt ttcctcacca tatgccacca 360

tcgacatccg tcgccgtcca tggtcgtcaa tctttagacg tttcacaagt agatcaaaga 420

gctcgaaacc attataataa tccggggaat aacaataata accggtcgta taacgttgtg 480

aacgttgata aaccggcgga tgacggttat aactggagga agtacggaca aaagcctatc 540

aaagggtgtg aatatccaag gagttattac aaatgtacac atgttaactg tccggtgaag 600

aagaaagtcg aacggtcatc ggatggacag atcactcaga tcatttacaa aggtcaacat 660

gatcacgaga ggcctcagaa tcgccgtggc ggtggaggca gagattccac tgaggttggt 720

ggtgcagggc aaatgatgga atctagtgat gatagtggtt atcgtaagga tcatgatgat 780

gatgatgatg atgatgaaga tgatgaagat cttccggctt caaagataag aagaatagac 840

ggtgtgtcga cgactcaccg gacggtgacc gagcctaaga ttatcgttca gacaaaaagt 900

gaagtcgatc ttctcgacga tggctatagg tggcgtaagt acggacaaaa agttgtcaaa 960

ggaaatcccc atccaaggag ctattataaa tgtacaacgc caaattgtac ggtccgtaaa 1020

catgtagaga gagcttccac ggatgctaag gctgtgatta caacttacga aggtaaacac 1080

aatcacgatg tccctgccgc tagaaacggt accgcggcag caaccgcagc tgcggtgggg 1140

ccgtctgacc accatcgtat gagatcaatg tcggggaaca atatgcaaca acatatgagt 1200

ttcggtaaca ataataacac aggccaatct ccggttcttt tgaggttgaa agaagagaaa 1260

atcacaattt ga 1272

<210> 2

<211> 423

<212> PRT

＜213〉Arabidopis thaliana (Arabidopsis thaliana)

<400> 2

Met Ala Val Glu Asp Asp Val Ser Leu Ile Arg Thr Thr Thr Leu Val

1 5 10 15

Ala Pro Thr Arg Pro Thr Ile Thr Val Pro His Arg Pro Pro Ala Ile

20 25 30

Glu Thr Ala Ala Tyr Phe Phe Gly Gly Gly Asp Gly Leu Ser Leu Ser

35 40 45

Pro Gly Pro Leu Ser Phe Val Ser Ser Leu Phe Val Asp Asn Phe Pro

50 55 60

Asp Val Leu Thr Pro Asp Asn Gln Arg Thr Thr Ser Phe Thr His Leu

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Leu Thr Ser Pro Met Phe Phe Pro Pro Gln Ser Ser Ala His Thr Gly

85 90 95

Phe Ile Gln Pro Arg Gln Gln Ser Gln Pro Gln Pro Gln Arg Pro Asp

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Thr Phe Pro His His Met Pro Pro Ser Thr Ser Val Ala Val His Gly

115 120 125

Arg Gln Ser Leu Asp Val Ser Gln Val Asp Gln Arg Ala Arg Asn His

130 135 140

Tyr Asn Asn Pro Gly Asn Asn Asn Asn Asn Arg Ser Tyr Asn Val Val

145 150 155 160

Asn Val Asp Lys Pro Ala Asp Asp Gly Tyr Asn Trp Arg Lys Tyr Gly

165 170 175

Gln Lys Pro Ile Lys Gly Cys Glu Tyr Pro Arg Ser Tyr Tyr Lys Cys

180 185 190

Thr His Val Asn Cys Pro Val Lys Lys Lys Val Glu Arg Ser Ser Asp

195 200 205

Gly Gln Ile Thr Gln Ile Ile Tyr Lys Gly Gln His Asp His Glu Arg

210 215 220

Pro Gln Asn Arg Arg Gly Gly Gly Gly Arg Asp Ser Thr Glu Val Gly

225 230 235 240

Gly Ala Gly Gln Met Met Glu Ser Ser Asp Asp Ser Gly Tyr Arg Lys

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Asp His Asp Asp Asp Asp Asp Asp Asp Glu Asp Asp Glu Asp Leu Pro

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Ala Ser Lys Ile Arg Arg Ile Asp Gly Val Ser Thr Thr His Arg Thr

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Val Thr Glu Pro Lys Ile Ile Val Gln Thr Lys Ser Glu Val Asp Leu

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Leu Asp Asp Gly Tyr Arg Trp Arg Lys Tyr Gly Gln Lys Val Val Lys

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Gly Asn Pro His Pro Arg Ser Tyr Tyr Lys Cys Thr Thr Pro Asn Cys

325 330 335

Thr Val Arg Lys His Val Glu Arg Ala Ser Thr Asp Ala Lys Ala Val

340 345 350

Ile Thr Thr Tyr Glu Gly Lys His Asn His Asp Val Pro Ala Ala Arg

355 360 365

Asn Gly Thr Ala Ala Ala Thr Ala Ala Ala Val Gly Pro Ser Asp His

370 375 380

His Arg Met Arg Ser Met Ser Gly Asn Asn Met Gln Gln His Met Ser

385 390 395 400

Phe Gly Asn Asn Asn Asn Thr Gly Gln Ser Pro Val Leu Leu Arg Leu

405 410 415

Lys Glu Glu Lys Ile Thr Ile

420

Claims (7)

1. plant transcription factor WRKY58 albumen, comprise with Arabidopis thaliana in the homology of AtWRKY58 be higher than 30% albuminoid; The protein of amino acid residue sequence shown in the SEQ ID NO:2, or with replacement, disappearance or the interpolation through one or several amino-acid residue of the amino acid residue sequence of SEQ ID NO:2, and has protein with the identical activity of amino acid residue sequence of SEQ ID NO:2.

2. an Arabidopis thaliana transcription factor AtWRKY58 encoding gene is characterized in that, is one of following Nucleotide:

(1) dna sequence dna shown in the SEQ ID NO:1;

(2) polynucleotide of aminoacid sequence shown in the coding SEQ ID NO:2.

3. Arabidopis thaliana transcription factor AtWRKY58 encoding gene according to claim 2 is characterized in that: the encoder block of this gene is the dna sequence dna from the 1st to the 1272nd at 5 ' end.

4. the expression vector that contains the encoding gene of the described plant transcription factor WRKY58 of claim 1 albumen.

5. the clone that contains the described plant transcription factor WRKY58 of claim 4 expression vector.

6. the application of the described plant transcription factor WRKY58 of claim 1 albumen, it is characterized in that, the expression vector of encoding gene that will contain the WRKY58 albumen of claim 1 changes paddy rice, wheat, cotton or seeding corn and other crops over to, thereby expresses this albumen and promote to grow and strengthen multiple resistance to cross.

7. the according to claim 6 application of described plant transcription factor WRKY58 albumen is characterized in that, the expression by suppressing the WRKY58 encoding gene is with the increase yield potential that delays senility, or strengthens the multiple resistance that it expresses to improve crop.

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