CN104357457A - Transcription factor TaWRKY gene sequence for cultivating wheat Brock and application thereof - Google Patents

Abstract

The invention relates to a TaWRKY gene sequence of WRKY transcription factor genes and a function thereof. The gene is 1242bp in total length and is capable of coding 354 amino acid polypeptides, relative molecular weight is 38.7kD and isoelectric point is 4.96, and homologies with wheat and WRKY genes in wheat are respectively above 90% and 80%. On the basis of phylogenetic analysis, the TaWRKY keeps the closet genetic relation with TaWRKY8 in WRKY family. Expression of the TaWRKY is induced by powdery mildew; TaWRKY in silent disease-resistant wheat Brock is mediated by viruses, and resistance of Brock to powdery mildew is remarkably reduced; compared with control group, proportion of abnormal adhesive cells on the leaf surface of silent plant is reduced, which is just 1/4 of the control group on 7d, while successful infection rate to the powdery mildew is improved by about 14 times. The results show that the TaWRKY has structural characteristics of WRKY family members, and plays an important role in powdery mildew-resistant response way of wheat.

Description

Cultivated wheat Brock transcription factor TaWRKY gene order and application thereof

The present invention obtains: state natural sciences fund (31071671) and Tianjin State Scientific and Technological Commission youth fund (14JCQNJC14900) subsidize.

Technical field:

The invention belongs to agricultural biological gene mend engineer technology field, relate to a kind of WRKY transcription factor of wheat taWRKYthe acquisition of gene order and the application in powdery mildew resistance gene in wheat functional study.

Background technology

Plant formed in very long evolutionary process series of complex and accurately mechanism, resist in growth and development process and be subject to various biology and abiotic stress.Under environment stress, plant adapts to environment stress by the change on a series of physiology, biochemistry and molecular level.Large quantity research surface, the response of plant to adverse circumstance is self multiple gene and the coefficient result of gene family, and these genes are mainly divided into functional gene and regulatory gene two class.Transcription factor is a very important class regulatory gene in plant.

WRKY transcription factor is the class transcription factor family be widely studied in plant in recent years, gains the name because it contains the WRKYGQK aminoacid sequence of one section of high conservative.Degeneration-resistant the coercing of WRKY transcription factor wide participation plant waits physiological and biochemical procedure with old and feeble, and the stimulation of physical abuse, invading pathogens, plant hormone substance all can cause the expression of WRKY gene to change.Tobacco WRKY3 and WRKY6 can respond the injury that insect's food-taking causes.In paddy rice, process LAN osWRKY13its resistance to bacterium and fungi epidemic disease can be improved.Arabidopis thaliana transcription factor WRKY2 has mediated the seed germination depending on ABA and the retardance process of growing after sprouting. hvWRKY38by the induction of arid, low temperature stress in barley.In paddy rice, in the leaf of drought-induced paddy rice osWRKY80expression amount significantly increase.1994, Ishiguro and Nakamura clones first WRKY Protein S PF1 first from sweet potato (Ipomoea batatas), and after this people are obtained for WRKY gene in succession in the various plants such as havergrass, parsley, Arabidopis thaliana, tobacco and paddy rice.At present, in Arabidopis thaliana and paddy rice, found 72 and 107 WRKY gene respectively, Wu etc. have also cloned 15 and 43 WRKY gene respectively with Niu etc. in wheat.

In previous work, we, when utilizing in cDNA-AFLP technical Analysis Brock gene expression profile under powdery mildew induction, find the fragment F16 of 367 bp, through Blast comparison, and wheat in this fragment and NCBI GenBank wRKY8(Genbank accession number: DQ323885.1), wheat wRKY71(Genbank accession number: EF368356.1), wheat wRKY80(Genbank accession number: JX679079.1), wheat cDNA library sequence (Genbank accession number: AK331823.1), barley wRKY1(Genbank accession number: AJ536667.1), barley wRKY38(Genbank accession number: AY541586.1) has the homology of 94%, 93%, 93%, 92%, 85% and 80% respectively.We have cloned a WRKY gene cDNA full length sequence, called after according to said gene sequences Design degenerate primer in powdery-mildew-resistance wheat Brock taWRKY.The expression pattern of fluorescent quantitative PCR technique to this gene is utilized to analyze, virus-mediated gene silencing (VIGS) technology is utilized to obtain reticent plant, observe statistics by infecting situation to powdery mildew before and after wheat leaf blade gene silencing, tentatively think that this WRKY gene take part in the early stage defensive raction process of wheat anti-powdery mildew.

Summary of the invention

The object of the invention is to disclose wheat transcription factor taWRKYthe acquisition of gene order and the application in wheat anti-powdery mildew correlation research.Main contents comprise:

Wheat WRKY transcription factor taWRKYgene order, this gene order total length 1242bp, has the nucleotide sequence shown in SEQ ID NO:1.Proteins encoded is containing 354 amino acid, and relative molecular weight is 38.7 kD, and theoretical iso-electric point is 4.96, has the aminoacid sequence shown in SEQ ID NO:1.

The present invention further discloses wheat WRKY transcription factor taWRKYgene order is resisting the application in Powdery Mildew for wheat.Phylogenetic analysis shows, taWRKYwith WRKY family taWRKY8sibship is nearest.The abduction delivering pattern analysis display of gene, taWRKYstrong by powdery mildew, rapid abduction delivering; With in the reticent anti-disease wheat Brock of virus-mediated gene silencing methods taWRKYgene, the opposing of wheat to powdery mildew obviously reduces, and shows two aspects: the first, and compared with the control, reticent strain blade surface deformity appressorium ratio obviously reduces, and is only 1/4th of contrast during 7d; The second, during powdery mildew inoculation wheat leaf blade 7 days, powdery mildew success infection rate contrast strain is about 6%, and gene silencing strain then reaches 86%, improves about 14 times.Above result display taWRKYtranscription factor has WRKY family member gene structure feature, plays an important role in wheat anti-powdery mildew response pathway.

Laboratory, the present inventor place early stage is when utilizing in cDNA-AFLP technical Analysis Brock gene expression profile under powdery mildew induction, find the EST F16 of 367 bp, through Blast comparison, in this fragment and NCBI GenBank, multiple plant WRKY gene has homology, therefore, we have cloned a WRKY gene cDNA full length sequence, called after according to said gene sequences Design degenerate primer in powdery-mildew-resistance wheat Brock taWRKY.In order to study the response situation of TaWRKY gene pairs powdery mildew induction, after we utilize quantitative fluorescence analysis method (qRT-PCR) to analyze powdery mildew induction taWRKYgene mRNA accumulation.In Brock, taWRKYgene is " W " type curvilinear characteristic by the expression trend after powdery mildew induction, when powdery mildew induction 2h taWRKYgenetic expression starts to rise, and is about 1.2 times that background is expressed; Be about 1.5 times during 4h, reach the highest during 8h, be about 3.75 times of background expression amount; Be returned to rapidly less than 2 times afterwards, start again after 24h to rise, during 48h, reach 3 times close to background expression amount.These results suggest that, taWRKYexpression level and the induction of powdery mildew closely related.Reticent taWRKYdNA triticum blade powdery mildew appressorium abnormal rate is low, and successful infection rate is high, test wheat disease resistance by dying down by force, illustrate target gene and mildew-resistance characteristic closely related.Above result display taWRKYtranscription factor has the feature of WRKY family member gene structure, plays an important role in wheat anti-powdery mildew response pathway.Therefore the present invention is completed.

 

Wheat transcription factor taWRKYgene belongs to WRKY gene family member, this gene order total length 1242bp, base sequence following (SEQ ID NO:1):

CCACGCGTCCGCGGAAATAGTTCTCCATCTCAACCTTCTCTTCTCCCTTCTCTTCTCTCCCGCGCGTTACCTCGAACCGGAAGCGAACTCTACATCCATCCTCGACCG ATGGATCCATGGGTCAGCAGCCAGCCTTCCCTTAGCCTCGACCTGCACGTCGGCCTCCCGCCGATGGGGCACCCGCACCACCACCAGGCGGCGCCCATGGTCGCGCTGGCCAAGCCCAAGGTCCTCGTCGAGGAGAACTTCATGCAGCTCAAGAAGGACCCTGAGGTTGCGGTTCTTGAGTCTGAGCTACAGCGGGTGAGCGAGGAGAACCGGCGGCTGGGCGAGATGCTCAGGGAGGTGGCCTCCAAGTACGAGACCCTGCAGGGCCAGTTCACCGACATGGTCACGGCCGGCGCCCACGCCGGCGGCAATAACCACTACAACAACCAGCCGTCCTCCGCGTCGGAGGGCGGGTCGGTGTCGCCGTCGAGGAAGCGCAAGAGCGAGGAGAGCAACGGCACGCCACCGCCGTCGCACCAGCAGCAGCAGCAGCACTACGCCGGCGGCCTCGCGTACGCGGCGGCGCCGGACCAGGCGGAGTGCACGTCCGGCGAGCCGTGCAAGCGCATCCGGGAGGAGTGCAAACCCGTCATCTCCAAGCGCTACGTCCACGCCGACCCCGCCGACCTCAGCCTGGTGGTGAAGGACGGGTACCAATGGCGCAAGTACGGGCAGAAGGTGACCAAGGACAACCCCTGCCCCAGAGCCTACTTCCGGTGCTCCTTCGCCCCCGGCTGCCCCGTCAAGAAGAAGGTGCAGAGGAGCGCCGAGGACAAGACCATACTCGTGGCGACGTACGAGGGCGAGCACAACCACAGCCAGCCCCCGTCGTCGCAGCCGCAGCAGCAGAACGACGGCTCCGGCGCGGGCAAGAACGCCGGGAAGCCACCCCAGGCGCCGGCCACGCCTCACCACCCGCAGCAGCATCAGCAGCAGCACAAGCAGGAAGCGCCAGCGGTAGCCGTCAGCGGCGAGTCCGCCGCCGCGGAATCCGAGATGATCCGTCGGAACCTGGCGGAGCAGATGGCCATGACGCTGACGAGGGACCCCAGCTTCAAGGCGGCGCTCGTCACCGCCCTCTCCGGCCGGATCCTCGAGCTATCGCCGACCAGGGACATCAAT TAAAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGATTCCAT

Shade list dashed part is initiator codon, and the two dashed part of shade is terminator codon.

Wheat transcription factor of the present invention taWRKYgene order, this gene contains the amino acid whose open reading code of coding 354, and the molecular weight of this albumen of theoretical prediction is 38.7kDa, sequence following (SEQ ID NO:2):

MDPWVSSQPSLSLDLHVGLPPMGHPHHHQAAPMVALAKPKVLVEENFMQLKKDPEVAVLESELQRVSEENRRLGEMLREVASKYETLQGQFTDMVTAGAHAGGNNHYNNQPSSASEGGSVSPSRKRKSEESNGTPPPSHQQQQQHYAGGLAYAAAPDQAECTSGEPCKRIREECKPVISKRYVHADPADLSLVVKDGYQ WRKYGQKVTKDNPCPRAYFRCSFAPGCPVKKKVQRSAEDKTILVATYEGEHNHSQPPSSQPQQQNDGSGAGKNAGKPPQAPATPHHPQQHQQQHKQEAPAVAVSGESAAAESEMIRRNLAEQMAMTLTRDPSFKAALVTALSGRILELSPTRDIN。

Shade list dashed part is WRKY conserved domain.

Cultivated wheat Brock transcription factor disclosed by the invention taWRKYthe positively effect that gene order has is:

(1) taWRKYfor the transcription factor gene obtained from disease-resistant cultivated wheat Brock, find that this gene plays an important role in wheat anti-powdery mildew in early days response by gene silent technology research;

(2) transcription factor gene has the expression of its downstream gene of regulation and control rapidly, thus improves the overall resistance of plant, therefore transcription factor taWRKYthe research of wheat anti-powdery mildew molecular breeding can be applied to as target gene.

Accompanying drawing illustrates:

Fig. 1. wheat taWRKYwith other species wRKYphylogenetic analysis;

Fig. 2. taWRKYthe expression trend of gene in Brock;

Fig. 3. taWRKYgene silencing plant leaf form; A:GKP-Buffer water diluent control group, B:BSMV γ: gFPcontrol group, C:BSMV γ: pDScontrol group, D:BSMV γ: taWRKYgene silencing group;

Fig. 4. taWRKYgene silencing Efficiency testing;

Fig. 5. taWRKYgene silencing plant (BSMV: taWRKY) and contrast (BSMV: gFP) powdery mildew infect situation;

Fig. 6. contrast and reticent plant on lopsided appressorium and successful infection rate compare, A for contrast and reticent plant on lopsided appressorium compare; B is that contrast and reticent plant powdery mildew success infection rate compare.

 

embodiment:

Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.

embodiment 1

1 materials and methods

1.1 experiment materials and reagent

Powdery mildew resistance kind Brock, is so kind as to give by Ray doctor Johnson.

Wheat powdery mildew No. 15 physiological strains, are provided by Plant Protection institute, Chinese Academy of Agricultral Sciences.

1.2 taWRKYthe clone of gene

Total serum IgE is extracted with reference to Roche company's T riPure Isolation Reagent test kit operational manual.Nanodrop1000 and agarose gel electrophoresis is utilized to detect RNA quality and concentration.Then with 4 μ g total serum IgE for template, utilize Oligo (dT) 18, under the effect of Transcriptor High Fidelity cDNA Synthesis Kit (Roche), synthesize cDNA.According to the Blast comparison result of F16, design a pair degenerate primer,

WRKYCDS-F: 5’-ATGGATCCATGGRTSRGCAGCC-3’;

WRKYCDS-R:5 '-TTAATTGATGTCCCTGGTCGGCGAK-3 ' carries out pcr amplification, amplification system is 5 × HF Buffer 10.0 μ l, 100mM dNTP Mixture 1.0 μ l, WRKYCDS-F 2.5 μ l, WRKYCDS-R 2.5 μ l, cDNA 1.0 μ l, DNA polymerase 0.5 μ l, ddH ₂o polishing to 50 μ l.PCR reaction parameter: 98 DEG C of denaturation 5 min; 98 DEG C of sex change 10s, 58 DEG C of annealing 30s, 72 DEG C extend 2 min; 35 circulations are carried out in reaction.72 DEG C extend 7 min eventually.Target stripe is reclaimed purifying, and purified product is connected on pGEM-T easy Vector, transformation of E. coli, blue hickie screening positive clone, after PCR checking and digestion verification, positive colony send company to check order, and sequencing result submits to NCBI GenBank to carry out homology analysis.

1.3 taWRKYgene Expression Profile Analysis

Adopt method high-density to inoculate fresh powdery mildew spores when wheat seedling grows to one heart stage of leaf, after inoculation 0,2,4,8,12,24 and 48h, get blade for extracting RNA and expressing trend analysis.

A pair fluorescent quantitation primer QTaWRKYF:5 '-AGTCCAATCCCGTCATCTCC-3 ' is designed according to the specific section of wheat TaWRKY gene

And QTaWRKYR:5 '-GTCGCCACGAGTATGGTCTT-3 ', size is 154bp.Use FastStart Universal SYBR Green Master (Roche) on quantitative real time PCR Instrument (Corbett Rotor Gene 6000), carry out gene amplification, fluorescent signal detects and solubility curve analysis.Reference gene is wheat tubulingene (GenBank:U76744.1), this gene amplification primer is TubulinF:5 '-CCGTCTCCTTCTGGCTGCTT-3 ' and TubulinR:5 '-GCCCGATGTGGATGCTTAT-3 ', and amplified fragments size is 186bp.PCR amplification system and program reference reagent box specification sheets carry out, and each sample arranges 3 repetitions, adopt 2 ^{-△ △ CT}method carries out data analysis ^[11].

1.4 VIGS vector constructions

For vector construction is barly strip mosaic virus (Barley stripe mosaic virus, BSMV) virus, and it is made up of BSMV α, BSMV β, BSMV γ: PDS/GFP 3 component.Terminator codon place, γ b open reading frame downstream on BSMV γ introduces nhei point of contact, for inserting cloned DNA fragments.BSMV γ-PDS warp nheafter I enzyme is cut, remove PDS gene fragment, reclaim carrier segments.According to taWRKYgene conserved regions sequences Design a pair two ends are added nhethe upstream and downstream primer of I restriction enzyme site, WRKY-V-F:5 '-AGCCGCAGCAGCAGAACG-3 ' and WRKY-V-R 5 '-CTTGAAGCTGGGGTCCCTC-3 '.Amplified fragments size is about 200bp.To contain taWRKYthe plasmid of full length gene carries out pcr amplification as template, amplified production through electrophoresis, purifying, nhei enzyme cut after with same warp nhei enzyme is cut and BSMV γ: the PDS carrier of phosphorylation connects.Connect product through clone, screening, successfully builds VIGS recombinant vectors after order-checking.

1.5 in-vitro transcription and virus inoculation

Carrier B SMV α, BSMV γ and BSMV β linearization for enzyme restriction, then use RiboMAX large Scale RNA Production-T7 kit (Promega) in-vitro transcription to obtain BSMV viral RNA.By 3 of virus RNA component balanced mix, after adding the mixing of equal-volume GKP damping fluid, be inoculated in wheat two leaf stage on the 2nd leaf.Using GKP damping fluid as negative control; Inoculation RNA α+RNA β+RNA γ: PDS/GFP (BSMV:PDS/GFP) also follows the tracks of gene silencing situation as positive control using blade bleaching symptom.Moisturizing 24 h after inoculation, then cultivates a couple of days in 20 DEG C in illumination box, routine observation virus symptoms and inoculation BSMV:PDS plant leaf bleaching situation.

1.6 gene silencing Efficiency testing

Extract inoculation BSMV: taWRKYand having wheat the 3rd leaf total serum IgE of virus symptoms, reverse transcription becomes cDNA, and qRT-PCR analyzes taWRKYthe expression level of gene, and then the silence efficiency determining gene.

1.7 gene silencings infect the impact of wheat leaf blade to powdery mildew

Brock the 3rd leaf inoculates powdery mildew, meets bacterium 48hr, 72hr's and 7d taWRKYthe blade of experimental group, GFP control group and GKP-Buffer water diluent control group is got the leaf section that 3-4cm is long, be soaked in destainer the 48h that decolours, period changes destainer until blade is transparent.Dye 4hr again in coomassie brilliant blue R_250 staining fluid.Wash away the staining fluid of leaf surface residual with distilled water, subsequently blade be placed in conserving liquid and preserve and examine under a microscope, statistics powdery mildew and blade make situation mutually.

experimental result

2.1 taWRKYthe clone of gene

Be separated the wheat Brock blade total serum IgE of powdery mildew induction 8h, with Oligo (dT) 18 for primer carries out reverse transcription, obtain cDNA, with this cDNA for masterplate, WRKYCDS-F and WRKYCDS-R is primer, pcr amplification goes out the gene fragment of an about 1.3kb, this fragment is connected to T-easy cloning vector, clones and check order, this sheet segment length 1242 bp, comprise F16 sequence section, with wheat wRKY8gene (the GenBank number of logging in: DQ323885.1) has 97% homology.Its ORF district is 1065bp altogether, and infer coding 354 amino acid (SEQ ID NO:2), the molecular weight of this albumen of theoretical prediction is 38.7 kD, iso-electric point 4.96.The analysis of aminoacid sequence finds, taWRKYpostmedian comprises WRKY gene superfamily conserved domain WRKYGQK, therefore by this unnamed gene is taWRKY(Fig. 1).

Wheat transcription factor of the present invention taWRKYgene order, this gene contains the amino acid whose open reading code of coding 354, and the molecular weight of this albumen of theoretical prediction is 38.7kDa, sees SEQ ID NO:2

Utilize ClustalW software to carry out the discovery of system pedigree analysis, clone in this research taWRKYin gene and WRKY family wRKY8sibship comparatively near (Fig. 1)

2.2 TaWRKY Gene Expression Profile Analysis

In order to study the response situation of TaWRKY gene pairs powdery mildew induction, after we utilize quantitative fluorescence analysis method (qRT-PCR) to analyze powdery mildew induction taWRKYgene mRNA adds up situation.In Brock, TaWRKY gene is " W " type curvilinear characteristic by the expression trend after powdery mildew induction, when powdery mildew induction 2h taWRKYgenetic expression starts to rise, and is about 1.2 times that background is expressed; Be about 1.5 times during 4h, reach the highest during 8h, be about 3.75 times of background expression amount; Be returned to rapidly less than 2 times afterwards, start again after 24h to rise, during 48h, reach 3 times (Fig. 2) close to background expression amount.These results suggest that, taWRKYexpression level and the induction of powdery mildew closely related.

wheat is reticent taWRKYthe acquisition of gene plant

Will taWRKYgene non-conservative district fragment inserts BSMV γ carrier, formation restructuring BSMV virus (BSMV γ: taWRKY); In contrast, build identical recombinant virus BSMV γ: PDS and BSMV γ: GFP with PDS and GFP simultaneously, inoculate wheat Brock second leaf respectively, found that obvious albinism (Fig. 3) appears in the plant leaf of inoculation recombinant virus BSMV:PDS, illustrate that this gene silencing experiments system is effective.Except the accumulated water spot that virus removal causes, in this research, gene silencing wheat and control group wheat do not have notable difference growing, in form etc.Then be extracted the 3rd leaf total serum IgE of the wheat of experimental group and control group respectively, utilize in Semi quantitative PCR analysis experiment material taWRKYthe mRNA level in-site of gene.As shown in Figure 4, in experimental group taWRKYthe expression amount of gene obviously declines, and illustrates that fragment that we design can effective silencing endogenous taWRKYgene.

taWRKYthe resistance change that after gene silencing, wheat leaf blade infects powdery mildew

To inoculate BSMV γ: gFPfor contrast, this research observes powdery mildew respectively and infects 48h, after 72h and 7d, and BSMV γ: taWRKYthe resistance change that gene silencing wheat plant infects powdery mildew.After inoculation powdery mildew 48h, BSMV γ: taWRKYgene silencing wheat plant blade starts to occur secondary hyphae, and control group BSMV γ: gFPin powdery mildew be substantially in appressorium state; After inoculation 72h, gene silencing strain blade there is the secondary mycelia of a large amount of multiple-limb, then just started in control group to occur secondary mycelia; After microbiological contamination 7d, there is a large amount of beading spore in gene silencing strain, the mycelia in control group starts to occur multiple-limb, but quantity is obviously less than gene silencing group (Fig. 5).Result implies, reticent taWRKYgene reduces the disease-resistant level of plant to a certain extent.

This research is added up, as statistical graph 6A and B respectively to the growing state of the lopsided appressorium often organized on 9 blades and mycelia altogether.Compared with control group, reticent strain blade surface deformity appressorium ratio is starkly lower than contrast, is only 1/4th (Fig. 6 A) of contrast, shows because gene silencing causes plant to decline to the resistivity of powdery mildew during 7d; Correspond, powdery mildew success infection rate significantly improves, and contrast strain during 7d and be about 6%, gene silencing strain then reaches 86%(Fig. 6 B), improve about 14 times, further illustrate this gene, in wheat opposing Powdery Mildew process, there is vital role.

SEQUENCE LISTING

<110> Tianjin Normal University

<120> cultivated wheat Brock transcription factor TaWRKY gene order and application thereof

<160> 2

<170> PatentIn version 3.5

 

<210> 1

<211> 1242

<212> DNA

<213> artificial sequence

 

<400> 1

ccacgcgtcc gcggaaatag ttctccatct caaccttctc ttctcccttc tcttctctcc 60

cgcgcgttac ctcgaaccgg aagcgaactc tacatccatc ctcgaccgat ggatccatgg 120

gtcagcagcc agccttccct tagcctcgac ctgcacgtcg gcctcccgcc gatggggcac 180

ccgcaccacc accaggcggc gcccatggtc gcgctggcca agcccaaggt cctcgtcgag 240

gagaacttca tgcagctcaa gaaggaccct gaggttgcgg ttcttgagtc tgagctacag 300

cgggtgagcg aggagaaccg gcggctgggc gagatgctca gggaggtggc ctccaagtac 360

gagaccctgc agggccagtt caccgacatg gtcacggccg gcgcccacgc cggcggcaat 420

aaccactaca acaaccagcc gtcctccgcg tcggagggcg ggtcggtgtc gccgtcgagg 480

aagcgcaaga gcgaggagag caacggcacg ccaccgccgt cgcaccagca gcagcagcag 540

cactacgccg gcggcctcgc gtacgcggcg gcgccggacc aggcggagtg cacgtccggc 600

gagccgtgca agcgcatccg ggaggagtgc aaacccgtca tctccaagcg ctacgtccac 660

gccgaccccg ccgacctcag cctggtggtg aaggacgggt accaatggcg caagtacggg 720

cagaaggtga ccaaggacaa cccctgcccc agagcctact tccggtgctc cttcgccccc 780

ggctgccccg tcaagaagaa ggtgcagagg agcgccgagg acaagaccat actcgtggcg 840

acgtacgagg gcgagcacaa ccacagccag cccccgtcgt cgcagccgca gcagcagaac 900

gacggctccg gcgcgggcaa gaacgccggg aagccacccc aggcgccggc cacgcctcac 960

cacccgcagc agcatcagca gcagcacaag caggaagcgc cagcggtagc cgtcagcggc 1020

gagtccgccg ccgcggaatc cgagatgatc cgtcggaacc tggcggagca gatggccatg 1080

acgctgacga gggaccccag cttcaaggcg gcgctcgtca ccgccctctc cggccggatc 1140

ctcgagctat cgccgaccag ggacatcaat taaaatcact agtgaattcg cggccgcctg 1200

caggtcgacc atatgggaga gctcccaacg cgttgattcc at 1242

 

 

<210> 2

<211> 354

<212> PRT

<213> artificial sequence

 

<400> 2

Met Asp Pro Trp Val Ser Ser Gln Pro Ser Leu Ser Leu Asp Leu His

1 5 10 15

Val Gly Leu Pro Pro Met Gly His Pro His His His Gln Ala Ala Pro

20 25 30

Met Val Ala Leu Ala Lys Pro Lys Val Leu Val Glu Glu Asn Phe Met

35 40 45

Gln Leu Lys Lys Asp Pro Glu Val Ala Val Leu Glu Ser Glu Leu Gln

50 55 60

Arg Val Ser Glu Glu Asn Arg Arg Leu Gly Glu Met Leu Arg Glu Val

65 70 75 80

Ala Ser Lys Tyr Glu Thr Leu Gln Gly Gln Phe Thr Asp Met Val Thr

85 90 95

Ala Gly Ala His Ala Gly Gly Asn Asn His Tyr Asn Asn Gln Pro Ser

100 105 110

Ser Ala Ser Glu Gly Gly Ser Val Ser Pro Ser Arg Lys Arg Lys Ser

115 120 125

Glu Glu Ser Asn Gly Thr Pro Pro Pro Ser His Gln Gln Gln Gln Gln

130 135 140

His Tyr Ala Gly Gly Leu Ala Tyr Ala Ala Ala Pro Asp Gln Ala Glu

145 150 155 160

Cys Thr Ser Gly Glu Pro Cys Lys Arg Ile Arg Glu Glu Cys Lys Pro

165 170 175

Val Ile Ser Lys Arg Tyr Val His Ala Asp Pro Ala Asp Leu Ser Leu

180 185 190

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

195 200 205

Lys Asp Asn Pro Cys Pro Arg Ala Tyr Phe Arg Cys Ser Phe Ala Pro

210 215 220

Gly Cys Pro Val Lys Lys Lys Val Gln Arg Ser Ala Glu Asp Lys Thr

225 230 235 240

Ile Leu Val Ala Thr Tyr Glu Gly Glu His Asn His Ser Gln Pro Pro

245 250 255

Ser Ser Gln Pro Gln Gln Gln Asn Asp Gly Ser Gly Ala Gly Lys Asn

260 265 270

Ala Gly Lys Pro Pro Gln Ala Pro Ala Thr Pro His His Pro Gln Gln

275 280 285

His Gln Gln Gln His Lys Gln Glu Ala Pro Ala Val Ala Val Ser Gly

290 295 300

Glu Ser Ala Ala Ala Glu Ser Glu Met Ile Arg Arg Asn Leu Ala Glu

305 310 315 320

Gln Met Ala Met Thr Leu Thr Arg Asp Pro Ser Phe Lys Ala Ala Leu

325 330 335

Val Thr Ala Leu Ser Gly Arg Ile Leu Glu Leu Ser Pro Thr Arg Asp

340 345 350

Ile Asn

 

Claims (3)

1. wheat WRKY transcription factor taWRKYgene order, this gene order total length 1242bp, has the nucleotide sequence shown in SEQ ID NO:1.

2. wheat WRKY transcription factor taWRKYgene order, proteins encoded is containing 354 amino acid, and relative molecular weight is 38.7 kD, and theoretical iso-electric point is 4.96, has the aminoacid sequence shown in SEQ ID NO:1.

3. the wheat WRKY transcription factor described in claim 1 or 2 taWRKYgene order is resisting the application in Powdery Mildew for wheat.