CN107058341A

CN107058341A - A kind of WRKY transcription factor gene related to cotton fiber development and its application

Info

Publication number: CN107058341A
Application number: CN201710417178.XA
Authority: CN
Inventors: 李学宝; 王娜娜; 李扬; 周立; 王瑶
Original assignee: Huazhong Normal University
Current assignee: Huazhong Normal University
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2017-08-18
Anticipated expiration: 2037-06-06
Also published as: CN107058341B

Abstract

The invention belongs to genetic engineering field, and in particular to a kind of WRKY transcription factor gene related to cotton fiber development and its application.The invention provides a kind of WRKY transcription factor gene related to cotton fiber development, its nucleotide sequence is as shown in SEQ ID NO.1；The present invention also provides the cDNA sequence of the WRKY transcription factor gene related to cotton fiber development, and nucleotide sequence is as shown in SEQ ID NO.2；The present invention also provides the protein of the WRKY transcription factor gene coding related to cotton fiber development, and amino acid sequence is as shown in SEQ ID NO.3.WRKY transcription factor genes high level expression in cotton fiber of the present invention, shows that the gene may play an important role during regulating cotton starting and Fiber elongation.

Description

A kind of WRKY transcription factor gene related to cotton fiber development and its application

Technical field

The invention belongs to genetic engineering field, and in particular to a kind of WRKY transcription factor gene related to cotton fiber development And its application.

Background technology

Cotton is one of topmost crops in the world, is also the maximum industrial crops of China.Cotton is to be related to state The specialty goods of the people's livelihood is counted, critical role is occupied in Chinese national economy.Cotton fiber, as important natural fiber, is weaving The main raw material(s) of industry, its yield and fiber quality directly determine the commercial value of cotton.With world textile industry Development, the requirement to interior qualities such as Cotton Fiber Strength, fineness and maturity is higher, but the cotton of domestic raw cotton is fine mostly at present Dimension length and intensity all do not reach the requirement of textile industry.Therefore, the yield of cotton fiber is improved, the quality of cotton fiber, one is improved It is directly the research direction and breeding objective of Cotton Industry.

The form generation of cotton fiber cell contains a highly regulated cell differentiation procedure.Each fibrocyte It is the unicellular hairy projection differentiated by the exocuticle on ovule surface.Fibre Development experience is several continuous and overlapping Starting, elongation, secondary wall thickening and the dehydration maturation in stage, i.e. fibrocyte.Cotton fibre cell walls last the form of about 50 days Generating process determines the quality parameter of cotton fiber.The initial number of ovule epidermal cell fiber determines the yield of mature fibers, The elongation of fiber and the thickened degree of secondary wall determine the length and intensity of mature fibers.Therefore, cotton fiber development is furtherd investigate Process and Regulation Mechanism, it is expected to open up new approach to improve production of cotton fibers and quality.

Cotton fiber development process is regulated and controled by transcriptional level.The central transcription factor family HD-ZIP eggs having now been found that In vain, MYB albumen, NAC albumen etc. all play vital effect in fibrocyte starting or growth course.WRKY transcription because Son is the peculiar new transcription regulatory factor found in recent years in plant, because containing the height being made up of WRKYGQK in its N-terminal Conservative amino acid sequence and gain the name.There are some researches show, WRKY transcription factors the growing of plant, aging, biology and Very important effect is played in abiotic stress response.But whether participate in regulation and control cotton fiber hair for WRKY transcription factors Educate and also there is no report.Therefore, effect and Regulation Mechanism of the WRKY transcription factors in cotton fiber development, separation identification phase are illustrated The important controlling gene of pass, is improved for cotton fiber quality, significant.

WRKY protein families belong to zinc finger type transcription regulatory factor, topmost to be structurally characterized in that in its DNA binding domain extremely Less containing a WRKY domain, the domain is one section of polypeptide being made up of about 60 highly conserved amino acid residues Sequence, has one to have 7 highly conserved peptide regions (WRKYGQK) in its N-terminal, is its nucleus, and its C-terminal is a zinc Refer to structure (zinc finger motif), general composition is CX_4~5CX_22~23HX₁H.According to WRKY motif numbers in WRKY domains The difference of mesh and zinc fingers feature, is divided into 3 classes by WRKY families：I class members contains 2 WRKY motifs and C₂H₂Type zinc finger Structure, ii class members contains 1 WRKY motif and C₂H₂Type zinc fingers, group iii member then containing 1 WRKY motif and C₂HC type zinc fingerses.Research finds that most of WRKY transcription factors all comprise only 1 WRKY domain, belongs to II class, and III Class WRKY transcription factors are existed only in higher plant, are but not present in some rudimentary plants such as bryophyte.Meanwhile, it is high Almost all of III class WRKY transcription factors are relevant all with the biotic responsing reaction of plant in plant, III class WRKY of this explanation Transcription factor is probably because plant is passed through produced by adaptive evolution by environmental pressure.

WRKY albumen special can be combined with (T) (T) TGAC (C/T) sequence of DNA lands, and this land is referred to as W- Box, wherein comprising a TGAC Conserved core sequence, it is essential for the combination of WRKY transcription factors.In addition, Zn²⁺Deng Participation and phosphorylation of metal ion etc. are also beneficial to WRKY transcription factors and W-box combination.Most of WRKY transcription because All containing the indefinite W-box of quantity in the target gene promoter of son, between them or palindrome is collectively aligned or formed. WRKY transcription factors are combined with W-box present in functional gene or other regulatory factor gene promoters, adjust these downstreams The expression of gene.

WRKY transcription factors participate in some growth and development processes of regulation and control plant.Such as soybean GmWRKY13 and arabidopsis AtWRKY75 is relevant with the growth of lateral root；Rice Os WRKY31 is also relevant with the formation and extension of lateral root.Arabidopsis AtWRKY6 and Rice Os WRKY23 can trigger the process of aging, and AtWRKY70 can be reacted with negative regulation aging.Arabidopsis AtTTG2 is one The gene of individual coding WRKY transcription factors, it has served important in the formation of arabidopsis epidermal trichome and kind skin. In addition, WRKY transcription factors also take part in some metabolic processes of plant, such as barley SUSIBA2 take part in internal sugared generation Thank, cotton GaWRKY1 regulates and controls the biosynthesis of sesquiterpene.It is thin that rice Os WRKY71 and barley HvWRKY38 can suppress aleuron The activity for the alpha-amylase that GA is induced in born of the same parents, so as to suppress the sprouting of seed.But, WRKY transcription factors are in cotton fiber development During adjusting function so far there are no research report.Therefore, research illustrates tune of the WRKY transcription factors in cotton fiber development Control is acted on, with important theory and practice meaning.

The content of the invention

The present invention is in view of the shortcomings of the prior art, it is therefore intended that provide a kind of WRKY related to cotton fiber development transcribe because Subbase because and its application.

The present invention provides the WRKY transcription factor gene related to cotton fiber development, and it has：

1) nucleotide sequence is as shown in SEQ ID NO.1；Or

2) nucleotide sequence as shown in SEQ ID NO.1 is substituted, lacks and/or increased one or several nucleotides；Or

3) nucleotide sequence under strict conditions with the DNA sequence dna hybridization of 1) restriction.

The present invention also provides the cDNA sequence of the WRKY transcription factor gene related to cotton fiber development, and nucleotide sequence is such as Shown in SEQ ID NO.2.

The present invention also provides the protein of the WRKY transcription factor gene coding related to cotton fiber development, amino acid sequence As shown in SEQ ID NO.3.

The above-mentioned WRKY transcription factor gene related to cotton fiber development answering in terms of improvement production of cotton fibers and quality With.

Beneficial effects of the present invention：The invention provides a new GhWRKY16 gene sequence related to cotton fiber development Row and cDNA sequence, gene high level expression in cotton fiber, show that the gene may be originated and extended in regulating cotton Played an important role in growth course；In GhWRKY16RNAi transgene cottons, important work is played during cotton fiber development The expression of the important controlling genes such as GhHOX3 and GhMYB109 is lowered, and Cellulose-synthase gene expression quantity also declines；With open country Raw type is compared, and GhWRKY16RNAi transgene cotton mature fibers length significantly shortens, and short flannel is less or lacks； GhWRKY16 albumen is positioned in nucleus, can be directly cis with W-box on GhHOX3 and GhMYB109 gene promoters Element is combined, and this shows that GhWRKY16 genes may adjust cotton fiber cell by the related downstream target gene of regulating cotton Starting and Fiber elongation process, influence production of cotton fibers and quality.

Brief description of the drawings

Fig. 1 is that fluorescence quantitative RT-RCR analyzes expression of the GhWRKY16 in cotton tissue, wherein R：Root；L：Blade；C： Cotyledon；H：Hypocotyl；P：Petal；A：Flower pesticide；F：Fiber；0d：Ovule on the day of blooming；3d：3 days fibers of Post flowering；6d：Post flowering 6 days fibers；9d：9 days fibers of Post flowering；12d：12 days fibers of Post flowering；15d：15 days fibers of Post flowering；18d：Post flowering 18 days Fiber, error line represents standard error.

Fig. 2 is Subcellular Localization of the GhWRKY16 albumen in arabidopsis lower plumular axis cell, wherein A：Under Arabidopsis plant GhWRKY16-GFP fluorescence signals in plumular axis cell；B：GFP fluoroscopic images are overlapped with homocellular brightfield image.

Fig. 3 is GhWRKY16 transgene cotton phenotypic analyses, wherein A：Fluorescence quantitative RT-RCR analyzes GhWRKY16 genes Expression in the fiber after wild type (WT) and transgene cotton strain (L1~L8) are bloomed 9 days；B：Wild type and GhWRKY16RNAi transgene cottons strain (L1, L3, L5) bloom the same day the external cultured in vitro of ovule；C：Wild type and The in vitro Ovule developments of GhWRKY16RNAi fibre length statistical analysis (n after 12 days, 15 days and 18 days>30)；D：Wild type and GhWRKY16RNAi transgene cotton mature fibers long wools and short flannel form compare；E：Wild type and GhWRKY16RNAi transgenosis The linear measure longimetry of cotton mature fibers and statistical analysis (n>50)；F：Wild type and GhWRKY16RNAi transgene cottons were bloomed the same day (0d) and Post flowering 1 day (1d), 2 days (2d) ovule transverse sections, the fibrocyte projection after display is bloomed 0 day, 1 day, 2 days are broken up Situation；Error line represents standard error, and * represents transgenosis and wild type the P values in t- inspections<0.05, there is significant difference；** Represent transgenosis and wild type the P values in t- inspections<0.01, there is pole significant difference.WT, wild type；L1~L8, GhWRKY16-RNAi transgene cotton strains.

Fig. 4 is that cellulose synthase GhCesA3, GhCesA5, GhCesA10 turn base in wild type (WT) and GhWRKY16RNAi Because of the expression in cotton (L1, L3, L5) 9 days fiber of Post flowering.WT, wild type；L1, L3 and L5, GhWRKY16-RNAi Transgene cotton strain.

Fig. 5 is at wild type (WT) with cotton fiber development related gene GhHOX3, GhRDL1, GhEXP1 and GhMYB109 With the expression in GhWRKY16RNAi transgene cottons (L1, L3, L5) 9 days fiber of Post flowering.WT, wild type；L1, L3 And L5, GhWRKY16-RNAi transgene cotton strain.

Fig. 6 is the EMSA analyses that GhWRKY16 albumen is combined with GhHOX3 and GhMYB109 promoters.

Embodiment

For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention Content is not limited solely to the following examples.

The clone identification of the cotton WRKY family gene GhWRKY16 full length sequences of embodiment 1 and functional analysis

The separation identification of 1.GhWRKY16 genes and cDNA sequence：

The related GhWRKY genes of examination and cDNA sequence, identify 22 cottons in cotton gene group and est database Flower WRKY gene, wherein there is gene (being named as GhWRKY16) predominant expression in cotton fiber, passes through DNA and protein sequence Row analysis, using the transcript (total serum IgE) of cotton fiber cell, separation obtains GhWRKY16 full length cDNA sequences.According to GhWRKY16cDNA primers, using cotton genomic dna as template, the DNA for obtaining the gene is expanded using round pcr Full length sequence.Sequence analysis shows that GhWRKY16 full length genes sequence (as shown in SEQ ID NO.1) starts comprising 5 '-upstream Sub-district (1~2000bp), 3 extrons, 2 intrones (underscore) and 3 '-UTR (3527-3649bp)；GhWRKY16 genes CDNA sequence as shown in SEQ ID NO.2, wherein containing 957bp ORFs (from initiation codon ATG to termination codon Sub- TAA), 318 amino acid (amino acid sequence is as shown in SEQ ID NO.3) are encoded, molecular weight is 34KDa.GhWRKY16 eggs Containing a WRKY domain in white DNA binding domain, the N-terminal of the domain has one to have highly conserved WRKYGQK7 peptides area Domain, is its nucleus, and its C-terminal zinc-finger motif is C₂H₂, belong to two class WRKY transcription factors.

2. the expression of quantitative RT PCR analysis GhWRKY16 genes：

Using RNA extracts kits (Qiagin) extraction purification cotton different tissues and the total serum IgE of fiber, real-time fluorescence is fixed The expression of RT-PCR research genes is measured (according to Li XB, Fan XP, Wang XL, Cai L, Yang WC, 2005.The Cotton ACTIN1gene is functionally expressed in fibers and participates in fiber elongation.Plant Cell 17:859-875 are carried out).First, by cotton different tissues (root, blade, cotyledon, lower embryo Axle, petal, flower pesticide, 0 day ovule of Post flowering, Post flowering 3,6,9,12,15 and 18 days fibers etc.) total serum IgE (2 μ g/ samples) use M- MLV RNase H^-Reverse Transcriptase (Promega) reverse transcriptions are into cDNA；Then, using cDNA as template, base is used Because of special primer (GhWRKY16RTP1：5 '-GAAGTGTAATTCCATGGATG-3 ' and GhWRKY16 RTP2：5’- TGACTAATCTGAACAACCCT-3 ') and Real-time PCR Master Mix (TOYOBO, Japan) carry out quantitative PCR it is anti- Should.The internal standard that cotton GhUBI1 genes react as RT-PCR, the amplification of each circulation of target gene is by SYBR-Green Fluoroscopic examination, calculates the expression relative value of target gene.Each experiment is repeated 3 times, statistical analysis experimental result.Fluorescence is determined Amount RT-PCR results show that GhWRKY16 respectively organizes have different degrees of expression with cotton fiber different developmental phases in cotton, Predominant expression (Fig. 1) in the Post flowering cotton fiber of 3~9 days, thus it is speculated that the gene may be played in the cotton fiber cell of elongation phase Regulating and controlling effect.

The Subcellar location analysis of 3.GhWRKY16 albumen

Build GhWRKY16:EGFP plant expression vectors, the carrier is transferred in GV3101 Agrobacteriums by electrotransformation, By flower-dipping method arabidopsis thaliana transformation, the T0 of the ripe arabidopsis of harvest screens positive plant for seed.By transgenic arabidopsis T2 generations Seed germination and growth on the MS culture mediums containing kanamycins after 5 days, is placed under Laser Scanning Confocal Microscope and observes transgenic arabidopsis The fluorescence signal of the green fluorescent protein (GFP) of the lower plumular axis cell of seedling.Subcellular Localization result shows, GFP fluorescence Signal is mainly distributed in the nucleus of lower plumular axis cell, and GhWRKY16 albumen is located in nucleus (Fig. 2).

4.GhWRKY16RNAi transgene cotton phenotypic analyses：

The PMD-GhWRKY16RNAi carriers of 35S promoter driving are built, Agrobacterium LBA4404 is converted.Utilize Agrobacterium Cotton Hypocotyl explant is contaminated, after co-culturing 2 days, hypocotyl is transferred on Selective agar medium and cultivated, induces and screens Transformed calli.By the callus after the squamous subculture of 8~10 months, induction differentiates embryo callus and body Blast, somatic embryo so that sprout be regenerated as transgene cotton seedling.Cotton seedling is transplanted into soil, grown to opening Spend solid.Cotton genomic dna is extracted, round pcr Testing and appraisal transgenic cotton plant is utilized.

(1) real-time fluorescence quantitative RT-PCR studies the expression of gene:According to Li XB, Fan XP, Wang XL, Cai L, Yang WC,2005.The Cotton ACTIN1gene is functionally expressed in fibers and participates in fiber elongation.Plant Cell 17:859-875 are carried out, and extract the Post flowering fibre of 9 days Tie up RNA, reverse transcription is into after cDNA, using cDNA as template, with the primer of gene specific (GhWRKY16RTP1 and GhWRKY16RTP2) and Real-time PCR Master Mix (TOYOBO, Japan) carry out quantitative PCR reaction.With GhUBI1 Expressions of the GhWRKY16 in wild type (WT) and each strain of transfer-gen plant is analyzed as internal reference.It is repeated 3 times, statistical Analyse experimental result.

(2) in vitro ovule culture experiment:Choose the wild type (WT) and 3 GhWRKY16RNAi transgenic cottons on the same day of blooming The ovule of flower strain (L1, L3 and L5), which is placed in BT culture mediums, distinguishes sterile culture (dark treatment, 30 DEG C) 12 days, 15 days and 18 days Afterwards, ovule surface fiber length is measured after being fixed with fixer and carries out statistical analysis, error line represents standard error, and * represents to turn Gene and wild type P values in t-test<0.05 has significant difference, and * * represent transgenosis and wild type P values in t-test< 0.01 has pole significant difference.

(3) ripe cotton fiber statistical measurement：After fiber maturation, to wild type (WT) and GhWRKY16RNAi transfer-gen plants Each strain mature fibers length is measured and statistics (n>50).CK is two nontransgenic plants separated, and is left all For different GhWRKY16RNAi transgenic lines.Error line represents standard error, and * represents transgenosis with wild type in t-test Middle P values<0.05 has significant difference, and * * represent transgenosis and wild type P values in t-test<0.01 has pole significant difference.

(4) ovule free-hand section：Wild type (WT) and GhWRKY16RNAi transfer-gen plants is taken to bloom the same day (0 day), 1 day Ovule with 2 days carries out free-hand section, and the thin slice for being cut into about 1mm is observed and taken pictures under the microscope.

The present invention constructs GhWRKY16RNA interference (RNAi) carriers, and converting cotton obtains transgenic cotton plant. The detection of expression quantity on rna level is carried out to GhWRKY16 transfer-gen plants first, is as a result shown in most of In GhWRKY16RNAi transfer-gen plants, the expression quantity of GhWRKY16 genes significantly lowers (Fig. 3 A).GhWRKY16RNAi The cotton boll of transfer-gen plant is compared smaller with wild type.To the GhWRKY16RNAi transgene cottons and wild on the same day of blooming Type ovule carries out in vitro culture, it has been observed that transgene cotton Ovule Development is slow after 12~18 days, fibre length is significantly shorter than Wild type (Fig. 3 B, C).Analysis is measured to the ripe cotton fiber length of RNAi transfer-gen plants, as a result shown, it is most of The mature fibers length of GhWRKY16RNAi transgenic cotton plants is considerably shorter than wild type (Fig. 3 E).Moreover, divesting long fibre After dimension, it is found that the short flannel on RNAi transgenic cotton flower seeds also tails off, the surface of the seed of some strains is almost without short flannel (figure 3D).The above results show that GhWRKY16 expression downwards may influence cotton fiber development process, cause ripe cotton fiber length Shorten, or even short flannel deficient phenomena.The trigger differentiation of cotton fiber cell whether is influenceed for further research GhWRKY16, to blooming The same day, the Post flowering ovule of 1 day and 2 days carry out free-hand section, and the number of ovule surface fiber cell process is observed under the microscope Whether mesh and arrangement mode change, it is found that the fibrocyte projection on RNAi transgene cotton ovules surface is significantly reduced, fine Tie up cell elongation hypoevolutism (Fig. 3 F).This explanation GhWRKY16 plays important in the starting of cotton fiber development and elongation stage Effect.

5.GhWRKY16RNAi cellulose synthase and the analysis of other related gene expressions in transgene cotton

According to Li XB, Fan XP, Wang XL, Cai L, Yang WC, 2005.The Cotton ACTIN1gene is functionally expressed in fibers and participates in fiber elongation.Plant Cell 17:859-875 are carried out.Extract wild type (WT) and the GhWRKY16RNAi transgenic cotton plants Post flowering fiber of 9 days RNA, the expression of Real-time PCR Analysis cellulose synthase and cotton fiber development related gene is carried out by internal reference of GhUBI1. Error line represents standard error, and * represents transgenosis and wild type P values in t-test<0.05 has significant difference, and * * represent to turn Gene and wild type P values in t-test<0.01 has pole significant difference.

The present invention analyzes the cellulose such as GhCesA3, GhCesA5 and GhCesA10 in GhWRKY16RNAi transgene cottons The expression (Fig. 4) of synthase gene.As a result show, compared with wild type, lower more significant two in GhWRKY16 and turn In gene strain L1, L3, L5, GhCesA3, GhCesA5 and GhCesA10 expression quantity are substantially lowered.Simultaneously to these three genes Promoter find there are WRKY family specificity binding members in GhCesA3 and GhCesA5 promoter when being analyzed “W-box”.It is concluded that GhWRKY16 may be by being combined with " W-box " element in GhCesA3 and GhCesA5 promoter And regulate and control their expression, so as to influence the early development of cotton fiber.

The combination of W-box elements on 6.EMSA experimental analysis GhWRKY16 albumen and GhHOX3, GhMYB109 promoter

The GhWRKY16 albumen with His labels is purified in expression in escherichia coli, is started from GhHOX3 and GhMYB109 The DNA sequencing fragment containing W-box is chosen on son, synthesis carries biotinylated probe.With with the above-mentioned of biotin labeling DNA sequence dna is probe, is competed using unlabelled identical DNA sequence as cold probe, carries out EMSA experiments.As a result show, GhWRKY16 albumen can be combined with the W-box cis elements in GhHOX3 and GhMYB109 promoters, add unlabelled visit Pin can weaken the fluorescence intensity of institute's label probe, the combination band in EMSA experiments is weakened or is disappeared.This shows GhWRKY16 may adjust cotton fiber development by regulating and controlling the expression of downstream GhHOX3 and GhMYB109 gene.

GhHOX3 is higher in cotton fiber development expression quantity at initial stage, and by regulation and control target gene GhEXP1 downstream and GhRDL1 expression influences cotton fiber to extend.GhMYB109 expression quantity in the cotton fiber for originating and extending is higher and regulates and controls Cotton fiber development, mature fibers length shortens in GhMYB109 expresses the transgene cotton lowered.Therefore these bases are analyzed Because of the expression (Fig. 5) in GhWRKY16RNAi transgene cotton fibers.As a result show, compared with wild type, GhHOX3, The expression quantity of GhRDL1, GhEXP1 and GhMYB109 in GhWRKY16RNAi transfer-gen plants is decreased obviously.Analyze them Promoter sequence find, WRKY family specificity binding members are contained in only GhHOX3 and GhMYB109 promoter sequence “W-box”.Therefore, it is presumed that GhWRKY16 may be by combining with " W-box " of GhHOX3, GhMYB109 promoter region To regulate and control its expression, so as to influence cotton fiber development.Further experiment confirm, GhWRKY16 transcription factors can with GhHOX3, The promoter of GhMYB25 and GhMYB109 genes is combined.As shown in fig. 6, ESMA experimental results show that GhWRKY16 albumen can be with Compound is combined to form with GhHOX3 and GhMYB109 DNA probe, and with the increase of competitive probe, the combination detected Compound is reduced or disappeared.It is above-mentioned test result indicates that, GhWRKY16 may be by regulating and controlling downstream GhHOX3 and GhMYB109 etc. To adjust cotton fiber development process, influence production of cotton fibers and quality.

Obviously, above-described embodiment is only intended to clearly illustrate made example, and the not limitation to embodiment.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change or change therefore amplified Move within still in the protection domain of the invention.

Sequence table

<110>Central China Normal University

<120>A kind of WRKY transcription factor gene related to cotton fiber development and its application

<160> 3

<210> 1

<211> 3649bp

<212> DNA

<213>CottonGhWRKY16Gene order

<400> 1

gagtttaatt tttattttga gtataaaata attttaaaat tttttattaa catttacctc 60

ttaataaatt aataaaatac gaaaaattaa ttattaaatt taccccaaga aataatttaa 120

aatataaaaa aattcgaact gtttatatta aatcaaaatg gctaagggtt ccactttttt 180

gaggaaaaaa aaagagaatc ccatgtgact tgtgggatga gactttgtca cattgaccgc 240

cgtttccagt ttccggtgaa aaagtagatc ggggccccac tttttattgt gggtcccact 300

aataacttta aaatttatta ttgtttatta attttgtttt taaggtcaac tctaatcact 360

gtctccaact ccaacctaaa ataaataaat aaaaggttta atttttttaa aattataata 420

atagagtaaa gttcgaataa caaacacgat tagctcgatt taaatatagg ctatacttga 480

ggcggtgaac acccttagca atcacatggg attcaaataa aaggtttaat tttataatcc 540

tatttttaaa ttttaaaata ttataattga gttattaaag tatgaatttt ttttgtcaat 600

ttaatcataa attcgagtat taaatgttat tttgaatgtg acgtggaata taactaaaac 660

acgtaacata aattattaaa tgaagtttga tacgattggt aaagaagaag attcaagacc 720

taccacatgt aaaagttaaa ttcgagtact aaatgttatt ttgaaagtga cgtaaaatat 780

aattaaaata cgtaaattat ttaatgaagt tttggcatga ttggtaaaga ggaagactta 840

ctgcatgcaa aagttagatt tgagtattaa atgttatttt gattgtgacg tgaaatataa 900

ttaaaacacg taaattatta aatgaataat ttgcacgaat ttttgagtct catggtaaag 960

ttttagtcca gttgtcagtt ttagcccgaa tcgtattagc tcttaatcta cttgtgttgt 1020

aataatttga tcctattttg tgattagttc ataattgtat tgtatttgta atacttaaaa 1080

aaacacgtaa tttaaattgt aggcaagtgt aaaatcaacc catacttaga tctaacctgt 1140

taaaaaaatt taacatcaat aaattttaag aaaattattt atttattttt gaacacgata 1200

tattcaaatt ttgtatgtaa ctgttttttc gtttttaaaa tgttttcaaa tatctttcgt 1260

gttaaattta aattggcatt taatatctac attaattatt aagttaccaa aatgatggga 1320

caacaaatta aaattatgac taaataagaa aaattaagaa atcaaaaatc gatacaaatt 1380

aggtaaaatt actacaaaac tccttatatc atacacacac tagatgagtt agcataacaa 1440

tttatctgtt aaaatttggt gtttatatat aaagtataat aatatattta gctatcaact 1500

tttacatatt tataaaattt agcctatact cttttattgg gtcgatatta ttattttaac 1560

agaagattgt tttactcacg catccaatgt aaatagaata ctttatttat tttttaaata 1620

aagaaaataa aatgcaattc ttggtacagt ttttagaata caattaaaaa gaaatcagaa 1680

tttatttatg agaaattaat attaaatatt tttttagaaa aaaaatccca aatttacaga 1740

gtcatcaatt tcaaacggct aatccccacc ttttgacctt atctcttgtc agtgacccaa 1800

tgagaaaggc atccctttga cgaaaactcg aatctctttc atcataaatc ataatcaatc 1860

tttcactttt tcttcgtaat taaatctcaa ttcatttcag ttcccccctc tttgttctgc 1920

ttcatctata taataaactc tgcttttatt tgggagtttc tcaaaagggc tgggtgttct 1980

ttgttttcgt atgagcagag atgacggtgg aattgatgat gggtcacggc ggcgttggcg 2040

gtggaggcaa cagttttaca gtcaaaatgg aggaaactgc tttgaaagaa gctgctaatg 2100

ctgggattca aggttttgaa gaactgataa gattgatgtc taaaagtcaa cagcttcgct 2160

cgcaagatgt ttgttttaat gccccttcaa gttcagaacc agctatggaa atccaagctg 2220

ttacagataa gactgtaagt tcctttaaaa aagttatttc gttattgggt cgacctagaa 2280

ctggtcatgc tcggttcaga cgagctcctc ttaatcatct cccacaacaa caacagattc 2340

aagtgagcaa agatgaagaa gtatcgactt ttaagccttt atgttcaacc ccaagttata 2400

agttacctcc tttgccaacc aaaagtagcc atgttttaaa gactggtgtt ttagagcctg 2460

ggaattctgt tatgtcttca ttgactgggg atactgatag catacaacac caaccgtgtt 2520

tctctattgg ttttcaattc actaaccctt cttctcatgg caaacctcct ttgtcttcaa 2580

aaaggaagtg taattccatg gatgatgttg ctaatctcaa gtgtggatca tcatcctctg 2640

ctcgttgcca ttgttccaag aagaggttgg tccttggtgc ttggttcttc actttttgat 2700

tgtgatcttt gatggtttca tttgattttg attatgtatg tgttcatgta ggaaatcaag 2760

agtgaaaaga gtcattagag tcccagctgt tagcaacaaa atggctgata ttcctcacga 2820

tgattattct tggagaaaat atggccaaaa accaatcaaa ggttctcctc atccaaggtg 2880

tgttttttcg attaaatttc gaatggattc taatagagtg aattctacac caaagtttca 2940

atttttttga agttgactaa agggttccaa agttttcaaa caataattca tgtatgtatt 3000

gaatgccttt ttgtttggtt ttcaactaca aacattaatc atattctatt tggcaaaaaa 3060

ttaattgtta atttgacttt ttgcggggaa gtgcatggag acttttgaat tagaaattct 3120

ttttactaga aaaatagata aattagtatt tagattaaag agtaaatttg tatgggatga 3180

aatttttaat aaaaataatt aatttactct tttatttagg actaatttat taaattttca 3240

taatagagat ttaaaataca atagtatttg tattataaca ttgaattatt gccttactac 3300

cgagttggtt cattgagttc gcttttttaa ctcagtccca tatgttttaa caaatttcag 3360

gggctattac aaatgtagca gtgtcagagg ttgtcctgca aggaagcatg tagaaagagc 3420

tgtcgaggat ccaaggatgt tgattgtaac atacgaaggt gatcacaatc atagccataa 3480

tatcaccaca gatgtcccat cggcgctagt tcttgaatca tcataaagat catcccatcc 3540

aacggctaaa atatctatag ggttgttcag attagtcaat ttagatccat aggttctgtc 3600

tccaacaaga caaaagaaga ttgattagtg ggtaagttta gctgtttct 3649

<210> 2

<211> 1109bp

<212> DNA

<213>CottonGhWRKY16The cDNA sequence of gene

<400> 2

atgacggtgg aattgatgat gggtcacggc ggcgttggcg gtggaggcaa cagttttaca 60

gtcaaaatgg aggaaactgc tttgaaagaa gctgctaatg ctgggattca aggttttgaa 120

gaactgataa gattgatgtc taaaagtcaa cagcttcgct cgcaagatgt ttgttttaat 180

gccccttcaa gttcagaacc agctatggaa atccaagctg ttacagataa gactgtaagt 240

tcctttaaaa aagttatttc gttattgggt cgacctagaa ctggtcatgc tcggttcaga 300

cgagctcctc ttaatcatct cccacaacaa caacagattc aagtgagcaa agatgaagaa 360

gtatcgactt ttaagccttt atgttcaacc ccaagttata agttacctcc tttgccaacc 420

aaaagtagcc atgttttaaa gactggtgtt ttagagcctg ggaattctgt tatgtcttca 480

ttgactgggg atactgatag catacaacac caaccgtgtt tctctattgg ttttcaattc 540

actaaccctt cttctcatgg caaacctcct ttgtcttcaa aaaggaagtg taattccatg 600

gatgatgttg ctaatctcaa gtgtggatca tcatcctctg ctcgttgcca ttgttccaag 660

aagaggaaat caagagtgaa aagagtcatt agagtcccag ctgttagcaa caaaatggct 720

gatattcctc acgatgatta ttcttggaga aaatatggcc aaaaaccaat caaaggttct 780

cctcatccaa ggggctatta caaatgtagc agtgtcagag gttgtcctgc aaggaagcat 840

gtagaaagag ctgtcgagga tccaaggatg ttgattgtaa catacgaagg tgatcacaat 900

catagccata atatcaccac agatgtccca tcggcgctag ttcttgaatc atcataaaga 960

tcatcccatc caacggctaa aatatctata gggttgttca gattagtcaa tttagatcca 1020

taggttctgt ctccaacaag acaaaagaag attgattagt gggtaagttt agctgtttct 1080

aaaaagtaaa aaaaaaaaaa aaaaaaaaa 1109

<210> 3

<211> 318

<212>PRT

<213>The protein of cotton GhWRKY16 gene codes

<400> 3

Met Thr Val Glu Leu Met Met Gly His Gly Gly Val Gly Gly Gly Gly Asn Ser Phe Thr

1 5 10 15 20

Val Lys Met Glu Glu Thr Ala Leu Lys Glu Ala Ala Asn Ala Gly Ile Gln Gly Phe Glu

25 30 35 40

Glu Leu IIe Arg Leu Met Ser Lys Ser Gln Gln Leu Arg Ser Gln Asp Val Cys Phe Asn

45 50 55 60

Ala Pro Ser Ser Ser Glu Pro Ala Met Glu IIe Gln Ala Val Thr Asp Lys Thr Val Ser

65 70 75 80

Ser Phe Lys Lys Val IIe Ser Leu Leu Gly Arg Pro Arg IIe Gly His Ala Arg Phe Arg

85 90 95 100

Arg Ala Pro Leu Asn His Leu Pro Gln Gln Gln Gln IIe Gln Val Ser Lys Asp Glu Glu

105 110 115 120

Val Ser Thr Phe Lys Pro Leu Cys Ser Thr Pro Ser Tyr Lys Leu Pro Pro Leu Pro Thr

125 130 135 140

Lys Ser Ser His Val Leu Lys Thr Gly Val Leu Glu Arg Gly Asn Ser Val Met Ser Ser

145 150 155 160

Leu Thr Gly Asp Thr Asp Ser IIe Gln His Gln Pro Cys Phe Ser Leu Gly Phe Gln Phe

165 170 175 180

Thr Asn Pro Ser Ser His Gly Lys Pro Pro Leu Ser Ser Lys Arg Lys Cys Asn Ser Met

185 190 195 200

Asp Asp Val Ala Asn Leu Lys Cys Gly Ser Ser Ser Ser Ala Arg Cys His Cys Ser Lys

205 210 215 220

Lys Arg Lys Ser Arg Val Lys Arg Val IIe Arg Val Pro Ala Val Ser Asn Lys Met Ala

225 230 235 240

Asp IIe Pho His Asp Asp Tyr Ser Trp Arg Lys Tyr Gly Gln Lys Pro IIe Lys Gly Ser

245 250 255 260

Pro His Pro Arg Gly Tyr Tyr Lys Cys Ser Ser Val Arg Gly Cys Pro Ala Arg Lys His

265 270 275 280

Val Glu Arg Ala Val Glu Asp Pro Arg Met Leu IIe Val Thr Tyr Glu Gly Asp His Asn

285 290 295 300

His Ser His Asn IIe Thr Thr Asp Val Pro Ser Ala Leu Val Leu Glu Ser Ser

305 310 315 318

Claims

1. a kind of WRKY transcription factor gene related to cotton fiber development, it is characterised in that it has：

（1）Nucleotide sequence is as shown in SEQ ID NO.1；Or

（2）The nucleotide sequence as shown in SEQ ID NO.1 is substituted, lacks and/or increased one or several nucleotides；Or

（3）Under strict conditions with（1）The nucleotide sequence of the DNA sequence dna hybridization of restriction.

2. a kind of cDNA sequence of the WRKY transcription factor gene related to cotton fiber development, nucleotide sequence such as SEQ ID Shown in NO.2.

3. a kind of protein encoded as WRKY transcription factor genes related to cotton fiber development described in claim 1, amino Acid sequence is as shown in SEQ ID NO.3.

4. the WRKY transcription factor gene related to cotton fiber development described in claim 1 is in improvement production of cotton fibers and quality side The application in face.