CN107058341B - WRKY transcription factor gene related to cotton fiber development and application thereof - Google Patents
WRKY transcription factor gene related to cotton fiber development and application thereof Download PDFInfo
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- CN107058341B CN107058341B CN201710417178.XA CN201710417178A CN107058341B CN 107058341 B CN107058341 B CN 107058341B CN 201710417178 A CN201710417178 A CN 201710417178A CN 107058341 B CN107058341 B CN 107058341B
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- cotton
- transcription factor
- cotton fiber
- fiber
- wrky
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Abstract
The invention belongs to the field of genetic engineering, and particularly relates to a WRKY transcription factor gene related to cotton fiber development and application thereof. The invention provides a WRKY transcription factor gene related to cotton fiber development, the nucleotide sequence of which is shown in SEQ ID NO. 1; the invention also provides a cDNA sequence of the WRKY transcription factor gene related to cotton fiber development, and the nucleotide sequence is shown as SEQ ID NO. 2; the invention also provides protein encoded by the WRKY transcription factor gene related to cotton fiber development, and the amino acid sequence is shown as SEQ ID NO. 3. The WRKY transcription factor gene is expressed at a high level in cotton fibers, and shows that the gene can play an important role in regulating and controlling the initiation and the elongation development of the cotton fibers.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a WRKY transcription factor gene related to cotton fiber development and application thereof.
Background
Cotton is one of the most important crops in the world and is also the largest economic crop in China. Cotton is a special commodity related to the national civilization and plays an important role in national economy in China. Cotton fiber is a major raw material in the textile industry as an important natural fiber, and its yield and fiber quality directly determine the commercial value of cotton. With the development of the world textile industry, the requirements on the internal quality of cotton fibers such as strength, fineness, maturity and the like are higher, however, the length and the strength of the cotton fibers of most domestic raw cotton can not meet the requirements of the textile industry at present. Therefore, increasing the yield of cotton fiber and improving the quality of cotton fiber are always the research direction and breeding target for the development of cotton industry.
Morphogenesis of cotton fiber cells involves a highly regulated cellular differentiation process. Each fiber cell is a single-cell hairy process differentiated from the outer epidermal cells on the surface of the ovule. Fiber development goes through several sequential and overlapping stages, namely initiation, elongation, secondary wall thickening and dehydration maturation of the fiber cells. The morphogenesis process of cotton fiber cell walls for about 50 days determines the quality parameters of cotton fibers. The initial number of ovule epidermal cell fibers determines the yield of mature fibers, and the degree of elongation of the fibers and thickening of the secondary wall determines the length and strength of the mature fibers. Therefore, the deep research on the development process and the regulation mechanism of the cotton fiber is expected to open up a new way for improving the yield and the quality of the cotton fiber.
The cotton fiber development process is regulated and controlled by the transcription level. The main transcription factor families HD-ZIP protein, MYB protein, NAC protein and the like which are found at present play a crucial role in the initiation or development process of fiber cells. The WRKY transcription factor is a special novel transcription regulation factor discovered in plants in recent years, and is named after the WRKYGQK transcription factor contains a highly conserved amino acid sequence consisting of WRKYGQK at the N end. Research has shown that the WRKY transcription factor plays a very important role in plant growth and development, senescence, biotic and abiotic stress response. However, no report is available on whether WRKY transcription factors are involved in regulating cotton fiber development. Therefore, the function and the regulation mechanism of the WRKY transcription factor in cotton fiber development are clarified, and the relevant important regulation genes are separated and identified, so that the method has important significance for improving the quality of cotton fibers.
The WRKY protein family belongs to zinc finger type transcription regulation factors, and has the main structural characteristics that at least one WRKY structural domain is contained in a DNA binding domain, the structural domain is a polypeptide sequence consisting of about 60 highly conserved amino acid residues, a 7-peptide region (WRKYGQK) with high conservation is arranged at the N end of the WRKY structural domain, the N end is a core region of the WRKYGQK, the C end of the WRKY structural domain is a zinc finger structure (zinc finger motif), and the general component of the WRKY structural domain is CX4～5CX22～23HX1H. According to the difference of the number of WRKY motifs and the characteristics of zinc finger structures in the WRKY domain, the WRKY family is divided into 3 types: class IMembers contain 2 WRKY motifs and C2H2Type II zinc finger structures, class II members containing 1 WRKY motif and C2H2Type III zinc finger structures containing 1 WRKY motif and C2HC type zinc finger structure. Research shows that most WRKY transcription factors only contain 1 WRKY structural domain and belong to the class II, while class III WRKY transcription factors only exist in higher plants and do not exist in some lower plants such as bryophytes. Meanwhile, almost all III-class WRKY transcription factors in higher plants are related to the biotic stress response reaction of the plants, which indicates that the III-class WRKY transcription factors are possibly generated by the plants through adaptive evolution under the environmental stress.
The WRKY protein can be specifically combined with the (T) (T) TGAC (C/T) sequence of a DNA binding region, and the binding region is called W-box and comprises a TGAC conserved core sequence which is essential for the binding of WRKY transcription factors. Furthermore, Zn2+The participation of plasma metal ions, phosphorylation and the like are also beneficial to the combination of WRKY transcription factors and W-box. Most promoters of target genes of WRKY transcription factors contain variable amounts of W-box, which are arranged in the same direction or form a palindromic structure. The WRKY transcription factor is combined with the W-box existing in the promoter of the functional gene or other regulatory factor genes to regulate the expression of the downstream genes.
For example, soybean GmWRKY13 and Arabidopsis AtWRKY75 are related to the growth of lateral roots, and rice OsWRKY31 is also related to the formation and extension of lateral roots, Arabidopsis AtWRKY6 and rice OsWRKY23 can initiate the senescence process, while AtWRKY70 can negatively regulate senescence reaction, Arabidopsis AtTTG2 is a gene encoding WRKY transcription factor, which plays an important role in the formation of Arabidopsis epidermoid and seed coat.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a WRKY transcription factor gene related to cotton fiber development and application thereof.
The invention provides a WRKY transcription factor gene related to cotton fiber development, which has the following characteristics:
1) the nucleotide sequence is shown as SEQ ID NO. 1; or
2) The nucleotide sequence shown as SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides; or
3) Nucleotide sequences which hybridize under stringent conditions with the DNA sequences defined in 1).
The invention also provides a cDNA sequence of the WRKY transcription factor gene related to cotton fiber development, and the nucleotide sequence is shown as SEQ ID NO. 2.
The invention also provides protein encoded by the WRKY transcription factor gene related to cotton fiber development, and the amino acid sequence is shown as SEQ ID NO. 3.
The WRKY transcription factor gene related to cotton fiber development is applied to improving the yield and quality of cotton fibers.
The invention has the beneficial effects that: the invention provides a new GhWRKY16 gene sequence and cDNA sequence related to cotton fiber development, wherein the gene is expressed at high level in cotton fibers, which indicates that the gene may play an important role in regulating and controlling the initial and elongation development processes of the cotton fibers; in GhWRKY16RNAi transgenic cotton, the expression of important regulatory genes such as GhHOX3 and GhMYB109 which play an important role in the development process of cotton fibers is reduced, and the expression level of cellulose synthase genes is also reduced; compared with the wild type, the mature fiber length of the GhWRKY16RNAi transgenic cotton is obviously shortened, and short fibers are fewer or are absent; the GhWRKY16 protein is located in cell nucleus and can be directly combined with W-box cis-element on GhHOX3 and GhMYB109 gene promoters, which shows that the GhWRKY16 gene can regulate the cotton fiber cell initiation and elongation development process by regulating cotton fiber related downstream target genes, and influences the yield and quality of cotton fibers.
Drawings
FIG. 1 shows the expression of GhWRKY16 in cotton tissue by fluorescent quantitative RT-PCR analysis, wherein R: a root; l: a blade; c: cotyledons; h: hypocotyls; p: flower petals; a: anther; f: fibers; 0 d: ovules on the day of flowering; 3 d: fiber 3 days after flowering; 6 d: fiber 6 days after flowering; 9 d: fiber 9 days after flowering; 12 d: fiber 12 days after flowering; 15 d: fiber 15 days after flowering; 18 d: fiber 18 days after flowering, error bars represent standard error.
FIG. 2 shows subcellular localization of GhWRKY16 protein in Arabidopsis thaliana hypocotyl cells, where A: GhWRKY16-GFP fluorescence signals in the hypocotyl cells of an arabidopsis plant; b: superposition of GFP fluorescence images with bright field images of the same cells.
Fig. 3 is a phenotype analysis of GhWRKY16 transgenic cotton, in which a: fluorescent quantitative RT-PCR analysis of the expression of the GhWRKY16 gene in the fiber 9 days after the Wild Type (WT) and transgenic cotton strains (L1-L8) bloom; b: in vitro culture of ovules of wild type and GhWRKY16RNAi transgenic cotton strains (L1, L3, L5) on the day of flowering; c: statistical analysis of fiber length after 12, 15 and 18 days of wild type and GhWRKY16RNAi ex vivo ovule culture (n > 30); d: comparing the shapes of the wild type cotton and the GhWRKY16RNAi transgenic cotton mature fiber long velvet and short velvet; e: measuring the length of mature fiber of wild type and GhWRKY16RNAi transgenic cotton and performing statistical analysis (n is more than 50); f: the ovule cross sections of wild type and GhWRKY16RNAi transgenic cotton on the day (0d) of flowering and 1 day (1d) and 2 days (2d) after flowering show the fibrocyte protuberant differentiation conditions after 0 day, 1 day and 2 days of flowering; error bars represent standard error, indicating a significant difference between transgene and wild type with P value <0.05 in t-test; indicates that there was a very significant difference in P value <0.01 in t-test between the transgene and the wild type. WT, wild type; L1-L8, GhWRKY16-RNAi transgenic cotton strain.
FIG. 4 shows the expression of cellulose synthases GhCesA3, GhCesA5 and GhCesA10 in the fiber 9 days after the Wild Type (WT) and GhWRKY16RNAi transgenic cotton (L1, L3 and L5) blossoms. WT, wild type; l1, L3 and L5, GhWRKY16-RNAi transgenic cotton lines.
FIG. 5 shows the expression of genes GhHOX3, GhRDL1, GhEXP1 and GhMYB109 related to cotton fiber development in the fibers 9 days after the Wild Type (WT) and GhWRKY16RNAi transgenic cotton (L1, L3 and L5) blossom. WT, wild type; l1, L3 and L5, GhWRKY16-RNAi transgenic cotton lines.
FIG. 6 is the EMSA analysis of the binding of GhWRKY16 protein to GhHOX3 and GhMYB109 promoter.
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1 cloning identification and functional analysis of full-Length sequence of WRKY family Gene GhWRKY16 of Cotton
1, separation and identification of GhWRKY16 gene and cDNA sequence:
related GhWRKY genes and cDNA sequences are screened in a cotton genome and EST database, 22 cotton WRKY genes are identified, one gene (named GhWRKY16) is preferentially expressed in cotton fibers, and a GhWRKY16 full-length cDNA sequence is obtained by DNA and protein sequence analysis and separation by using transcripts (total RNA) of cotton fiber cells. Designing a primer according to the GhWRKY16cDNA sequence, and amplifying by adopting a PCR technology by taking cotton genome DNA as a template to obtain the DNA full-length sequence of the gene. Sequence analysis shows that the full-length sequence (shown as SEQ ID NO. 1) of the GhWRKY16 gene comprises a5 '-upstream promoter region (1-2000 bp), 3 exons, 2 introns (underlined) and 3' -UTR (3527-3649 bp); the cDNA sequence of the GhWRKY16 gene is shown in SEQ ID NO.2, wherein the cDNA sequence contains 957bp open reading frame (from initiation codon ATG to termination codon TAA), 318 amino acids are coded (the amino acid sequence is shown in SEQ ID NO. 3), and the molecular weight is 34 KDa. The GhWRKY16 protein DNA binding domain contains a WRKY structural domain, the N end of the structural domain is provided with a WRKYGQK7 peptide region with high conservation as the core region, and the C-terminal zinc finger motif of the domain is C2H2Belonging to the class II WRKY transcription factors.
2. Quantitative RT-PCR analysis of the expression of the GhWRKY16 gene:
total RNA of different tissues and fibers of purified cotton was extracted using RNA extraction kit (Qiagin) and gene expression was studied by real-time fluorescent quantitative RT-PCR (performed according to Li XB, Fan XP, Wang XL, Cai L, Yang WC,2005.The CottonACTIN1gene is functional expressed in fibers and particulate in fibrous growth. plant Cell 17: 859-875). First, total RNA (2. mu.g/sample) of various tissues of cotton (root, leaf, cotyledon, hypocotyl, petal, anther, 0-day ovule after flowering, 3, 6, 9, 12, 15 and 18-day fiber after flowering, etc.) was treated with M-MLV RNase H-Reverse transcription of Reverse Transcriptase (Promega) into cDNA; then, quantitative PCR reaction was carried out using cDNA as a template, gene-specific primers (GhWRKY16RTP 1: 5'-GAAGTGTAATTCCATGGATG-3' and GhWRKY16RTP 2: 5'-TGACTAATCTGAACAACCCT-3') and Real-time PCR Master Mix (TOYOBO, Japan). The cotton GhUBI 1gene is used as an internal standard of RT-PCR reaction, the amplification of each cycle of the target gene is detected by SYBR-Green fluorescence, and the expression level relative value of the target gene is calculated. Each experiment was repeated 3 times and the results were statistically analyzed. Fluorescent quantitative RT-PCR results show that GhWRKY16 has different degrees of expression in different tissues of cotton and different development stages of cotton fibers, and is preferentially expressed in cotton fibers 3-9 days after flowering (figure 1), and the gene is presumed to possibly play a regulating role in cotton fiber cells in the elongation stage.
Cell sub-localization analysis of GhWRKY16 protein
Constructing a GhWRKY16 eGFP plant expression vector, transferring the vector into GV3101 agrobacterium by an electric transformation method, transforming arabidopsis thaliana by a flower soaking method, harvesting T0 generation seeds of mature arabidopsis thaliana, and screening positive plants. After the transgenic arabidopsis T2 generation seeds were germinated and grown for 5 days on a kanamycin-containing MS medium, the transgenic arabidopsis seedlings were observed under a confocal microscope for fluorescence signals of Green Fluorescent Protein (GFP) of hypocotyl cells. The result of subcellular localization analysis shows that the GFP fluorescence signal is mainly distributed in the nucleus of the hypocotyl cell, and the GhWRKY16 protein is located in the nucleus (FIG. 2).
GhWRKY16RNAi transgenic cotton phenotypic analysis:
constructing 35S promoter-driven PMD-GhWRKY16RNAi vector, and transforming Agrobacterium LBA 4404. The cotton hypocotyl explant is infected with agrobacterium and cultured for 2 days, and the hypocotyl is transferred to selective culture medium for culture, induced and screened to obtain transformed callus. After the callus is subjected to subculture for 8-10 months, embryogenic callus and somatic embryos are induced and differentiated, and the somatic embryos further germinate and regenerate into transgenic cotton seedlings. Transplanting the cotton seedling into soil, and growing until the cotton seedling blooms and fruits. Extracting cotton genome DNA, and detecting and identifying transgenic cotton plants by using a PCR technology.
(1) Real-time fluorescent quantitative RT-PCR studies gene expression according to Li XB, Fan XP, Wang XL, Cai L, Yang WC,2005.The Cotton ACTIN1gene is functional expressed in fibers and fibers in fiber interaction. plant Cell 17: 859-875, fiber RNA 9 days after flowering was extracted, cDNA was reverse transcribed, and then quantitative PCR reaction was performed using gene-specific primers (GhWRKY16RTP1 and GhWRKY16RTP2) and Real-time PCR Master Mix (TOYOBO, Japan). The expression condition of GhWRKY16 in Wild Type (WT) and each strain of transgenic plants is analyzed by taking GhUBI1 as an internal reference. And repeating the steps for 3 times, and carrying out statistical analysis on the experimental results.
(2) In-vitro ovule culture experiment, selecting ovules of a Wild Type (WT) and 3 GhWRKY16RNAi transgenic cotton strains (L1, L3 and L5) on the day of flowering, placing the ovules in a BT culture medium for aseptic culture (dark treatment, 30 ℃) for 12 days, 15 days and 18 days respectively, fixing the ovules by using a fixing solution, measuring the length of fibers on the surfaces of the ovules, and performing statistical analysis, wherein an error line represents a standard error, and indicates that the P value of the transgenic and the wild type in t-test is 0.05 significant difference, and indicates that the P value of the transgenic and the wild type in t-test is 0.01 significant difference.
(3) Statistical measurement of mature cotton fibers: after the fiber is mature, the length of the mature fiber of each strain of Wild Type (WT) and GhWRKY16RNAi transgenic plants is measured and counted (n > 50). CK is two isolated non-transgenic plants, and the rest are different GhWRKY16RNAi transgenic strains. Error bars represent standard errors, indicating significant differences between the transgene and the wild type at t-test with P values <0.05, and very significant differences between the transgene and the wild type at t-test with P values < 0.01.
(4) Carrying out bare-handed slicing on ovules: wild Type (WT) and GhWRKY16RNAi transgenic plants were sliced by hand on day (0), 1 and 2 of flowering, cut into 1mm slices and photographed under a microscope.
The invention constructs GhWRKY16RNA interference (RNAi) vector, transforms cotton and obtains transgenic cotton plants. Firstly, the detection of the expression level of GhWRKY16 transgenic plants on the RNA level shows that the expression level of GhWRKY16 gene is greatly reduced in most GhWRKY16RNAi transgenic plants (FIG. 3A). The cotton bolls of the GhWRKY16RNAi transgenic plants are smaller than the wild type. The GhWRKY16RNAi transgenic cotton and wild ovules on the day of flowering are cultured in vitro, and observation after 12-18 days shows that the transgenic cotton ovules are slow in development and the fiber length is obviously shorter than that of the wild type cotton (figure 3B, C). The length of the mature cotton fiber of the RNAi transgenic plant is measured and analyzed, and the result shows that the length of the mature fiber of most GhWRKY16RNAi transgenic cotton plants is obviously shorter than that of the wild type (figure 3E). Furthermore, after stripping long fibers, it was found that there was less flock on the seeds of RNAi transgenic cotton, and there was almost no flock on the surface of the seeds of some lines (FIG. 3D). The results show that the cotton fiber development process can be influenced by the expression down-regulation of GhWRKY16, so that the length of mature cotton fibers is shortened, and even the phenomenon of short fiber deletion is caused. To further study whether GhWRKY16 affects the initial differentiation of cotton fiber cells, the ovules on the day of flowering, 1 day after flowering and 2 days were sliced by hand, and the number and arrangement of fiber cell protrusions on the ovules were observed under a microscope to find that the fiber cell protrusions on the surface of the ovules of RNAi transgenic cotton were significantly reduced and the fiber cells were retarded in elongation and development (FIG. 3F). This indicates that GhWRKY16 plays an important role in both the initial and elongation stages of cotton fiber development.
Analysis of expression of cellulose synthase and other related genes in GhWRKY16RNAi transgenic cotton
According to Li XB, Fan XP, Wang XL, Cai L, Yang WC,2005, The Cotton ACTIN1gene isoflavone expressed in fibers and particulate in fiber interaction, plant cell 17: 859-. Fiber RNA of Wild Type (WT) and GhWRKY16RNAi transgenic cotton plants 9 days after flowering is extracted, and real-time quantitative PCR analysis is carried out on the expression of cellulose synthase and cotton fiber development related genes by taking GhUBI1 as an internal reference. Error bars represent standard errors, indicating significant differences between the transgene and the wild type at t-test with P values <0.05, and very significant differences between the transgene and the wild type at t-test with P values < 0.01.
The expression conditions of cellulose synthase genes such as GhCosA 3, GhCosA 5 and GhCosA 10 in GhWRKY16RNAi transgenic cotton are analyzed (figure 4). The results show that the expression levels of GhCESA3, GhCESA5 and GhCESA10 are obviously reduced in two transgenic lines L1, L3 and L5 with more remarkable GhWRKY16 reduction compared with the wild type. When the promoters of the three genes are analyzed simultaneously, WRKY family specific binding elements W-box are found in the promoters of GhCESA3 and GhCESA 5. We conclude that GhWRKY16 may influence early development of cotton fibers by regulating their expression by binding to the "W-box" element in the promoters of GhCesA3 and GhCesA 5.
EMSA experiment analysis of binding of GhWRKY16 protein to W-box element on GhHOX3 and GhMYB109 promoters
Expressing and purifying the His-tagged GhWRKY16 protein in escherichia coli, selecting a DNA sequence fragment containing a W-box from GhHOX3 and GhMYB109 promoters, and synthesizing a probe with a biotin label. The EMSA experiment was performed by using the DNA sequence labeled with biotin as a probe and the same unlabeled DNA sequence as a cold probe for competition. The result shows that the GhWRKY16 protein can be combined with the W-box cis-element on the GhHOX3 and GhMYB109 promoters, the fluorescence intensity of the marked probe can be weakened by adding an unlabeled probe, and the combined band in an EMSA experiment is weakened or disappears. This suggests that GhWRKY16 may regulate cotton fiber development by regulating the expression of downstream GhHOX3 and GhMYB109 genes.
The expression level of GhHOX3 is higher at the early development stage of cotton fibers, and the cotton fiber elongation is influenced by regulating the expression of downstream target genes GhEXP1 and GhRDL 1. The expression level of GhMYB109 in the initial and elongated cotton fibers is high, the cotton fiber development is regulated, and the mature fiber length in the transgenic cotton with the GhMYB109 expression down-regulated is shortened. Therefore, the expression of the genes in GhWRKY16RNAi transgenic cotton fibers is analyzed (FIG. 5). The results show that the expression levels of GhHOX3, GhRDL1, GhEXP1 and GhMYB109 in GhWRKY16RNAi transgenic plants are all obviously reduced compared with the wild type. Analysis of their promoter sequences revealed that only the promoter sequences of GhHOX3 and GhMYB109 contained the WRKY family specific binding element "W-box". Therefore, we speculate that GhWRKY16 may regulate its expression by binding with "W-box" of GhHOX3 and GhMYB109 promoter regions, thereby influencing cotton fiber development. Further experiments prove that the GhWRKY16 transcription factor can be combined with the promoters of GhHOX3, GhMYB25 and GhMYB109 genes. As shown in fig. 6, the ESMA experimental results show that the GhWRKY16 protein can bind to the DNA probes of GhHOX3 and GhMYB109 to form a complex, and the detected binding complex decreases or disappears with the increase of competitive probes. The experimental results show that GhWRKY16 can regulate the development process of cotton fibers by regulating downstream GhHOX3, GhMYB109 and the like, and the yield and quality of the cotton fibers are influenced.
It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Sequence listing
<110> university of Master in China
<120> WRKY transcription factor gene related to cotton fiber development and application thereof
<160>3
<210>1
<211>3649bp
<212>DNA
<213>CottonGhWRKY16Gene sequences
<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 actttttgat2700
tgtgatcttt gatggtttca tttgattttg attatgtatg tgttcatgta ggaaatcaag2760
agtgaaaaga gtcattagag tcccagctgt tagcaacaaa atggctgata ttcctcacga 2820
tgattattct tggagaaaat atggccaaaa accaatcaaa ggttctcctc atccaaggtg2880
tgttttttcg attaaatttc gaatggattc taatagagtg aattctacac caaagtttca2940
atttttttga agttgactaa agggttccaa agttttcaaa caataattca tgtatgtatt3000
gaatgccttt ttgtttggtt ttcaactaca aacattaatc atattctatt tggcaaaaaa3060
ttaattgtta atttgacttt ttgcggggaa gtgcatggag acttttgaat tagaaattct3120
ttttactaga aaaatagata aattagtatt tagattaaag agtaaatttg tatgggatga3180
aatttttaat aaaaataatt aatttactct tttatttagg actaatttat taaattttca3240
taatagagat ttaaaataca atagtatttg tattataaca ttgaattatt gccttactac3300
cgagttggtt cattgagttc gcttttttaa ctcagtccca tatgttttaa caaatttcag3360
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>CottonGhWRKY16cDNA sequence of Gene
<400>2
atgacggtgg aattgatgat gggtcacggc ggcgttggcg gtggaggcaa cagttttaca60
gtcaaaatgg aggaaactgc tttgaaagaa gctgctaatg ctgggattca aggttttgaa120
gaactgataa gattgatgtc taaaagtcaa cagcttcgct cgcaagatgt ttgttttaat180
gccccttcaa gttcagaacc agctatggaa atccaagctg ttacagataa gactgtaagt240
tcctttaaaa aagttatttc gttattgggt cgacctagaa ctggtcatgc tcggttcaga300
cgagctcctc ttaatcatct cccacaacaa caacagattc aagtgagcaa agatgaagaa360
gtatcgactt ttaagccttt atgttcaacc ccaagttata agttacctcc tttgccaacc420
aaaagtagcc atgttttaaa gactggtgtt ttagagcctg ggaattctgt tatgtcttca480
ttgactgggg atactgatag catacaacac caaccgtgtt tctctattgg ttttcaattc540
actaaccctt cttctcatgg caaacctcct ttgtcttcaa aaaggaagtg taattccatg600
gatgatgttg ctaatctcaa gtgtggatca tcatcctctg ctcgttgcca ttgttccaag660
aagaggaaat caagagtgaa aagagtcatt agagtcccag ctgttagcaa caaaatggct720
gatattcctc acgatgatta ttcttggaga aaatatggcc aaaaaccaat caaaggttct780
cctcatccaa ggggctatta caaatgtagc agtgtcagag gttgtcctgc aaggaagcat840
gtagaaagag ctgtcgagga tccaaggatg ttgattgtaa catacgaagg tgatcacaat900
catagccata atatcaccac agatgtccca tcggcgctag ttcttgaatc atcataaaga960
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> protein coded by cotton GhWRKY16 gene
<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
205210 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 (4)
1. A WRKY transcription factor gene related to cotton fiber development is characterized in that a nucleotide sequence is shown as SEQ ID NO. 1.
2. A cDNA sequence of WRKY transcription factor gene related to cotton fiber development is shown in SEQ ID No. 2.
3. A protein encoded by the WRKY transcription factor gene related to cotton fiber development as claimed in claim 1, wherein the amino acid sequence is shown as SEQ ID No. 3.
4. The use of the cotton fiber development-related WRKY transcription factor gene as claimed in claim 1 for improving cotton fiber yield and quality.
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| CN104004773A (en) * | 2014-06-19 | 2014-08-27 | 天津农学院 | Wheat WRKY transcription factor gene and application thereof to transforming arabidopsis root development |
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| CN104004773A (en) * | 2014-06-19 | 2014-08-27 | 天津农学院 | Wheat WRKY transcription factor gene and application thereof to transforming arabidopsis root development |
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| Title |
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| XM_017788325.1;佚名;《GenBank》;20160809;第200-1156位 * |
| 棉花（Gossypium hirsutum）WRKY基因分离与鉴定;周立;《中国优秀硕士学位论文全文数据库_农业科技辑》;20140815;摘要 * |
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