CN113106105A - Cotton gene and its use - Google Patents
Cotton gene and its use Download PDFInfo
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Abstract
本发明提供了一种棉花基因及其用途，所述棉花基因的核苷酸序列如SEQID NO:1所示。该基因对于棉花对黄萎病的抗性具有负调控作用。
The present invention provides a cotton gene and use thereof. The nucleotide sequence of the cotton gene is shown in SEQ ID NO: 1. This gene has a negative regulatory effect on the resistance of cotton to Verticillium wilt.
Description
Technical Field
The invention relates to a cotton gene and application thereof.
Background
Cotton Verticillium wilt, known as "cancer" of cotton, is a soil-borne fungal vascular bundle disease caused by Verticillium dahliae (Verticillium dahliae). The verticillium dahliae infects cotton to cause yellowing, necrosis, falling off and plant death of leaves, seriously affects the yield and quality of cotton and causes huge economic loss. Although a breeder breeds a batch of cotton varieties with stronger resistance to verticillium wilt and excellent comprehensive characters by a conventional breeding method, the breeding progress of the cotton verticillium wilt resistance is slow due to the lack of verticillium wilt resistance sources, the verticillium wilt variation speed and the long period of the traditional breeding method. Therefore, the disease-resistant key genes are excavated and identified by applying the modern genetic engineering technology, so that the disease-resistant gene resources can be provided for cotton, and the method has important significance for breeding the cotton for resisting verticillium wilt.
The WRKY transcription factor is a plant-specific transcription factor, usually exists in a gene family form, and 74 WRKY gene family members exist in arabidopsis thaliana only. 238 WRKY transcription factors (plantaTFDB) were found in the Gossypium hirsutum genome. Research shows that the WRKY transcription factor not only participates in the growth and development of plants, but also plays an important role in the disease-resistant process of the plants. Wherein GhWRKY3, GhWRKY4, GhWRKY7, GhWRKY22, GhWRKY33 and GhWRKY40 are induced by verticillium wilt to be up-regulated and expressed. Recent researches find that GbWRKY1, GhWRKY70 and GhWRKY70D13 negatively regulate the disease resistance of cotton to verticillium wilt by inhibiting Jasmonic Acid (JA) signal channels, and the regulation and control effect of Salicylic Acid (SA) is also reported.
Disclosure of Invention
The invention aims to provide a cotton gene and application thereof.
In order to realize the purpose, the technical scheme is as follows: a cotton gene has a nucleotide sequence shown as SEQ ID NO. 1. The cotton gene is named GhWRKY 55.
The invention also provides a protein coded by the cotton gene, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The invention also provides a VIGS vector for enhancing the resistance of cotton to verticillium wilt, which is a tobacco rattle virus vector pTRV2 containing the DNA fragment for silencing the cotton gene. Preferably, the primers for amplifying the DNA fragments include those shown as SEQ ID NO. 9 and SEQ ID NO. 10.
The invention also provides a VIGS system for enhancing the resistance of cotton to verticillium wilt, which comprises the VIGS vector and a tobacco rattle virus vector pTRV 1.
The invention also provides a method for enhancing the resistance of cotton to verticillium wilt, which comprises reducing the expression of the cotton gene in cotton.
The present invention also provides a method of enhancing resistance to verticillium wilt in cotton, said method comprising silencing a cotton gene as described above in cotton; preferably, the cotton gene silencing is achieved by introducing the VIGS vector or the VIGS system into cotton.
The invention also provides a breeding method of the verticillium wilt resistant cotton, which comprises the step of reducing the expression of the cotton gene in the cotton.
The invention also provides a breeding method of the verticillium wilt-resistant cotton, which comprises the step of silencing the cotton gene in the cotton. Preferably, the cotton gene silencing is achieved by introducing the VIGS vector or the VIGS system into cotton.
The invention also provides the application of the cotton gene, the protein, the VIGS vector or the VIGS system in enhancing the resistance of cotton to verticillium wilt.
Preferably, the cotton resistance to verticillium wilt is enhanced by silencing a cotton gene as described above in cotton.
Has the advantages that:
the invention provides a cotton gene which has negative regulation and control effect on the resistance of cotton to verticillium wilt.
Drawings
FIG. 1 shows that the disease-resistant variety Shimadong resists inoculation of Verticillium dahliae 12(A) and 24h (B) differentially expressed genes from 18-1 (HR). HR 0: treating a sample with 18-1 root system resistance of the stone continental land by using distilled water; HR12 and HR24, Shilangen 18-1 resistant inoculated verticillium wilt bacteria 12 and 24h root system samples.
FIG. 2 shows a GhWRKY55 phylogenetic tree analysis diagram. GhWRKY55 has higher homology with AtWRKY 55. Wherein AtWRKY (Arabidopsis thaliana, NP), AtWRKY (NP), AtWRKY NP (AFKY), AtWRKY (NP), AtWRUCWRKY (NP), GlankyunVnayu (NPNXP), Glankyu (NPVnaupx, Naccu), Gluchi (NPVnaxu), Navie (NP), Naccu (Narco).
FIG. 3 shows the result of alignment analysis of multiple sequences of GhWRKY 55. GhWRKY55 contains typical WRKY and C2HC zinc finger domains. Wherein the sequences of NaWRKY55, DcWRKY55, GmWRKY55, HaWRKY55, JcWRKY55, GhWRKY55 and AtWRKY55 are consistent with those in FIG. 2.
FIG. 4 shows the result of analysis of expression pattern of GhWRKY55 gene in cotton anti-infection material. In the figure, A is the analysis result of the expression pattern in each tissue of cotton; b is the result of expression pattern analysis after inoculation of verticillium wilt; c is an expression pattern analysis result after MeJA treatment; d is the expression pattern analysis result after SA treatment.
FIG. 5 shows the phenotype of GhCHLI two weeks after GhCHLI injection of GJUN cotton No. 1.
FIG. 6 shows the result of the functional analysis of the GhWRKY55 gene silencing. In the figure, A is the result of analyzing the expression pattern of GhWRKY55 inoculated with verticillium wilt bacteria in TRV GhWRKY55 and TRV 00; b is the result of the phenotype analysis of TRV GhWRKY55 and TRV 00 after inoculation of verticillium wilt bacteria 21 d; c is the disease index analysis result of 14 days and 21 days after verticillium wilt bacteria inoculation; d is the incidence analysis result of TRV GhWRKY55 and TRV 00; e is the recovery culture of TRV GhWRKY55 and TRV 00 plant stem pathogenic bacteria after inoculation of verticillium dahliae 14 d; f is the result of measuring the relative content of pathogenic bacteria in plant stalks of TRV GhWRKY55 and TRV 00 after inoculation of verticillium dahliae for 14 days and 21 days.
FIG. 7 shows that overexpression of GhWRKY55 reduces resistance of Arabidopsis to verticillium dahliae. In the figure, A is the expression level of GhWRKY55 in a overexpression arabidopsis strain; b is the phenotype of transgenic arabidopsis after inoculation of verticillium dahliae 21 d; c is the coloration of 14d trypan blue of Verticillium wilt bacteria inoculated with GhWRKY55 overexpression transgenic Arabidopsis; d is disease index statistics of 14, 21 and 28D transgenic arabidopsis thaliana after inoculation with verticillium wilt bacteria (n is 30); e is the determination of the relative content of pathogenic bacteria in the stem of the GhWRKY55 overexpression transgenic arabidopsis plant 14 days after verticillium wilt bacteria inoculation. The statistical method was T-test (P < 0.05;. P <0.01), and the data were mean ± standard deviation.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
The applicant analyzes the gene expression profiles of different disease-resistant upland cotton materials responding to verticillium wilt infection through comparison of transcriptomics, and finds that the expression quantity of the GhWRKY55 gene is remarkably reduced after the gene is induced by verticillium wilt bacteria in the disease-resistant variety Shidai 18-1, and the time of the gene is induced to be subjected to down-regulation expression in the disease-sensitive variety Jichong 1 is later than that of the disease-resistant variety and the down-regulation expression degree is remarkably lower than that of the disease-resistant variety. Recent studies indicate that WRKY55 belongs to type III WRKY transcription factor, and the gene negatively regulates the resistance of Arabidopsis to Pseudomonas syringae. The invention utilizes VIGS method to inoculate verticillium wilt bacteria after the expression of GhWRKY55 gene is instantly silenced in cotton, and finds that the gene has negative regulation and control effect on cotton verticillium wilt resistance. In addition, the GhWRKY55 gene is over-expressed in Arabidopsis thaliana, and the GhWRKY55 gene is further proved to negatively regulate the disease resistance of cotton to verticillium wilt. The invention provides a new method for breeding verticillium wilt-resistant cotton.
The quantitative tests in the following examples, all set up three biological replicates and three technical replicates.
Example 1 cloning of the GhWRKY55 Gene
Transcriptome analysis found that the expression level of GhWRKY55 gene in the disease-resistant variety Shilangen 18-1 was significantly reduced 12h and 24h after inoculation of Verticillium dahliae, and reached 17.5 times and 20.1 times respectively (see FIGS. 1A and B). The cDNA sequence of GhWRKY55 is amplified by using the 18-1 root cDNA of the continental stone as a template.
The primers required for cloning were as follows:
GhWRKY55-F：ATGGACCAACAACAACAACAACA
GhWRKY55-R：TCAACAATGCTTCTTGTCCCCT
connecting the amplified sequence to a PMDT-19 vector, screening positive clones, and sending the positive clones to Shanghai biological engineering Co., Ltd for sequencing. The nucleotide sequence is shown as SEQ ID NO. 1, and the protein sequence is shown as SEQ ID NO. 2.
Phylogenetic tree analysis was performed on GhWRKY55 with some known WRKY transcription factors in arabidopsis and other species.
As a result, as shown in FIG. 2, the sequence has the highest homology with Arabidopsis AtWRKY55, so that the applicant named it GhWRKY 55.
Except GhWRKY55, other representative plant WRKY55 amino acid sequences are selected to be subjected to multi-sequence alignment analysis through DANMAN software.
As a result, as shown in FIG. 3, GhWRKY55 contains typical WRKY and C2HC zinc finger domains, and thus this sequence is a member of type III WRKY transcription factors.
Example 2 analysis of expression Pattern of GhWRKY55
Respectively extracting root, stem and leaf RNA of Shiluzhuan 1 (verticillium wilt resistant variety, HR) and Junmian No. 1 (verticillium wilt susceptible variety, HS). The extraction method refers to a plant polysaccharide polyphenol total RNA extraction kit of Tiangen Biochemical technology Co., Ltd (Beijing).
After extraction, RNA integrity was analyzed by 1.2% agarose gel electrophoresis and RNA concentration and purity measurements were determined by Nanodrop ND-2000 microspectrophotometer.
Mu.g of the RNA template was used to synthesize the first strand of cDNA by Reverse transcription using M-MLV Reverse Transcriptase kit from Novozam Biotechnology Ltd (Nanjing).
Designing an RT-qPCR primer, and analyzing the expression specificity of the GhWRKY55 in roots, stems and leaves of different disease-resistant varieties by adopting an RT-qPCR method. Relative expression amount of genes according to 2-ΔΔCTAnd (4) calculating.
Reaction system: mu.L of 10-fold diluted cDNA, 5. mu.L of 2 XSSYBR Green Mix, 0.2. mu.L of each forward and reverse primer of 10. mu.mol/L, and 3.6. mu.L of ddH2O 3.6.
qPCR procedure: pre-denaturation at 95 ℃ for 3min, 10s at 95 ℃, 15s at 60 ℃ and 15s at 72 ℃ for 40 cycles.
The RT-qPCR primer sequence is as follows:
GhWRKY55-qF：GTTCGGGTTCAGGTGGAAGAGG
GhWRKY55-qR：GCCACTGCCGGATACTCAACAT
the primers used for the gene GhUBQ7 were as follows:
GhUBQ7-F：GAAGGCATTCCACCTGACCAAC
GhUBQ7-R：CTTGACCTTCTTCTTCTTGTGCTTG
as shown in FIG. 4A, GhWRKY55 is expressed predominantly in the root of Shi Da Ji, which is resistant to No. 18-1 and Jun Cotton No. 1.
1. Expression analysis of GhWRKY55 gene after inoculation of verticillium wilt
1.1 activation and culture of Verticillium dahliae
200 mu L of verticillium dahliae strain V991 spore liquid is evenly smeared on a PDA culture medium, after being cultured for 7 days in a dark place, a proper amount of mycelium blocks are selected and inoculated into a Czapek's culture medium, and then the culture medium is placed on a shaking bed of 150 r/min. Culturing at 25 deg.C in dark for 5-7 days. Regulating spore concentration to 106And each ml is ready for use.
1.2PDA solid culture medium formula:
cutting peeled fresh potato 200g into pieces, adding 500mL ddH2O allowed to boil on high fire for 10min in a microwave oven. Decocting, filtering with gauze, adding glucose 15g, agar powder 20g, and ddH into the filtrate2And O is metered to 1L. Sterilizing and subpackaging into culture dishes.
1.3 Czapek's Medium formulation:
30g of sucrose, 3g of sodium nitrate, 1g of magnesium sulfate, 1g of potassium chloride, 1g of monopotassium phosphate, 0.02g of ferrous sulfate and ddH2And (4) metering the volume of O to 1L, adjusting the pH value to 6.0, and sterilizing for later use.
1.4 inoculation of Verticillium wilt bacteria
Adopting a root-damaging method to inoculate verticillium wilt germs on the Shi mainland 18-1-resistant cotton seedlings and the Jun cotton No. 1 cotton seedlings in the first-heart period of two leaves, wherein the culture conditions after inoculation are 16h of illumination/8 h of darkness, and the day and night temperatures are 25 ℃ and 23 ℃ respectively.
Extracting the total RNA of cotton roots at different time points (0, 0.5, 1, 3, 6, 9, 12 and 24h) after inoculation, and carrying out expression characteristic analysis of GhWRKY55 under the stress of verticillium wilt.
The result is shown in FIG. 4B, GhWRKY55 is induced by verticillium dahliae in 18-1 resistant variety in the Shilang, while the induction time in Junmian No. 1 is obviously lagged behind 18-1 resistant variety in the Shilang, and the expression is only reduced in 12h and 24h after inoculation of verticillium dahliae. The down-regulated expression of GhWRKY55 is related to the disease resistance of cotton to verticillium wilt.
2. Expression analysis of GhWRKY55 gene after hormone treatment
1mM SA (salicylic acid) and 100 mu M MeJA (methyl jasmonate) are respectively sprayed on the two-leaf one-heart-stage stone continental 18-1 and army cotton No. 1 cotton seedlings, the total RNA of the cotton roots at different time points (0, 0.5, 1, 3, 6, 9, 12 and 24h) after treatment is extracted, and the expression mode of GhWRKY55 after SA and MeJA treatment is detected by an RT-qPCR method.
Results as shown in 4C and 4D, in the Shilandia resistance 18-1, GhWRKY55 significantly up-regulated expression after both SA and methyl jasmonate (MeJA) treatment; in cotton army number 1, the gene was not induced by MeJA, but was up-regulated only 6 and 12h after SA treatment. GhWRKY55 may be involved in SA and MeJA mediated disease resistance signaling pathways in cotton.
Example 3 VIGS technology for identifying the function of the GhWRKY55 gene in cotton verticillium wilt resistance
Designing a primer for amplifying a silencing fragment of the GhWRKY55 gene according to the coding sequence of the GhWRKY55 gene:
GhWRKY55-VIGS-F：CGGAATTCACAACAACAACAACACACCACC
GhWRKY55-VIGS-R：GGGGTACCGTTGTCATCGGGTGGAAGGT
the cDNA of disease-resistant variety of the 18-1 root of the continental fossa is taken as a template, and a GhWRKY55 gene silencing fragment is amplified by using the primer and is connected to a pTRV2 vector. Then agrobacterium GV3101 is transformed by electric shock. The magnesium ion chelated protein (GhCHLI) of cotton is used as a positive control, TRV: GhCHLI (positive control), TRV:00 (no-load), TRV: GhWRKY55 and pTRV1 are mixed in equal volume, then the mixture is stood for 3 hours, and the bacterial liquid is injected into cotton cotyledon No. 1 of army cotton by a 1ml syringe with a needle removed. The true leaves of TRV: GhCHLI plants showed obvious yellowing phenotype after VIGS injection for 14d (FIG. 5).
This indicates that the construction of the VIGS system is successful and the expression of the target gene can be effectively inhibited.
RT-qPCR is used for detecting the expression of GhWRKY55 in TRV:00 and TRV: GhWRKY 55.
The results are shown in fig. 6A, where GhWRKY55 is significantly lower in TRV: GhWRKY55 than in TRV:00 two weeks after VIGS injection. The result shows that the expression of GhWRKY55 in TRV GhWRKY55 is obviously inhibited.
When the TRV:00 and TRV: GhWRKY55 cotton seedlings grow to the two-leaf one-heart stage, the verticillium wilt bacteria V991 is inoculated by a root injury method.
The results are shown in FIG. 6B: after inoculation of verticillium wilt bacteria 21d, the TRV GhWRKY55 plants have lighter morbidity, and the TRV 00 plants are more serious. And after 14d and 21d of verticillium wilt pathogen inoculation, counting the disease index of cotton according to the disease index grading standard.
Grading standard of disease index:
level 0: the cotton leaves have no obvious yellowing and wilting, and the whole leaves grow normally.
Level 1: less than 25% of the leaves in the plant are yellow and wilted.
And 2, stage: more than 25% but less than 50% of the leaves in the plant are yellow and wilted.
And 3, level: more than 50% but less than 75% of the leaves in the plants are yellow and wilted, and a few leaves are shed.
4, level: more than 75% of the leaves in the plants yellow, withered or shed.
Disease index ∑ (number of diseased plants × number of stages)/(total number of survey cotton plants × 4) × 100
The results are shown in FIG. 6C, after inoculation for 21d, the TRV:00 plants have a large amount of leaf abscission and plant necrosis, the disease index reaches 0.72, while the TRV: GhWRKY55 silent plants have a lighter disease index, the disease index is 0.45 and is significantly lower than that of the TRV:00 plants.
The result is shown in FIG. 6D, the TRV GhWRKY55 plant stem browning degree is less, and the verticillium wilt resistance is obviously higher than that of the control.
In order to further observe the recovery condition of the fungi, 10 cotton plants of TRV:00 and TRV: GhWRKY55 are randomly selected respectively, a stem section at the position of 2cm above the cotyledon is cut, the stem section is disinfected and rinsed and then placed in a sterilized culture dish, the stem section of 2cm is cut into 1cm by using a scalpel, then the stem section is placed in a PDA culture medium containing the cefamycin to be cultured in a dark place, and the growth condition of the fungi is observed after 7 days.
The results of pathogen recovery culture are shown in FIG. 6E, in which the number of verticillium wilt bacteria colonies isolated from TRV:00 stems is significantly greater than that of TRV: GhWRKY 55.
10 strains of TRV:00 and TRV: GhWRKY55 are randomly selected respectively, stem sections at 2cm positions of the upper parts of cotyledons are cut, liquid nitrogen is used for fully grinding, and stem genome DNA is extracted through a CTAB method. The content of verticillium wilt is identified by qPCR.
GhUBQ7 is used as an internal reference gene, and the primer sequences are as follows:
GhUBQ7-F：GAAGGCATTCCACCTGACCAAC
GhUBQ7-R：CTTGACCTTCTTCTTCTTGTGCTTG
the specific primers for verticillium wilt are as follows:
ITS1-F：AAAGTTTTAATGGTTCGCTAAGA
ST-Ve1-R：CTTGGTCATTTAGAGGAAGTAA
the result of the measurement of the relative content of pathogenic bacteria is shown in FIG. 6F, and the relative content of pathogenic bacteria in the TRV GhWRKY55 plant is obviously lower than that in the TRV 00 plant.
Example 4 overexpression identification of GhWRKY55 function in Arabidopsis thaliana
A plant overexpression vector pGWB17-GhWRKY55 is constructed by using the Gateway technology, wild type Arabidopsis is transformed by a flower dipping method to serve as a T0 generation plant, and the collected seeds are T1 generation plants. And (3) placing the seeds in a hygromycin-resistant 1/2MS culture medium, culturing for 15 days, counting the survival rate of the arabidopsis thaliana, and selecting a separation ratio of 3: 1, transplanting the transgenic arabidopsis seedlings, and collecting seeds of T2 generation after the pods are mature. The method is carried out until a GhWRKY55 homozygous over-expression transgenic Arabidopsis strain is obtained.
The expression modes of GhWRKY55 in over-expression plants and WT are identified by respectively extracting total RNA of GhWRKY55 homozygous over-expression transgenic Arabidopsis strains AO55-1, AO55-2, AO55-3 and WT leaves and by RT-qPCR. The result is shown in FIG. 7A, the expression level of GhWRKY55 in the overexpression transgenic Arabidopsis strain is obviously higher than that of the wild type.
After inoculation of Verticillium dahliae 21d, the results are shown in FIG. 7B, and the leaf in the overexpression transgenic Arabidopsis lines has obvious yellowing and wilting symptoms. While wild plants are less diseased. Collecting the overexpression transgenic arabidopsis lines and wild plant leaves inoculated with verticillium wilt bacteria 14d, and soaking the overexpression transgenic arabidopsis lines and wild plant leaves in a boiling trypan blue solution for 1-1.5 min. Trypan blue solution was mixed with equal volumes of phenol, lactic acid, glycerol and 1mg/ml Trypan blue. Followed by washing with sterile water, destaining with 95% Ethanol/L actinohenol solution and rinsing with 50% Ethanol. Finally, the cells were immersed in 50% glycerol and observed under a microscope for dead cells.
Trypan blue staining results are shown in fig. 7C: the overexpression transgenic arabidopsis strains are deeply colored and have more dead cells. Wild type plants are less colored and die less.
After inoculation of verticillium dahliae 14, 21 and 28d, the disease index of arabidopsis is counted according to the yellowing number of leaves.
The disease indices are shown in FIG. 7D, and after inoculation of 28D, the disease indices of A055-1, AO55-2, and AO55-3 reached 0.69, 0.71, and 0.65, respectively; the WT plants are relatively light in disease, the disease index is only 0.52, and the disease index is obviously lower than that of transgenic Arabidopsis plants.
Selecting 10 GhWRKY55 overexpression transgenic Arabidopsis strains AO55-1, AO55-2, AO55-3 and WT respectively, shearing the whole leaf, fully grinding by using liquid nitrogen, and extracting genome DNA by a CTAB method. The content of verticillium wilt is identified by qPCR, and AtEF-la and verticillium wilt specific primers (ITS1-F and ST-Ve1-R) are respectively used as reference genes of arabidopsis thaliana and verticillium wilt.
AtEF-la-F：AACGGTGCCAGTGGGACG
AtEF-la-R：CCTTGACAGCAACATTCTTGACAT
The specific primers of verticillium wilt are as follows:
ITS1-F：AAAGTTTTAATGGTTCGCTAAGA
ST-Ve1-R：CTTGGTCATTTAGAGGAAGTAA
the result of the relative content of pathogenic bacteria in the stalks is shown in FIG. 7E, and the relative content of pathogenic bacteria in the GhWRKY55 overexpression transgenic arabidopsis strain is obviously higher than that of a wild plant.
The invention discovers that GhWRKY55 negatively regulates the disease resistance of cotton to verticillium wilt, and the gene can be used as a candidate gene for breeding verticillium wilt-resistant cotton.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Sequence listing
<110> Shenzhen agricultural genome institute of Chinese agricultural science institute
<120> a cotton gene and its use
<130> WK21-HCP-CN1-0182
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1101
<212> DNA
<213> upland cotton (Gossypium hirsutum)
<400> 1
atggaccaac aacaacaaca acacaccacc ttgtctctaa tccttgatgg gtgtaagtta 60
gctaaagagc ttgaagaatg catcgggaat ttgggtaacc agctacagcc tgaaatcctt 120
tccaagtctt gtgatgatat cataaatatc tttgttactg caaagcaaag gttaaacaat 180
aatgcccatc atcatcatca tcatcatcat caagacccct ctttgtttac ccatcactta 240
ctccacccac ctcaggattc atctgcacac cacatgcaga ctgatcccag tttgcaggaa 300
tggttaaagt atggtgttat tacacaagca gtggacatga ttcaatcttg tagacccagc 360
attagcatgg ggggagaaat ccaggctatg gatgtgtcag attctggtaa agctgcttct 420
tcttcctcat ctcaacgttc ccatagaagt aggaaggatg atgaagagaa atgcaaaacg 480
agagtagctg ctcctcagat gggaaatacc gaccttccac ccgatgacaa ctacacttgg 540
agaaagtatg gacagaaaga aattctaggt tcaaagtacc ccagggcata ctacaggtgt 600
acacaccaaa aaatgtacaa ctgccctgca aagaagcaag tgcaacgtct agacaatgat 660
ttttacacat ttgaagtaac ctacattggt cagcacacgt gcaccatgtc ctccaccgcg 720
ccctcaattc cgcagccacc cccgctatta catgatcaga tggttatgac tcaagctatg 780
gtgtctcagc ctgcactacc tcccatttct tcttctacta cttcatcatc aatagctcct 840
tttggaagct ggctttcaat ggaatttagc cttggttcgg gttcaggtgg aagaggtagc 900
ggcggtgctg gttcatcatc aggaggaggc tcagctacag gcagtcgata tggaagggat 960
gttgagtatc cggcagtggc ggatatggct gatgtaatgt ttaattcagg aagcagcagc 1020
agcaatagca tggattttat ttttccatgt gcagaagaca aatgggaggc ccccggctca 1080
ggggacaaga agcattgttg a 1101
<210> 2
<211> 366
<212> PRT
<213> Cotton (Gossypium hirsutum)
<400> 2
Met Asp Gln Gln Gln Gln Gln His Thr Thr Leu Ser Leu Ile Leu Asp
1 5 10 15
Gly Cys Lys Leu Ala Lys Glu Leu Glu Glu Cys Ile Gly Asn Leu Gly
20 25 30
Asn Gln Leu Gln Pro Glu Ile Leu Ser Lys Ser Cys Asp Asp Ile Ile
35 40 45
Asn Ile Phe Val Thr Ala Lys Gln Arg Leu Asn Asn Asn Ala His His
50 55 60
His His His His His His Gln Asp Pro Ser Leu Phe Thr His His Leu
65 70 75 80
Leu His Pro Pro Gln Asp Ser Ser Ala His His Met Gln Thr Asp Pro
85 90 95
Ser Leu Gln Glu Trp Leu Lys Tyr Gly Val Ile Thr Gln Ala Val Asp
100 105 110
Met Ile Gln Ser Cys Arg Pro Ser Ile Ser Met Gly Gly Glu Ile Gln
115 120 125
Ala Met Asp Val Ser Asp Ser Gly Lys Ala Ala Ser Ser Ser Ser Ser
130 135 140
Gln Arg Ser His Arg Ser Arg Lys Asp Asp Glu Glu Lys Cys Lys Thr
145 150 155 160
Arg Val Ala Ala Pro Gln Met Gly Asn Thr Asp Leu Pro Pro Asp Asp
165 170 175
Asn Tyr Thr Trp Arg Lys Tyr Gly Gln Lys Glu Ile Leu Gly Ser Lys
180 185 190
Tyr Pro Arg Ala Tyr Tyr Arg Cys Thr His Gln Lys Met Tyr Asn Cys
195 200 205
Pro Ala Lys Lys Gln Val Gln Arg Leu Asp Asn Asp Phe Tyr Thr Phe
210 215 220
Glu Val Thr Tyr Ile Gly Gln His Thr Cys Thr Met Ser Ser Thr Ala
225 230 235 240
Pro Ser Ile Pro Gln Pro Pro Pro Leu Leu His Asp Gln Met Val Met
245 250 255
Thr Gln Ala Met Val Ser Gln Pro Ala Leu Pro Pro Ile Ser Ser Ser
260 265 270
Thr Thr Ser Ser Ser Ile Ala Pro Phe Gly Ser Trp Leu Ser Met Glu
275 280 285
Phe Ser Leu Gly Ser Gly Ser Gly Gly Arg Gly Ser Gly Gly Ala Gly
290 295 300
Ser Ser Ser Gly Gly Gly Ser Ala Thr Gly Ser Arg Tyr Gly Arg Asp
305 310 315 320
Val Glu Tyr Pro Ala Val Ala Asp Met Ala Asp Val Met Phe Asn Ser
325 330 335
Gly Ser Ser Ser Ser Asn Ser Met Asp Phe Ile Phe Pro Cys Ala Glu
340 345 350
Asp Lys Trp Glu Ala Pro Gly Ser Gly Asp Lys Lys His Cys
355 360 365
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
atggaccaac aacaacaaca aca 23
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
tcaacaatgc ttcttgtccc ct 22
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 5
gttcgggttc aggtggaaga gg 22
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 6
gccactgccg gatactcaac at 22
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (Artificial)
<400> 7
gaaggcattc cacctgacca ac 22
<210> 8
<211> 25
<212> DNA
<213> Artificial sequence (Artificial)
<400> 8
cttgaccttc ttcttcttgt gcttg 25
<210> 9
<211> 30
<212> DNA
<213> Artificial sequence (Artificial)
<400> 9
cggaattcac aacaacaaca acacaccacc 30
<210> 10
<211> 28
<212> DNA
<213> Artificial sequence (Artificial)
<400> 10
ggggtaccgt tgtcatcggg tggaaggt 28
Claims (10)
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Citations (4)
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| US20030182684A1 (en) * | 2002-03-14 | 2003-09-25 | Yale University | Tobacco rattle virus vectors and related compositions and methods |
| CN109112149A (en) * | 2018-02-12 | 2019-01-01 | 华中农业大学 | Regulate and control cotton Calcium-dependent protein kinase GhCPK33 gene and the application of cotton verticillium wilt resistance |
| CN112322628A (en) * | 2020-09-27 | 2021-02-05 | 湖北大学 | Transcription factor GhWRKY1-like gene for regulating and controlling verticillium wilt and drought resistance of cotton and application thereof |
| CN112410350A (en) * | 2020-12-14 | 2021-02-26 | 中国农业科学院植物保护研究所 | Upland cotton GhWRKY74 protein and coding gene and application thereof |
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2021
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Patent Citations (4)
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|---|---|---|---|---|
| US20030182684A1 (en) * | 2002-03-14 | 2003-09-25 | Yale University | Tobacco rattle virus vectors and related compositions and methods |
| CN109112149A (en) * | 2018-02-12 | 2019-01-01 | 华中农业大学 | Regulate and control cotton Calcium-dependent protein kinase GhCPK33 gene and the application of cotton verticillium wilt resistance |
| CN112322628A (en) * | 2020-09-27 | 2021-02-05 | 湖北大学 | Transcription factor GhWRKY1-like gene for regulating and controlling verticillium wilt and drought resistance of cotton and application thereof |
| CN112410350A (en) * | 2020-12-14 | 2021-02-26 | 中国农业科学院植物保护研究所 | Upland cotton GhWRKY74 protein and coding gene and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| NONE: "ACCESSION NO：XM_016899980，PREDICTED: Gossypium hirsutum WRKY transcriptionfactor 55-like (LOC107963474), mRNA", 《GENBANK》 * |
| NONE: "ACCESSION NO：XP_016755469，WRKY transcription factor 55-like [Gossypium hirsutum]", 《GENBANK》 * |
| YIQIAO WANG ET AL.: "WRKY55 transcription factor positively regulates leaf senescence and the defense response by modulating the transcription of genes implicated in the biosynthesis of reactive oxygen species and salicylic acid in Arabidopsis", 《DEVELOPMENT》 * |
| 周之楠 等: "大麦黄花叶病相关基因WRKY55 的克隆及功能研究", 《植物遗传资源学报》 * |
| 赵君等: "棉花抗黄萎病相关基因的序列和表达分析", 《棉花学报》 * |
| 赵曾强等: "棉花GhWRKY44基因的克隆与表达分析", 《西北植物学报》 * |
| 郑娜等: "VIGS技术高通量筛选棉花抗黄萎病相关基因", 《中国科学:生命科学》 * |
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