CN113106105B - Cotton gene and its use - Google Patents

Abstract

The invention provides a cotton gene and application thereof, wherein the nucleotide sequence of the cotton gene is shown as SEQID NO. 1. The gene has negative regulation and control effect on the resistance of cotton to verticillium wilt.

Description

A kind of cotton gene and its application

technical field

The invention relates to a cotton gene and its application.

Background technique

Cotton Verticillium wilt, known as the "cancer" of cotton, is a soil-borne fungal vascular disease caused by Verticillium dahliae. The infection of cotton by Verticillium dahliae can cause leaf yellowing, necrosis, shedding and plant death, seriously affecting the yield and quality of cotton, and causing huge economic losses. Although breeders have bred a batch of cotton varieties with strong resistance to Verticillium wilt and excellent comprehensive traits through conventional breeding methods, due to the lack of sources of resistance to Verticillium wilt, the rapid variation of Verticillium dahliae and the long cycle of traditional breeding methods , cotton breeding for Verticillium wilt resistance progressed slowly. Therefore, the use of modern genetic engineering technology to mine and identify key genes for disease resistance can provide cotton with disease-resistant gene resources, which is of great significance for cotton breeding against Verticillium wilt.

WRKY transcription factors are plant-specific transcription factors, usually in the form of gene families, and there are 74 WRKY gene family members in Arabidopsis alone. There are 238 WRKY transcription factors found in the upland cotton genome (PlantTFDB). Studies have shown that WRKY transcription factors not only participate in plant growth and development, but also play an important role in plant disease resistance. Among them, GhWRKY3, GhWRKY4, GhWRKY7, GhWRKY22, GhWRKY33 and GhWRKY40 were up-regulated by Verticillium dahliae. The latest study found that GbWRKY1, GhWRKY70 and GhWRKY70D13 all negatively regulate the resistance of cotton to Verticillium wilt by inhibiting the jasmonic acid (JA) signaling pathway, and the regulation of salicylic acid (SA) has also been reported.

Contents of the invention

The object of the present invention is to provide a cotton gene and its application.

To achieve the above purpose, the technical solution adopted: a cotton gene, the nucleotide sequence of the cotton gene is shown in SEQ ID NO:1. The cotton gene was named GhWRKY55.

The present invention also provides a protein encoded by the aforementioned cotton gene, the amino acid sequence of which is shown in SEQ ID NO:2.

The present invention also provides a VIGS vector for enhancing the resistance of cotton to Verticillium wilt, the VIGS vector is the tobacco rattle virus vector pTRV2 containing the DNA fragment for silencing the above-mentioned cotton gene. Preferably, the primers for amplifying the DNA fragments include primers as shown in SEQ ID NO:9 and SEQ ID NO:10.

The present invention also provides a VIGS system for enhancing the resistance of cotton to Verticillium dahliae, including the above-mentioned VIGS vector and tobacco rattle virus vector pTRV1.

The present invention also provides a method for enhancing the resistance of cotton to Verticillium wilt, the method comprising reducing the expression of the above-mentioned cotton gene in cotton.

The present invention also provides a method for enhancing the resistance of cotton to Verticillium wilt, said method comprising silencing the above-mentioned cotton gene in cotton; preferably, said cotton gene silencing is the above-mentioned VIGS vector Or the aforementioned VIGS system is introduced into cotton and realized.

The present invention also provides a breeding method for Verticillium wilt-resistant cotton, the breeding method includes reducing the expression of the above-mentioned cotton gene in cotton.

The present invention also provides a breeding method for Verticillium wilt-resistant cotton, the breeding method comprising silencing the above-mentioned cotton gene in cotton. Preferably, the cotton gene silencing is achieved by introducing the aforementioned VIGS vector or the aforementioned VIGS system into cotton.

The present invention also provides the use of the above-mentioned cotton gene, the above-mentioned protein, the above-mentioned VIGS vector or the above-mentioned VIGS system in enhancing the resistance of cotton to Verticillium wilt.

Preferably, the resistance of cotton to Verticillium wilt is enhanced by silencing the aforementioned cotton genes in cotton.

Beneficial effect:

The invention provides a cotton gene, which has a negative regulatory effect on the resistance of cotton to verticillium wilt.

Description of drawings

Figure 1 shows the differentially expressed genes of the resistant variety Shidaikang 18-1 (HR) inoculated with Verticillium dahliae 12 (A) and 24h (B). HR0: distilled water treated root samples of Shidaikang 18-1; HR12 and HR24: root samples of Shidaikang 18-1 inoculated with Verticillium dahliae 12 and 24h.

Figure 2 shows the phylogenetic tree analysis diagram of GhWRKY55. GhWRKY55 has higher homology with AtWRKY55. Among them, AtWRKY44(Arabidopsis thaliana, NP_181263.2), AtWRKY33(NP_181381.2), AtWRKY4(NP_172849.1), AtWRKY31(NP_567644.1), AtWRKY42(NP_192354.1), AtWRKY6(NP_16487W92) 1), AtWRKY60(NP_180072.1), AtWRKY18(NP_567882.1), AtWRKY64(NP_176829.1), AtWRKY66(NP_178174.1), AtWRKY67(NP_564877.1), AtWRKY41(NP_192845.1), AtWRKY41(NP_192845.1), AtWRKY41(NP_192845.1) )，AtWRKY30(NP_568439.1)，AtWRKY46(NP_182163.1)，AtWRKY54(NP_181607.1)，AtWRKY57(NP_177090.1)，AtWRKY75(NP_196812.1)，AtWRKY55(NP_181606.1)，JcWRKY55(Jatropha curcas，AGQ04249 .1)，HaWRKY55(Helianthusannuus，XP_021975670.1)，DcWRKY55(Dendrobium catenatum，XP_020690062.1)，NaWRKY55(Nicotiana attenuata，XP_019246449.1)，GmWRKY55(Glycine max，ABS18448)，HvWRKY55(Hordeum vulgare，AFV67807)。

Figure 3 shows the results of multiple sequence alignment analysis of GhWRKY55. GhWRKY55 contains typical WRKY and C2HC zinc finger domains. The sequences of NaWRKY55, DcWRKY55, GmWRKY55, HaWRKY55, JcWRKY55, GhWRKY55 and AtWRKY55 are consistent with those in Figure 2.

Figure 4 shows the analysis results of the expression pattern of GhWRKY55 gene in cotton resistant and sensitive materials. In the figure, A is the analysis result of expression pattern in various tissues of cotton; B is the result of analysis of expression pattern after inoculation with Verticillium dahliae; C is the result of analysis of expression pattern after treatment with MeJA; D is the result of analysis of expression pattern after treatment with SA.

Figure 5 shows the phenotype of Junmian No. 1 injected with TRV:GhCHLI two weeks later.

Figure 6 shows the results of functional analysis of GhWRKY55 gene silencing. In the figure A is the expression pattern analysis result of GhWRKY55 after inoculation with Verticillium dahliae in TRV:GhWRKY55 and TRV:00; B is the phenotype analysis result of TRV:GhWRKY55 and TRV:00 after inoculation with Verticillium dahliae for 21 days; C is the result of inoculation with Verticillium dahliae Analysis results of disease index 14 and 21 days after Verticillium dahliae; D is the analysis result of the incidence of TRV:GhWRKY55 and TRV:00; E is the recovery culture of TRV:GhWRKY55 and TRV:00 plant stem pathogenic bacteria 14 days after inoculation with Verticillium dahliae; F is 14 and 21 days after inoculation with Verticillium dahliae, the relative content of pathogenic bacteria in the stems of TRV:GhWRKY55 and TRV:00 plants was measured.

Figure 7 shows that overexpression of GhWRKY55 reduces Arabidopsis resistance to Verticillium dahliae. In the figure, A is the expression level of GhWRKY55 in overexpressed Arabidopsis lines; B is the phenotype of transgenic Arabidopsis 21 days after inoculation with Verticillium dahliae; C is the expression level of GhWRKY55 overexpressed transgenic Arabidopsis 14 days after inoculation with Verticillium dahliae Pan blue staining; D is the disease index statistics of transgenic Arabidopsis thaliana 14, 21 and 28 days after inoculation with Verticillium dahliae (n=30); E is the stem of GhWRKY55 overexpressed transgenic Arabidopsis plants 14 days after inoculation with Verticillium dahliae Determination of the relative content of pathogenic bacteria. The statistical method was T test (*P<0.05; **P<0.01), and the obtained data were mean ± standard deviation.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples.

The applicant analyzed the gene expression profiles of different disease-resistant upland cotton materials in response to Verticillium dahliae infection by comparative transcriptomics, and found that the expression level of the GhWRKY55 gene was significantly reduced after being induced by Verticillium dahliae in the disease-resistant variety Shidaikang 18-1 , while the down-regulated expression in the susceptible variety Junmian 1 was later than that in the resistant variety, and the degree of down-regulated expression was significantly lower than that in the resistant variety. Recent studies have shown that WRKY55 belongs to type III WRKY transcription factors, and this gene negatively regulates Arabidopsis resistance to Pseudomonas syringae. The present invention utilizes the VIGS method to inoculate Verticillium dahliae after transiently silencing the expression of the GhWRKY55 gene in cotton, and finds that the gene has a negative regulatory effect on cotton Verticillium dahlias resistance. In addition, the GhWRKY55 gene was overexpressed in Arabidopsis, further confirming that the GhWRKY55 gene negatively regulates the resistance of cotton to Verticillium wilt. The invention provides a new method for breeding cotton resistant to Verticillium wilt.

In the quantitative experiments in the following examples, three biological repetitions and three technical repetitions were set.

Embodiment 1: GhWRKY55 gene cloning

Transcriptome analysis found that the expression of GhWRKY55 gene in the disease-resistant variety Shidalu Kang 18-1 decreased significantly 12 and 24 hours after inoculation with Verticillium dahliae, reaching 17.5 and 20.1 times, respectively (see Figure 1A, B). The cDNA sequence of GhWRKY55 was amplified using the root cDNA of Shidalu Kang 18-1 as a template.

The primers required for cloning are as follows:

GhWRKY55-F: ATGGACCAACAACAACAACAACA

GhWRKY55-R: TCAACAATGCTTCTTGTCCCCT

The amplified sequence was connected to the PMDT-19 carrier, and positive clones were screened and sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. Its nucleotide sequence is shown in SEQ ID NO:1, and its protein sequence is shown in SEQ ID NO:2.

A phylogenetic tree analysis of GhWRKY55 was performed with some known WRKY transcription factors in Arabidopsis and other species.

The results are shown in Figure 2, the sequence has the highest homology with Arabidopsis AtWRKY55, so the applicant named it GhWRKY55.

Except for GhWRKY55, other representative plant WRKY55 amino acid sequences were selected for multiple sequence alignment analysis by DANMAN software.

The results are shown in Figure 3, GhWRKY55 contains typical WRKY and C2HC zinc finger domains, so this sequence is a member of type III WRKY transcription factors.

Embodiment 2: GhWRKY55 expression pattern analysis

RNA was extracted from the roots, stems and leaves of Shidaikang 18-1 (Verticillium wilt-resistant variety, HR) and Junmian 1 (Verticillium wilt-susceptible variety, HS) at the two-leaf one-center stage. The extraction method refers to the 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 were detected by Nanodrop ND-2000 micro-spectrophotometer.

Take 1 μg of RNA template and reverse transcribe the first strand of cDNA with reference to the M-MLV Reverse Transcriptase Kit of Novozyme Biotechnology Co., Ltd. (Nanjing).

RT-qPCR primers were designed, and the expression specificity of GhWRKY55 in roots, stems and leaves of different disease-resistant varieties was analyzed by RT-qPCR method. The relative expression of genes was calculated according to the 2 ^-ΔΔCT method.

Reaction system: 1 μL of 10-fold diluted cDNA, 5 μL of 2×SYBR Green Mix, 0.2 μL of 10 μmol/L forward and reverse primers, and 3.6 μL of ddH2O.

qPCR program: pre-denaturation at 95°C for 3min, 10s at 95°C, 15s at 60°C, 15s at 72°C, a total of 40 cycles.

The RT-qPCR primer sequences are as follows:

GhWRKY55-qF: GTTCGGGTTCAGGTGGAAGAGG

GhWRKY55-qR:GCCACTGCCGGATACTCAACAT

Using GhUBQ7 as an internal reference gene, the primers are as follows:

GhUBQ7-F: GAAGGCATTCCACCTGACCAAC

GhUBQ7-R: CTTGACCTTTCTTCTTCTTGTGCTTG

The results were shown in Figure 4A, GhWRKY55 was predominantly expressed in the roots of Shidalukang 18-1 and Junmian 1.

1. Expression analysis of GhWRKY55 gene after inoculation with Verticillium dahliae

1.1 Activation and cultivation of Verticillium dahliae

Spread 200 μL of Verticillium dahliae strain "V991" spore liquid evenly on the PDA medium, and after cultivating in the dark for 7 days, select an appropriate amount of hyphae to inoculate into Czapek's medium, and then place the medium on a shaker at 150r/min . Incubate at 25°C in the dark for 5-7 days. Adjust the spore concentration to 10 ⁶ /ml for later use.

1.2 PDA solid medium formula:

200 g of peeled fresh potatoes were chopped, and 500 mL of ddH ₂ O was added to boil it in a microwave oven on high heat for 10 min. After boiling, filter with gauze, add 15g of glucose, 20g of agar powder to the filtrate, and dilute to 1L with ddH ₂ O. After sterilization, dispense into Petri dishes.

1.3 Czapek's medium formula:

Sucrose 30g, sodium nitrate 3g, magnesium sulfate 1g, potassium chloride 1g, potassium dihydrogen phosphate 1g, ferrous sulfate 0.02g, ddH ₂ O to 1L, pH to 6.0, sterilized for later use.

1.4 Inoculation of Verticillium dahliae

The cotton seedlings of Shidaikang 18-1 and Junmian 1 at the two-leaf and one-core stage were inoculated with Verticillium dahliae by root injury method. After inoculation, the culture conditions were 16 hours of light/8 hours of darkness, and the day and night temperatures were 25°C and 23°C, respectively.

The total RNA of cotton roots was extracted at different time points (0, 0.5, 1, 3, 6, 9, 12 and 24 hours) after inoculation, and the expression characteristics of GhWRKY55 under the stress of Verticillium dahliae were analyzed.

The results are shown in Figure 4B. GhWRKY55 was significantly down-regulated in Shidalukang 18-1 resistant variety induced by Verticillium dahliae, while the induction time in Junmian 1 was significantly later than that of Shidalukang 18-1. Verticillium dahliae down-regulated expression at 12 and 24h. The down-regulated expression of GhWRKY55 is related to the resistance of cotton to Verticillium wilt.

2. Expression analysis of GhWRKY55 gene after hormone treatment

Spray 1mM SA (salicylic acid) and 100μM MeJA (methyl jasmonate) on cotton seedlings of Shidaikang 18-1 and Junmian 1 at the two-leaf one-center stage, respectively, and at different time points (0, 0.5, 1 , 3, 6, 9, 12 and 24h) cotton root total RNA, and the expression pattern of GhWRKY55 after SA and MeJA treatment was detected by RT-qPCR method.

The results were shown in 4C and 4D. In Shidalukang 18-1, the expression of GhWRKY55 was significantly up-regulated after SA and methyl jasmonate (MeJA) treatment; in Junmian 1, the gene was not induced by MeJA, only The expression was upregulated after 6 and 12h of SA treatment. GhWRKY55 may be involved in the SA and MeJA-mediated signal pathway of cotton disease resistance.

Embodiment 3: VIGS technology identifies the function of GhWRKY55 gene in cotton resistance to Verticillium wilt

According to the coding sequence of GhWRKY55 gene, the primers for amplifying the GhWRKY55 gene silence fragment are designed:

GhWRKY55-VIGS-F: CGGAATTCACAACAACAACAACACACCACC

GhWRKY55-VIGS-R: GGGGTACCGTTGTCATCGGGTGGAAGGT

The GhWRKY55 gene silencing fragment was amplified with the above primers using the root cDNA of the disease-resistant variety Shidalu Kang 18-1 as a template, and connected to the pTRV2 vector. Then Agrobacterium GV3101 was transformed by electric shock method. Taking cotton magnesium ion chelating protein (GhCHLI) as a positive control, mix TRV:GhCHLI (positive control), TRV:00 (empty load), TRV:GhWRKY55 and pTRV1 in equal volumes respectively, let it rest for 3 hours, and use 1ml of the needle removed The syringe injects the bacterial solution into the cotyledon of Cotton No. 1 cotton. 14 days after VIGS injection, the true leaves of TRV:GhCHLI plants showed obvious yellowing phenotype (Fig. 5).

This indicates that the VIGS system was successfully constructed and can effectively inhibit the expression of the target gene.

The expression of GhWRKY55 in TRV:00 and TRV:GhWRKY55 was detected by RT-qPCR.

The results are shown in Figure 6A, two weeks after VIGS injection, the expression of GhWRKY55 in TRV:GhWRKY55 was significantly lower than that in TRV:00. It indicated that the expression of GhWRKY55 was significantly inhibited in TRV:GhWRKY55.

When TRV:00 and TRV:GhWRKY55 cotton seedlings grew to the stage of two leaves and one heart, they were inoculated with Verticillium dahliae V991 by root injury method.

The results are shown in 6B: 21 days after inoculation with Verticillium dahliae, TRV:GhWRKY55 plants were less diseased, while TRV:00 plants were more severely affected. After 14 and 21 days of inoculation with Verticillium dahliae, according to the disease index grading standard, the cotton disease index was counted.

Disease index grading standard:

Grade 0: Cotton leaves have no obvious yellowing and wilting, and the leaves grow normally as a whole.

Grade 1: Less than 25% of the leaves on the plant are yellow and wilted.

Grade 2: more than 25% but less than 50% of the leaves of the plant are yellow and wilted.

Grade 3: More than 50% but less than 75% of the leaves of the plants are yellow and wilted, and a few leaves fall off.

Grade 4: More than 75% of the leaves on the plant turn yellow and wither or fall off.

Disease index = ∑ (number of disease-grade plants × series) / (total number of investigated cotton plants × 4) × 100

The results are shown in Figure 6C. After inoculation for 21 days, TRV:00 plants had a large number of leaves shedding, plant necrosis, and the disease index reached 0.72, while TRV:GhWRKY55 silenced plants had a milder disease, and the disease index was 0.45, which was significantly lower than TRV: 00 plants.

The results were shown in Fig. 6D, TRV:GhWRKY55 plants had less browning degree of stems, and their resistance to Verticillium dahliae was significantly higher than that of the control.

In order to further observe the recovery of the fungus, 10 TRV:00 and TRV:GhWRKY55 cotton plants were randomly selected, and the stem segments at the upper 2cm of the cotyledons were cut, disinfected, rinsed, and placed in a sterilized petri dish, and cut with a scalpel. The 2cm stem section was cut into 1cm, and then the stem section was placed in the PDA medium containing cephalosporin and cultured in the dark, and the growth of the fungus was observed after 7 days.

The results of the recovery culture of pathogenic bacteria are shown in Figure 6E, the number of Verticillium dahliae colonies isolated from the stems of TRV:00 was significantly more than that of TRV:GhWRKY55.

Ten plants of TRV:00 and TRV:GhWRKY55 were randomly selected respectively, and the stem segment at the upper 2cm of the cotyledons was cut off, fully ground with liquid nitrogen, and the genomic DNA of the stem was extracted by the CTAB method. The content of Verticillium dahliae was identified by qPCR.

GhUBQ7 was used as an internal reference gene, and the primer sequences were as follows:

GhUBQ7-F: GAAGGCATTCCACCTGACCAAC

GhUBQ7-R: CTTGACCTTTCTTCTTCTTGTGCTTG

Specific primers for Verticillium dahliae are as follows:

ITS1-F: AAAGTTTTTAATGGTTCGCTAAGA

ST-Ve1-R: CTTGGTCATTTAGAGGAAGTAA

As shown in FIG. 6F , the relative content of pathogenic bacteria in TRV:GhWRKY55 plants was significantly lower than that in TRV:00.

Embodiment 4: Overexpression identifies the function of GhWRKY55 in Arabidopsis

The plant overexpression vector pGWB17-GhWRKY55 was constructed by using Gateway technology, and the wild-type Arabidopsis was transformed by the flower dipping method, which was used as the T0 generation plant, and the harvested seeds were the T1 generation. The seeds were cultured in hygromycin-resistant 1/2MS medium for 15 days, and the survival rate of Arabidopsis was counted. Transgenic Arabidopsis seedlings with a segregation ratio of 3:1 were selected for transplanting, and the T2 generation was harvested after the fruit pods matured. seed. Follow this method until the GhWRKY55 homozygous overexpression transgenic Arabidopsis line is obtained.

The total RNA of leaves of GhWRKY55 homozygous overexpression transgenic Arabidopsis lines AO55-1, AO55-2, AO55-3 and WT were extracted, and the expression pattern of GhWRKY55 in overexpression plants and WT was identified by RT-qPCR. Results As shown in Figure 7A, the expression level of GhWRKY55 in the overexpression transgenic Arabidopsis lines was significantly higher than that in the wild type.

21 days after being inoculated with Verticillium dahliae, the results are shown in Figure 7B, the leaves of the overexpressed transgenic Arabidopsis lines showed obvious symptoms of yellowing and wilting. The wild-type plants were mildly affected. The leaves of overexpressed transgenic Arabidopsis lines and wild plants 14 days after inoculation with Verticillium dahliae were collected, and soaked in boiled trypan blue solution for 1-1.5 min. The trypan blue solution was prepared by mixing equal volumes of phenol, lactic acid, glycerol and 1 mg/ml trypan blue. Then wash with sterile water, decolorize with 95% Ethanol/Lactophenol solution, and rinse with 50% ethanol. Finally soaked in 50% glycerol, microscopic observation of dead cells.

The results of trypan blue staining are shown in Figure 7C: the overexpression transgenic Arabidopsis lines were darker and had more dead cells. There was lighter coloration and less cell death in wild-type plants.

14, 21 and 28 days after inoculation with Verticillium dahliae, according to the number of yellow leaves, the Arabidopsis disease index was calculated.

As shown in Figure 7D, the disease index of A055-1, AO55-2, and AO55-3 reached 0.69, 0.71, and 0.65 after 28 days of inoculation; while the disease index of WT plants was relatively mild, and the disease index was only 0.52, which was significant lower than transgenic Arabidopsis plants.

10 transgenic Arabidopsis lines AO55-1, AO55-2, AO55-3, and WT overexpressing GhWRKY55 were selected, and the whole leaves were cut out, fully ground with liquid nitrogen, and genomic DNA was extracted by CTAB method. The content of Verticillium dahliae was identified by qPCR, and AtEF-la and Verticillium dahlia-specific primers (ITS1-F and ST-Ve1-R) were used as internal reference genes of Arabidopsis and Verticillium dahliae, respectively.

AtEF-la-F: AACGGTGCCAGTGGGACG

AtEF-la-R: CCTTGACAGCAACATTCTTGACAT

Specific primers for Verticillium dahliae are:

ITS1-F: AAAGTTTTTAATGGTTCGCTAAGA

ST-Ve1-R: CTTGGTCATTTAGAGGAAGTAA

The relative content of pathogenic bacteria in stems was shown in Figure 7E, the relative content of pathogenic bacteria in GhWRKY55 overexpressed transgenic Arabidopsis lines was significantly higher than that in wild-type plants.

The invention discovers that GhWRKY55 negatively regulates the resistance of cotton to Verticillium wilt, and the gene can be used as a candidate gene for cotton breeding against Verticillium wilt.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

sequence listing

<110> Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences

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ggggtaccgt tgtcatcggg tggaaggt 28

Claims (10)

1. The application of a cotton gene in enhancing the resistance of cotton to verticillium wilt is characterized in that the nucleotide sequence of the cotton gene is shown in SEQ ID NO. 1, and the resistance of cotton to verticillium wilt is enhanced by silencing the cotton gene in cotton.

2. Use of a protein encoded by the cotton gene of claim 1 for enhancing resistance of cotton to verticillium wilt, wherein the protein has the amino acid sequence shown in SEQ ID NO. 2, and wherein the cotton resistance to verticillium wilt is enhanced by silencing the cotton gene of claim 1 in cotton.

3. A method of enhancing the resistance of cotton to verticillium wilt, comprising reducing the expression of the cotton gene of claim 1 in cotton.

4. A method of enhancing the resistance of cotton to verticillium wilt, comprising silencing the cotton gene of claim 1 in cotton.

5. The method according to claim 4, wherein the VIGS vector is tobacco rattle virus vector pTRV2 containing a DNA fragment for silencing the cotton gene of claim 1, or the VIGS system comprising the VIGS vector and tobacco rattle virus vector pTRV1, wherein the cotton gene silencing is achieved by introducing the VIGS vector or the VIGS system into cotton.

6. A method of breeding verticillium wilt resistant cotton, comprising reducing the expression of the cotton gene of claim 1 in cotton.

7. A method of breeding cotton for resistance to verticillium wilt, comprising silencing cotton gene as claimed in claim 1 in cotton.

8. The method according to claim 7, wherein the tobacco rattle virus vector pTRV2 containing the DNA fragment for silencing the cotton gene of claim 1 is used as the VIGS vector, or the VIGS system comprising the VIGS vector and the tobacco rattle virus vector pTRV1, and the cotton gene silencing is achieved by introducing the VIGS vector or the VIGS system into cotton.

9. The application of a VIGS vector or a VIGS system in enhancing the resistance of cotton to verticillium wilt is characterized in that the VIGS vector is a tobacco rattle virus vector pTRV2 containing a DNA segment for silencing a cotton gene as claimed in claim 1, and primers for amplifying the DNA segment are shown as SEQ ID NO. 9 and SEQ ID NO. 10; the VIGS system comprises the VIGS vector and a tobacco rattle virus vector pTRV1.

10. Use according to claim 9, wherein the resistance of cotton to verticillium wilt is enhanced by silencing the cotton gene of claim 1 in cotton.

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