Application of cotton GbGELP21A gene in plant disease resistance

Abstract

The invention discloses application of a cotton GbGELP21A gene in plant disease resistance, and belongs to the field of gene engineering. According to the invention, a GbGELP21A protein in a GDSL lipase family is identified in gossypium barbadense, the amino acid sequence of the GbGELP21A protein is as shown in SEQ ID NO.2, and the nucleotide sequence of the coding gene GbGELP21A of the GbGELP21A protein is as shown in SEQ ID NO.1. An overexpression vector and a silent vector of the gene are constructed, arabidopsis thaliana and cotton are transformed, and transgenic experiment results show that after being infected by verticillium wilt, the condition of a GbGELP21A overexpression plant is obviously superior to that of a wild type plant, the condition of a silent plant is more serious, and the GbGELP21A gene is capable of regulating the disease resistance of the plant. The invention lays a foundation for verticillium wilt resistance breeding, and has great economic benefits and wide application prospects for plant production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to the application of cotton GbGELP21A gene in plant disease resistance. Background Art

[0002] Cotton is one of the main economic crops and has a wide range of uses in agriculture, textile industry, medical care, national defense and military industry.

[0003] Verticillium wilt is a soil-borne vascular disease of cotton caused by fungi. The main pathogens are Verticillium dahliae Kleb and Verticillium alboatrum, belonging to the subdivision Deuteromycetes, family Pallensaceae, and genus Verticillium. The Verticillium wilt fungus spreads and colonizes cotton plants not only within the vascular bundles but also infects other organs and tissues, even spreading throughout the plant through the surfaces and interiors of organs. As a soil-borne fungal disease, Verticillium wilt is characterized by its wide spread, rapid infection, long infection cycle, and hidden nature, making its prevention and control in agriculture extremely difficult. Verticillium wilt has a wide host range and, in unsuitable growing environments, forms microsclerotia that can survive in the soil for long periods and maintain their infectious capacity. The pathogenic mechanism of Verticillium wilt is highly complex and regulated by intricate signaling pathways. Xinjiang Sea Island cotton is a long-staple cotton variety independently developed in my country for long-term cultivation in the cotton-producing regions of Xinjiang. Verticillium wilt is a major disease that seriously threatens my country's cotton agricultural production, so it is of great significance to study and discover disease-resistance-related functional genes.

[0004] GDSL-type lipases are a class of hydrolases that hydrolyze a variety of substrates, including thioesters, aryl esters, phospholipids, and amino acids, exhibiting broad substrate specificity. Their structural hallmark is the conserved GDSL motif at the N-terminus of the protein. One subgroup, SGNH hydrolases, contains five conserved blocks (I-V), with the four catalytic residues Ser, Gly, Asn, and His located in Blocks I, II, III, and V, respectively. The SGNH hydrolase superfamily encompasses two subfamilies: GDSL-type esterases or lipases and GDSL-type lipase / acetylhydrolases. GDSL-type esterases or lipases are a larger gene family encompassing plant-specific GDSL-type esterases or lipases. Currently, 105, 80, 114, 194, 112, 126, 96, and 130 GDSL gene family members have been identified in plants such as Arabidopsis thaliana, Sedum alfredii, rice, soybean, Chinese cabbage, poplar, grape, and sorghum, respectively. Many GDSL lipase members have been shown to play important roles in plants. The functions of plant GDSL lipases are mainly involved in regulating plant growth and development, morphogenesis, participating in stress response and participating in oil metabolism.

[0005] At present, there are no reports on whether GDSL-type lipase in cotton is related to disease resistance. Therefore, there is an urgent need to provide a cotton GDSL-type esterase and its encoding gene for use in regulating plant disease resistance. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of the cotton GbGELP21A gene in plant disease resistance to solve the problems existing in the above-mentioned prior art. The GbGELP21A gene provided by the present invention positively regulates the plant's resistance to Verticillium wilt, which is of great significance for the cultivation of disease-resistant plants and has great economic benefits and broad application prospects for plant production.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an application of cotton lipase GbGELP21A in any of the following:

[0009] (1) Application in regulating plant resistance to Verticillium wilt;

[0010] (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt;

[0011] (3) Application in the preparation of products for improving plant resistance to Verticillium wilt;

[0012] The amino acid sequence of the lipase GbGELP21A is shown in SEQ ID NO.2.

[0013] The present invention also provides a use of the above-mentioned lipase GbGELP21A encoding gene in any of the following:

[0014] (1) Application in regulating plant resistance to Verticillium wilt;

[0015] (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt;

[0016] (3) Application in the preparation of products for improving plant resistance to Verticillium wilt;

[0017] The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0018] Preferably, the expression level of the encoding gene is up-regulated in the plant to improve the plant's resistance to Verticillium wilt.

[0019] Preferably, the step of increasing the expression of the coding gene in the plant comprises the following steps: constructing an overexpression vector comprising the coding gene, transforming the overexpression vector into Agrobacterium, and then infecting the plant;

[0020] The plant is cotton or Arabidopsis thaliana.

[0021] The present invention also provides a use of an overexpression vector comprising the above-mentioned encoding gene in any of the following:

[0022] (1) Application in regulating plant resistance to Verticillium wilt;

[0023] (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt;

[0024] (3) Application in the preparation of products for improving plant resistance to Verticillium wilt.

[0025] The present invention also provides a use of an engineered bacterium comprising the above-mentioned overexpression vector in any of the following:

[0026] (1) Application in regulating plant resistance to Verticillium wilt;

[0027] (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt;

[0028] (3) Application in the preparation of products for improving plant resistance to Verticillium wilt.

[0029] The present invention also provides a method for improving plant resistance to Verticillium wilt, comprising the steps of increasing the expression level of a gene encoding lipase GbGELP21A in a plant to improve the plant's resistance to Verticillium wilt;

[0030] The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0031] Preferably, the method of increasing the expression of the gene encoding lipase GbGELP21A comprises the following steps:

[0032] An overexpression vector comprising the gene encoding the lipase GbGELP21A is constructed, and the overexpression vector is transformed into Agrobacterium, which is then used to infect plant plants.

[0033] Preferably, the plant is cotton or Arabidopsis thaliana.

[0034] The present invention also provides a method for breeding transgenic plants with improved resistance to Verticillium wilt, comprising the following steps:

[0035] Overexpressing the gene encoding the lipase GbGELP21A in plant cells, then cultivating the plant cells, and obtaining regenerated plants using the plant cells, thereby obtaining the transgenic plants with improved resistance to Verticillium wilt;

[0036] The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

[0037] The present invention discloses the following technical effects:

[0038] The present invention identifies a protein, GbGELP21A, from the GDSL lipase family in sea island cotton. The amino acid sequence is shown in SEQ ID NO. 2, and the nucleotide sequence of the gene encoding it, GbGELP21A, is shown in SEQ ID NO. 1. Overexpression and silencing vectors for this gene were constructed and transformed into Arabidopsis thaliana and cotton. Transgenic experiments showed that after infection with Verticillium wilt, GbGELP21A-overexpressing plants showed significantly better disease progression than wild-type plants, while silencing plants showed more severe disease progression, indicating that the GbGELP21A gene positively regulates plant disease resistance. This invention lays a foundation for Verticillium wilt-resistant breeding and is of great significance for revealing cotton's disease resistance mechanisms, enriching the molecular biology theory of plant stress resistance, and improving plant stress tolerance. It also offers significant economic benefits and broad application prospects for plant production. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 The expression statistics of GbGELP21A in Xinhai 7 (XH7) and Xinhai 21 (XH21) after gene silencing;

[0041] Figure 2 This is the phenotypic observation result of 8 cotton plants infected with Verticillium wilt related to GbGELP21A silencing;

[0042] Figure 3 This figure shows the phenotypic observation results of four Arabidopsis plants related to GbGELP21A overexpression after being treated with Verticillium wilt. DETAILED DESCRIPTION

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0045] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0046] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0047] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0048] The Verticillium wilt pathogen V592 strain of the present invention is Verticillium dahliae Kleb, which is preserved in the inventor's laboratory and has been published in "Duan Lisheng. Research on the molecular mechanism and disease resistance application of GDSL lipase gene in cotton resistance to Verticillium wilt [D]. Shihezi University, 2024.".

[0049] The Arabidopsis thaliana glip1-2 mutant of the present invention was purchased from the AraShare platform.

[0050] The cotton materials Xinhai No. 7 (XH7) and Xinhai No. 21 (XH21) of the present invention were provided by the Anyang Cotton Research Institute of the Chinese Academy of Agricultural Sciences, and were artificially self-pollinated in the experimental field of Shihezi University Farm in 2016. They have been published in "Duan Lisheng. Research on the molecular mechanism and disease resistance application of GDSL lipase gene in cotton resistance to Verticillium wilt [D]. Shihezi University, 2024."

[0051] The applicant promises to distribute the above biological materials to the public within 20 years from the date of filing this application.

[0052] Example 1 Planting of cotton and Arabidopsis

[0053] First, the cotton seeds were germinated in a 28°C culture room. After the seeds turned white, they were planted in culture soil. Then, they were grown in a greenhouse at 30°C with a photoperiod of 16 hours of light and 8 hours of darkness. After about a week, VIGS infection could be carried out after the two cotyledons were fully expanded.

[0054] A small amount of wild-type Arabidopsis seeds were placed in a 1.5 mL EP tube and sterilized in a clean bench. The sterilization steps were as follows: first, rinse the seeds repeatedly with sterile distilled water five times, constantly pipetting with a 1 mL pipette; then, disinfect with 75% alcohol for 3 minutes and rinse five times with sterile distilled water; then, disinfect with 10% sodium hypochlorite for 3 minutes and rinse again five times with sterile distilled water until all residual sodium hypochlorite was removed. Finally, the seeds were sown on 1 / 2 MS solid culture medium plates and placed in a 4°C refrigerator for vernalization for 3 days. They were then placed in a 22°C light incubator under conditions of 16 h light and 8 h dark for about 10 days. After the seedlings grew four rosette leaves, they were transplanted to culture soil (nutrient soil: vermiculite: perlite volume ratio of 3:1:1) and continued to be cultured under conditions of 16 h light and 8 h dark.

[0055] Example 2 Extraction of cotton total RNA and synthesis of cDNA

[0056] Tender sections of cotton seedling root, stem, and leaf tissue were excised, immediately wrapped in tin foil, and quickly frozen in liquid nitrogen for 10 minutes. Grind thoroughly in a pre-chilled sterile mortar. Total RNA was extracted using the Tiangen Biotech Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441), following the manufacturer's instructions. Total RNA was reverse transcribed using the Tiangen Biotech FastKing cDNA First-Strand Synthesis Kit (Genomic Depleted) (KR116).

[0057] Example 3 Cloning of cotton GbGELP21A

[0058] The CDS sequence of cotton GDSL lipase GbGELP21A gene (shown as SEQ ID NO.1) was retrieved from the cotton genome database as a reference sequence for primer design. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0059] GbGELP21A-F:ATGGACACTCCCACCCCTCTCTTCTCCTTGC (SEQ ID NO.3);

[0060] GbGELP21A-R: CCAGAATTGCTTGAGACTGCTTTGAAGAGC (SEQ ID NO. 4).

[0061] SEQ ID NO.1:

[0062]

[0063] The amino acid sequence of the GDSL lipase GbGELP21A expressed by the GbGELP21A gene is shown in SEQ ID NO.2.

[0064] SEQ ID NO.2:

[0065] MDTPTPLFSLLCFFLFSLFLGEEQHVVEGSRVWTFGFRPKMLFVFGDSYADTGNNRKALASSWKLPYGITFPGKPAGRFSDGRVLTDFIAGYLAIKTPVPYRYRKELGGRLKYGLNFAYGGTGVFDTPAPEPNMTTQIDFLQQLLNDSVYPKRALKTSVALVSLAGNDYSNYIATNG STAGFPVFIGRVVNQMKVNLKRIHNLGVRKIAASALQPLGCLPRSTAQFSFEQCNESENALVGLHNQLLAQAVNDLNKETNSSSFFVLDIYNAFWNVFNKKQAHQVSPTFVNPFEPCCVGVSAAFSCGSVDENGVKQYTLCSNPKSKFFWDTVHPTEQGWRAVYSTPALQSSLKQFW.

[0066] PCR amplification was performed using cotton cDNA as a template. The sample addition system was: 10 μL of 2×Taq PCR MasterMixⅡ, 1 μL of cDNA, and 0.5 μL of upstream and downstream primers. After the sample was added, it was centrifuged briefly to mix and then amplified on a PCR instrument. The amplification program was: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 2 minutes, and final extension at 72°C for 10 minutes.

[0067] After amplification, perform agarose gel electrophoresis and recover the target band using an agarose gel recovery kit, following the kit's instructions. After gel recovery, the target product is ligated with pMD-19T. Transformation is performed using Tiangen Biopharmaceuticals' DH5a competent medium, following the instructions. After plating, perform PCR analysis and send the identified monoclonal clone to Shanghai Biotechnology for sequencing.

[0068] Example 4 Construction of GbGELP21A gene plant expression vector

[0069] Primers containing homology arms were designed, and the plant expression vectors pCAMBIA1300 and pCLCrVA were double-digested with restriction endonucleases to obtain the digested vector fragments. The target gene fragment and the vector fragment were then recovered. The target gene fragment and the vector fragment were ligated in vitro. Upon identification, the correct recombinant plasmids were named pCAMBIA1300-GbGELP21A and pCLCrVA-GbGELP21A, respectively. In this embodiment, Arabidopsis thaliana was used as the transgenic plant material, but other plants can also be used as transgenic materials.

[0070] Among them, the primer sequences for constructing the plant expression vector pCAMBIA1300-GbGELP21A are shown in SEQ ID NO.5 and SEQ ID NO.6; the primer sequences for constructing the plant silencing vector pCLCrVA-GbGELP21A are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0071] pCAMBIA1300-GbGELP21A-F: attcatttggagaggacagggtaccATGGACACTCCCACCCCTCT CTTCTCCTTGC (SEQ ID NO. 5);

[0072] pCAMBIA1300-GbGELP21A-R: accatggtactagtgtcgactctagaCCAGAATTGCTTGAGACTGC TTTGAAGAGC (SEQ ID NO. 6);

[0073] pCLCrVA-GbGELP21A-F: caaaatggcatgcctgcagactagtCATTGCCACCAACGGCTCTA (SEQ ID NO. 7);

[0074] pCLCrVA-GbGELP21A-R: gaattcactagacctaggggcgcgccTCGCCATCCTTGTTCCGTAG (SEQ ID NO. 8);

[0075] Example 5 Transformation of Agrobacterium

[0076] Under sterile conditions in a clean bench, the successfully identified bacterial plasmid was transformed into Agrobacterium GV1301. The transformation steps are as follows:

[0077] (1) Take 5 μL of the identified plasmid and add it to 50 μL of Agrobacterium competent cells, mix well, and let it stand on ice for 5 minutes;

[0078] (2) Place in liquid nitrogen for 5 minutes;

[0079] (3) Heat shock in a 37°C water bath for 5 min, followed by rest on ice for 5 min;

[0080] (4) Add 600 μL of antibiotic-free LB medium, mix well, and incubate at 28°C, 200 rpm, with shaking for 1-2 h;

[0081] (5) Centrifuge at 6000 rpm for 5 min;

[0082] (6) Discard the supernatant and spread 100 μL of the bacterial solution on LB solid medium containing rifamycin (100 mg / L), gentamicin (50 mg / L), and kanamycin (50 mg / L) and culture in a constant temperature incubator at 28°C for 2 days.

[0083] (7) Single clones were picked from the LB solid plate and identified by PCR. Agrobacterium with the correct band size was identified as the target gene successfully transferred into the Agrobacterium.

[0084] Example 6: Infection of Arabidopsis thaliana by the Flower Drop Method

[0085] When the transplanted Arabidopsis thaliana reaches its peak flowering stage one week after its first flowering, the previous siliques are cut off and then infected. Preparation of infection solution for Arabidopsis thaliana: The correctly activated and identified Agrobacterium GV3101 is inoculated into 10mL of LB liquid culture medium containing three antibodies (50μg / mL rifampicin, 50μg / mL gentamicin and 50μg / mL kanamycin) for expansion culture for 6-8h. After the color of the bacterial culture turns orange, centrifuge at 4000rpm for 5min, discard the supernatant in the clean bench to collect the bacteria, and then use the infection solution (5% sucrose + 0.02% Silwet L-77) to resuspend the bacterial solution and make the OD 600 When the pH reaches 0.8, incubate in the dark for 3 hours before infecting the blooming flowers. After infection, incubate in the dark for 8 hours, then recover under normal conditions. Infect once a week for a total of three times until the T0 generation seeds are harvested.

[0086] Example 7 Identification of transgenic Arabidopsis

[0087] The collected infected Arabidopsis seeds were sown on a 1 / 2 MS solid culture medium (containing 50 mg / L Kan) on a sterile workbench according to the planting method described above. After two to three weeks of growth in a climatic chamber, untransformed Arabidopsis seedlings gradually turned white and died, while successfully transformed Arabidopsis seedlings grew normally. Resistant seedlings were transplanted and managed normally until the T3 generation seed was harvested. Normally growing Arabidopsis were transferred to culture soil for further cultivation. DNA from the initially screened Arabidopsis was then extracted and identified by PCR.

[0088] Example 8 Cotton VIGS gene silencing experiment

[0089] Preparation of VIGS infection solution: Add Agrobacterium LBA4404 containing pCLCrVB (helper plasmid), pCLCrVA (empty vector), pCLCrVA-PDS (positive control) and pCLCrVA-GbGELP (containing target gene fragment) at a ratio of 1:100 to 5 mL LB liquid medium (containing 50 μg / mL kanamycin, 50 μg / mL streptomycin and 50 μg / mL rifampicin), and place in a 28°C constant temperature shaker at 200 rpm for overnight culture. Add the Agrobacterium culture to 45 mL LB liquid medium containing 50 μg / mL kanamycin, 50 μg / mL streptomycin, 10 mM MES and 20 μM acetosyringone for overnight culture, transfer the bacterial solution to a 50 mL centrifuge tube for bacterial enrichment, centrifuge at 5000 rpm for 10 minutes, discard the supernatant and gently pipette the bacteria to resuspend them with permeation solution to make the OD 600 Reach 1.0. Incubate in the dark at room temperature for 4 hours. After incubation, mix the Agrobacterium infection solution containing pCLCrVB (helper plasmid) with the other carrier Agrobacterium in a 1:1 ratio. Use a needle to scratch the underside of the cotyledon, and then use a needleless syringe to fill the entire leaf with the bacterial solution. After infection, incubate in the dark overnight, then transfer to a greenhouse at 25°C with 16 hours of light and 8 hours of darkness. After about 15 days, the true leaves of the positive control PDS-silenced plants will show albinism symptoms, indicating that the VIGS experiment was successful.

[0090] Example 9 Phenotypic Observation of Cotton and Arabidopsis

[0091] 1. Cotton phenotype observation

[0092] Virus-mediated gene silencing (VIGS) was used to silence the homologous gene GbGELP21A in the susceptible material Xinhai No. 7 and the resistant material Xinhai No. 21, respectively. The root materials infected with Verticillium dahliae V592 for 10 days were tested before and after silencing.

[0093] like Figure 1 As shown, the results showed that the GbGELP21A gene was significantly upregulated in both the susceptible material Xinhai No. 7 and the resistant material Xinhai No. 21 after infection with Verticillium dahliae, and the relative change fold in the resistant material was greater than that in the susceptible material; the relative expression level of the GbGELP21A gene after silencing was significantly downregulated in Xinhai No. 7 and Xinhai No. 21, by 60% and 51%, respectively; and the relative expression level of GbGELP21A in the silenced Xinhai No. 7 and Xinhai No. 21 was significantly upregulated after infection with Verticillium dahliae.

[0094] In addition, if Figure 2As shown in the figure, through phenotypic observation, it was found that the disease resistance of Xinhai No. 7 and Xinhai No. 21 with silenced GbGELP21A gene was significantly reduced after infection with Verticillium dahliae compared with the wild type, especially Xinhai No. 7, which developed the disease the fastest.

[0095] 2. Arabidopsis Phenotypic Observation

[0096] The wild type WT, GbGELP21A gene transgenic (1300-GbGELP21A), glip1-2 mutant (AtGELP21A) and GbGELP21A transgenic AtGELP21A (AtGELP21A complementation) were infected with Verticillium dahliae and their phenotypes were observed. Figure 3 As shown, the glip1-2 mutant showed the most severe etiolation, followed by wild-type Arabidopsis and AtGELP21A-complemented Arabidopsis. Arabidopsis overexpressing the GbGELP21A genotype showed the best resistance to Verticillium wilt, and compared with the glip1-2 mutant, the GbGELP21A-transfected AtGELP21A Arabidopsis showed significantly less etiolation.

[0097] In summary, it was shown that overexpression of the GbGELP21A gene could indeed enhance the plant's resistance to Verticillium wilt.

[0098] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of cotton lipase GbGELP21A in any of the following: (1) Application in regulating plant resistance to Verticillium wilt; (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt; (3) Application in the preparation of products for improving plant resistance to Verticillium wilt; The amino acid sequence of the lipase GbGELP21A is shown in SEQ ID NO.

2.

2. Use of the gene encoding the lipase GbGELP21A as claimed in claim 1 in any of the following: (1) Application in regulating plant resistance to Verticillium wilt; (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt; (3) Application in the preparation of products for improving plant resistance to Verticillium wilt; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that The expression level of the coding gene is up-regulated in the plant, thereby improving the resistance of the plant to Verticillium wilt.

4. The use according to claim 3, characterized in that The method of increasing the expression of the coding gene in plants comprises the following steps: constructing an overexpression vector comprising the coding gene, transforming the overexpression vector into Agrobacterium, and then infecting the plant; The plant is cotton or Arabidopsis thaliana.

5. Use of an overexpression vector comprising the coding gene according to any one of claims 2 to 4 in any of the following: (1) Application in regulating plant resistance to Verticillium wilt; (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt; (3) Application in the preparation of products for improving plant resistance to Verticillium wilt.

6. Use of an engineered bacterium comprising the overexpression vector according to claim 5 in any of the following: (1) Application in regulating plant resistance to Verticillium wilt; (2) Application in the cultivation of transgenic plants with improved resistance to Verticillium wilt; (3) Application in the preparation of products for improving plant resistance to Verticillium wilt.

7. A method for improving plant resistance to Verticillium wilt, characterized in that: The method comprises the steps of up-regulating the expression level of the gene encoding the lipase GbGELP21A in the plant to improve the resistance of the plant to Verticillium wilt; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, wherein The method of increasing the expression of the gene encoding lipase GbGELP21A comprises the following steps: An overexpression vector comprising the gene encoding the lipase GbGELP21A is constructed, and the overexpression vector is transformed into Agrobacterium, which is then used to infect plant plants.

9. The method according to claim 7 or 8, wherein The plant is cotton or Arabidopsis thaliana.

10. A method for breeding transgenic plants with improved resistance to Verticillium wilt, characterized in that: The following steps are involved: Overexpressing the gene encoding the lipase GbGELP21A in plant cells, then cultivating the plant cells, and obtaining regenerated plants using the plant cells, thereby obtaining the transgenic plants with improved resistance to Verticillium wilt; The nucleotide sequence of the coding gene is shown in SEQ ID NO.1.

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