Application of cotton GbABH132 gene in plant verticillium wilt resistance

Abstract

The invention discloses a cotton gene GbABH132 and application of the cotton gene GbABH132 in plant verticillium wilt resistance. The nucleotide sequence of the gene is as shown in SEQ ID No. 1, and the coded amino acid sequence is as shown in SEQ ID No. 2. After the gene is silenced by using a VIGS technology, the susceptibility of a cotton plant to verticillium wilt is increased; a cotton gene GbABH132 is introduced into arabidopsis thaliana, and a transgenic plant with higher verticillium wilt resistance than wild arabidopsis thaliana is obtained. Therefore, the invention has important significance in breeding and research for improving the verticillium wilt resistance of cotton, and can be applied to breeding disease-resistant cotton varieties.

Description

Technical Field

[0001] This invention specifically relates to the application of the cotton GbABH132 gene in plant resistance to Verticillium wilt, and belongs to the field of cotton genetic engineering technology. Background Technology

[0002] Verticillium wilt, a typical soil-borne fungal vascular disease caused by Verticillium dahliae, is predominantly caused by this fungus. In my country, Verticillium dahliae is the dominant pathogen, affecting over 400 plant species and causing significant crop losses annually. Verticillium wilt is extremely difficult to control, primarily because Verticillium dahliae is a xylem-colonizing pathogen, and there is currently no effective treatment to cure infected plants. Another reason is that Verticillium dahliae produces micronuclei, or blackened dormant structures, which can survive in the soil for decades. Morphological and physiological symptoms in cotton infected with Verticillium dahliae include leaf wilting, yellowing and curling, and browning of the vascular bundles. The pathogen has multiple physiological forms, mutates rapidly, can survive in the soil for several years, and can repeatedly germinate, making complete control difficult and severely harming crop production. my country has a wide cotton-growing area, accounting for nearly a quarter of global cotton production. However, cotton losses caused by Verticillium wilt account for 32.49% of the total losses from other diseases. Coupled with my country's monoculture cotton planting structure, scarcity of cotton resistance resources, and the rapid mutation rate of Verticillium wilt, the control of cotton Verticillium wilt is extremely difficult. Currently, breeding superior cotton varieties resistant to Verticillium wilt is the most economical and effective strategy to solve this dilemma.

[0003] The alpha / beta hydrolase superfamily (ABH) is a widely distributed and functionally plastic group of hydrolases, renowned for their diverse biochemical activities across three domains of life. ABHs are typically associated with the breakdown and recycling of cellular metabolites, the handling of external nutrients, and the detoxification of exogenous substances. Furthermore, ABHs play crucial regulatory roles in metabolism; for example, carboxypeptidase II regulates protein lifespan, function, and turnover, while phospholipases produce second messengers such as diacylglycerols and phosphatidic acids to regulate cellular function and respond to environmental stimuli. The ABH family also supports a variety of unique catalytic functions for defense and hormone regulation. Studies related to cotton have reported that salicylate-binding protein 2 (SABP2) can regulate the response to salicylate, participating in the regulation of plant immune responses. Gibberellin receptors are members of the ABH family; GID1 acts as a receptor for gibberellins, activating plant immune responses. In this study, a cotton gene GbABH132 was cloned and identified. Through sequence structure, functional analysis, and expression pattern analysis, it was found that the gene is expressed in large quantities in the roots when infected by Verticillium wilt. Subsequently, transgenic plants were created using gene silencing and overexpression methods, which verified its important role in resisting Verticillium wilt. Summary of the Invention

[0004] The purpose of this invention is to identify the cotton gene GbABH132 and to study its application in improving plant resistance to Verticillium wilt.

[0005] In a first aspect, the present invention provides a cotton gene GbABH132, the nucleotide sequence of which is shown in SEQ ID No. 1 and the encoded amino acid sequence is shown in SEQ ID No. 2.

[0006] Secondly, this invention provides the application of the cotton gene GbABH132 in plant resistance to Verticillium wilt.

[0007] Furthermore, the plant is cotton or Arabidopsis thaliana.

[0008] Furthermore, the application involves introducing the cotton gene GbABH132 into Arabidopsis thaliana to obtain transgenic plants with stronger resistance to Verticillium wilt compared to wild Arabidopsis thaliana.

[0009] This invention identifies for the first time a cotton gene, GbABH132, that can enhance plant disease resistance. Silencing the expression level of this gene in the cotton variety Zhongzhimian 36 and the inland-sea infiltration line MBI8255 increased the susceptibility of cotton to Verticillium wilt, while overexpression in Arabidopsis thaliana enhanced resistance to Verticillium wilt.

[0010] Thirdly, the present invention also provides a method for obtaining Verticillium wilt-resistant crops, comprising the following steps: (1) Obtain the GbABH132 gene as described in claim 1 and ligate it with the expression vector plasmid to construct a recombinant vector containing GbABH132; (2) The recombinant vector containing GbABH132 was transformed into competent Agrobacterium tumefaciens cells by freeze-thaw method, and positive transformants were obtained by screening. (3) Transfect wild-type crops with bacterial solutions containing positive transformants, harvest the T0 generation after the crops mature, and screen the T0 generation to obtain transgenic crops resistant to Verticillium wilt; (4) Self-pollinate the transgenic crop, screen and harvest transgenic plants or seeds with stable traits; select homozygotes of the T3 generation GbABH132 gene with stable traits in the self-pollination offspring and retain them to obtain crops resistant to Verticillium wilt.

[0011] The advantages and positive effects of this invention are as follows: (1) To systematically analyze the evolutionary relationship, sequence structure and expression pattern of the GbABH132 gene, and to clarify the important role of the GbABH132 gene in plant disease resistance; (2) After the cotton plants with silenced GbABH132 gene were obtained by transgenic technology, the number of diseased fingers increased significantly and the number of pathogens colonized in the stems increased after infection with Verticillium wilt. This indicates that the gene positively regulates the disease resistance of cotton. (3) A homozygous Arabidopsis thaliana line overexpressing the GbABH132 gene was obtained through transgenic technology. After infection by Verticillium wilt, the disease index was significantly reduced, fewer pathogens colonized, and the degree of cell damage was milder.

[0012] Therefore, this invention is of great significance in breeding and research to improve plant resistance to Verticillium wilt, and can be applied to the selection and breeding of cotton varieties and other plant varieties resistant to Verticillium wilt. Attached Figure Description

[0013] Figure 1 Evolutionary relationships and motif analysis of the cotton GbABH132 gene in different species: (A) Phylogenetic evolutionary relationship of the GbABH132 gene in dicotyledonous and monocotyledonous plants; (B) Motif analysis of the GbABH132 gene in dicotyledonous and monocotyledonous plants.

[0014] Figure 2 Evolutionary relationships and gene structure analysis of the cotton GbABH132 gene in different cotton species, including: (A) Phylogenetic tree of gene GbABH132 in sea island cotton, upland cotton, Asian cotton and Gossypium ramondii; (B) Conserved domains of gene GbABH132 in sea island cotton, upland cotton, Asian cotton and Gossypium ramondii; (C) Schematic diagram of potential Motif site distribution of GbABH132 gene in the four cotton species; (D) Schematic diagram of the structure of GbABH132 gene in the four cotton species.

[0015] Figure 3 Secondary and tertiary structure analysis of cotton GbABH132 gene: (A) Secondary structure analysis of GbABH132 gene protein; (B) Tertiary structure display of GbABH132 gene protein.

[0016] Figure 4 Expression sites and expression levels of the cotton GbABH132 gene after infection: (A) Expression sites of the cotton GbABH132 gene in plants; (B) Changes in the expression level of the cotton GbABH132 gene at 0, 1 and 2 days after infection of cotton roots.

[0017] Figure 5Phenotypic analysis of cotton GbABH132 gene silencing in Zhongzhi cotton Z36: (A) Phenotypic diagrams of silent and control plants 15 days after infection with Verticillium wilt in Zhongzhi cotton Z36 materials; (B) GbABH132 albino phenotype after PDS gene silencing; (C) Stem pathogen recovery culture of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt; (D) Stem browning phenotype of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt; (E) Analysis of cotton GbABH132 gene silencing efficiency; (F) Statistical analysis of disease index of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt.

[0018] Figure 6 Phenotypic analysis of cotton GbABH132 gene silencing in the land-sea infiltration line MBI8255: (A) Phenotypic diagrams of silent plants and control plants 15 days after infection with Verticillium wilt in the land-sea infiltration line MBI8255; (B) Albinism phenotype of GbABH132 after PDS gene silencing; (C) Pathogen recovery culture of stems of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt; (D) Phenotypic diagram of stem browning of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt; (E) Analysis of GbABH132 gene silencing efficiency; (F) Statistical analysis of disease index of cotton plants with silent GbABH132 gene and control plants 15 days after infection with Verticillium wilt.

[0019] Figure 7 Disease resistance phenotype identification of Arabidopsis thaliana lines overexpressing the GbABH132 gene: (A) Phenotypic images of wild-type plants and transgenic lines 15 days after infection with Verticillium wilt; (B) PCR detection of T3 generation transgenic lines, well 1 is the maker, and wells 2-9 are transgenic lines; (C) Statistical analysis of disease index of wild-type plants and transgenic lines 15 days after infection with Verticillium wilt.

[0020] Figure 8 The interaction between the promoter of the cotton GbABH132 gene and the transcription factor ERF054 luciferase was verified by a complementary experiment.

[0021] Figure 9 A schematic diagram of the distribution of cis-acting elements in the promoter region of the cotton GbABH132 gene.

[0022] Figure 10 The expression levels of related hormones after silencing the GbABH132 gene in cotton. Detailed Implementation

[0023] The present invention will now be described in detail with reference to embodiments. Unless otherwise specified, the methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0024] Example 1 1.1 Evolutionary relationship and gene structure of cotton GbABH132 gene The evolutionary relationships of the GbABH132 gene in dicotyledons and monocotyledons were analyzed. Homology alignment of the GbABH132 gene protein sequence was performed in the Plant Genome Database (https: / / phytozome-next.jgi.doe.gov), identifying homologous genes in four cotton species (upland cotton, sea island cotton, Asian cotton, and Ramond cotton), monocotyledons (maize, rice, and wheat), and dicotyledons (soybean and Arabidopsis). A phylogenetic tree was constructed using MEGA software. The results showed that the GbABH132 gene is more closely related within the same genus *Gossypium*, and it is also widely distributed across different angiosperms. Figure 1 A). The Motif1, Motif5, and Motif8 of this gene are highly conserved in dicotyledonous and monocotyledonous plants, but in tobacco, the motif of this gene differs significantly from that of other plants, consistent with the characteristics of the ABHs family in terms of conservation and diversification in adapting to the environment during evolution. Figure 1 B).

[0025] Further analysis was conducted on the evolutionary relationships of the GbABH132 gene in the four Gossypium genera. During evolution, the GbABH132 gene is more closely related to the tetraploid *Gossypium hainanense*. Furthermore, the domains and motifs of the GbABH132 gene are highly conserved across the four Gossypium genera. Figure 2 ).

[0026] Using Zhongzhimian 36 and the infiltration line MBI8255 as research materials, the full-length sequence (969 bp) of the GbABH132 gene was obtained from their cDNA. This gene encodes a protein composed of 322 amino acid residues. Homology alignment of the obtained full-length sequence with the genome files of upland cotton, Gossypium Raymondii, Gossypium Asiatum, and Gossypium Islandum revealed that all of them contain an α / β core domain (a β sheet composed of 8 strands surrounded by an α helix), and also contain an additional 310 helix (…). Figure 3 ).

[0027] 1.2 Expression pattern of GbABH132 in cotton To understand the expression pattern of GbABH132 in tissues and organs, under normal growth conditions, when Sea Island cotton H1, Zhongzhi cotton Z36, and the introgression line MBI8255 reached the three-leaf-one-heart stage, appropriate samples of roots, stems, and leaves were collected from seedlings. After quick-freezing in liquid nitrogen, RNA was extracted and reverse-transcribed into cDNA. The expression level of the GbABH132 gene in different organs was then detected using qRT-PCR. The results showed that the expression level of GbABH132 did not differ significantly among roots, stems, and leaves. Figure 4 A). Transcriptome analysis of root tissues from cotton plant Z36 after Verticillium wilt infection showed that its expression level was rapidly upregulated after infection with Verticillium wilt pathogen, especially reaching a peak at 24 hours after infection. Figure 4 B), which suggests that GbABH132 may play a role in the early stages of resistance to Verticillium wilt.

[0028] Example 2 2.1 Construction of the GbABH132 gene plant VIGS vector Zhongzhi cotton Z36 and the introgressive growth line MBI8255 were used as VIGS injection materials. Seeds were soaked for 24 hours. After germination, seeds with uniform growth were selected and planted in sandy soil sterilized at 121℃. Growth conditions included a temperature of 28℃, a photoperiod of 16 hours of light and 8 hours of darkness, and 68% relative humidity. When the cotyledons of the cotton seedlings were fully expanded, VIGS injection solution was prepared and the seedlings were injected with VIGS. TRV2:00 was used as a negative control, and TRV2:PDS as a positive control. During injection, the needle tip was gently stroked on the underside of the cotyledons, and then the bacterial solutions containing the target gene, PDS, and control (TRV:00) were injected into the leaf, filling the entire leaf. After injection, the cotton seedlings were covered with a film and cultured in the dark for 24 hours, followed by normal growth culture. The leaves of the PDS-injected cotton seedlings began to turn white after approximately 12 days of growth. Figure 5 B Figure 6 B) Once the albino phenotype was relatively stable, small amounts of true leaves were taken from cotton plants containing the target gene (TRV: GbABH132) and the control group (TRV: 00) for RNA extraction and reverse transcription into cDNA for RT-qPCR detection. The results showed that the expression level of the GbABH132 gene was significantly lower than that in the TRV: 00 control plants, indicating that GbABH132 gene silencing was successful. Figure 5 E, Figure 6 E). Until the plants grow to the two-leaf-one-heart stage, the Verticillium wilt pathogen strain V991 is activated using Czapek medium, the spore suspension concentration is adjusted to 1×10 spores / mL, the inoculation amount is 2 mL / plant, and at the same time, the roots of silent plants and control plants are treated with inoculation.

[0029] 2.2 Phenotypic identification of cotton resistance to Verticillium wilt after GbABH132 gene silencing.

[0030] Fifteen days after inoculation, the disease incidence was observed in two different cotton materials: the Zhongzhi cotton Z36 and the land-sea infiltration line MBI8255. The results showed that plants with the silenced GbABH132 gene exhibited significant leaf yellowing, wilting, and abscission. Figure 5 A, Figure 6 A), compared to the control group, the TRV:00 index was significantly higher ( Figure 5 F, Figure 6 F). Furthermore, after inoculation, stem dissection and observation revealed that the browning of vascular tissue in plants with silenced GbABH132 genes was significantly more severe than in the control group (TRV:00). Figure 5 D、 Figure 6 D). Subsequently, the colonization of Verticillium wilt was observed through pathogen recovery culture. Stem tissue from the same location was disinfected with 0.1% mercuric chloride and then placed in PDA medium for incubation at 25°C in the dark. After one week, the silent GbABH132 plants produced a large amount of white mycelium, while the control group plants TRV:00 produced less mycelium ( Figure 5 (C, 6C). These results all indicate that knocking down the expression of the GbABH132 gene makes cotton more susceptible to the disease, thus preliminarily identifying that the GbABH132 gene positively regulates cotton resistance to Verticillium wilt.

[0031] 2.3 Silencing GbABH132 gene-related hormone expression We obtained the upstream 2000 bp sequence of the GbABH132 gene and performed predictive analysis using the PlantCare website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The results showed that, in addition to a large number of cis-regulatory elements related to light response, the promoter region of the gene also contains cis-regulatory elements related to salicylic acid (SA) and methyl jasmonate (JA) hormone responses. Figure 9 This indicates that the GbABH132 gene may play a potential role in the mechanism of resistance to Verticillium wilt, and these plant resistance-related hormones may play a role. Subsequently, corresponding hormone detection primers were designed to detect the changes in salicylic acid, methyl jasmonate, and ethylene (ETH) in plants with silenced GbABH132 genes and control plants. The results showed that compared with the control plants, the contents of methyl jasmonate and ethylene in plants with silenced GbABH132 genes were significantly reduced, while the content of salicylic acid did not change significantly. Figure 10 Therefore, it can be inferred that the GbABH132 gene may resist Verticillium wilt infection by regulating methyl jasmonate and ethylene-related hormones.

[0032] Example 3 3.1 Construction of plant overexpression vector pCAMBIA3301-GbABH132 Gene cloning primers (F: gaacacgggggactcttgacATGGAAGGAATTCAACACAA; R: gatcggggaaattcgagctggtcaTTAATGGAACTTGGAGAAGAAG) were designed based on the CDS coding region sequence of the GbABH132 gene. Using cDNA from *Cotton Z36* as a template, the target fragment of the GbABH132 gene was amplified by PCR and recovered. The plant overexpression vector pCAMBIA3301 was digested with restriction endonucleases NcoI and BstEII, purified, and ligated with the target gene fragment. The ligation product was then transformed into *E. coli* DH5α competent cells, thus obtaining the recombinant vector pCAMBIA3301-GbABH132. After confirming correct sequencing results, the plasmid containing the target gene was extracted and transformed into *Agrobacterium* GV3101 competent cells. After the Agrobacterium tumefaciens transformed with the recombinant plasmid grew into single colonies, colony PCR was performed on each colony. Single colonies with PCR products of the same size as the target gene were streaked, cultured by shaking, and then stored in 60% glycerol at -80°C. Thus, the plant overexpression recombinant vector pCAMBIA3301-GbABH132 was successfully constructed.

[0033] 3.2 Screening of transformed Arabidopsis thaliana and transgenic Arabidopsis thaliana In a clean bench, Arabidopsis seeds were placed in 2.0 mL centrifuge tubes, and 0.1% HgCl2 was added to cover the seeds. The tubes were shaken up and down for 5 min, centrifuged at 12000 r / min for 1 min, and the HgCl2 was carefully removed and discarded. Deionized water was added and the tubes were gently shaken for 7-8 min. The deionized water was discarded, and the seeds were washed with deionized water 3-4 times. The seeds were then vernalized at 4℃ for 72 h. Vernalized Arabidopsis seeds were sown on MS solid medium and cultured in an Arabidopsis greenhouse under the following conditions: 22℃, 16 h light, 8 h dark. When the Arabidopsis had two true leaves, they were transplanted into potting soil. During peak flowering, open flowers and pods were removed, leaving only the flower buds. The flowers were then dipped in the buds for infection. The resuspended solution used for infection contained Silweet 77 200 μL / L, MS 2.15 g / L, sucrose 50 g / L, AS 200 mmol / mL, and pH 5.7-5.8. To improve transformation efficiency, infection was performed once every 7 days, for a total of 3 infections. After infection, the plants were covered and cultured in the dark for 24 hours, after which they were allowed to grow normally in a greenhouse. Once the seeds on the infected Arabidopsis plants matured, mature T0 seeds were harvested and dried in a 37 ℃ incubator. The T0 seeds were then sown evenly in the nutrient soil and covered with plastic wrap to retain moisture. After 2-4 days, green shoots appeared on the Arabidopsis plants. The plastic wrap was then removed, and herbicide was sprayed twice daily. The herbicide preparation was: 10% solution-Basta 750 μL, water 500 mL. After continuous herbicide spraying for one week, the plants were cultured normally for 5-6 days. Non-overexpression plants produced yellowish, smaller leaves and failed to grow normally, while overexpression plants had larger, light green leaves. T1 generation positive Arabidopsis were harvested. For T1 generation positive Arabidopsis identification, healthy Arabidopsis seedlings were transplanted into pots filled with nutrient soil and cultured for 3-4 weeks. DNA was extracted from rosette leaves. Specific overexpression identification primers were designed (OE-GbABH132-F: gaacacgggggactcttgacATGGAAGGAATTCAACACAA; OE-GbABH132-R: gatcggggaaattcgagctggtcaTTAATGGAACTTGGAGAAGAAG). After PCR amplification of DNA, positive seedlings were identified by gel agarose gel electrophoresis. False positives were removed based on the PCR results, and positive plants were retained. Seeds from the normally grown and matured T1 generation positive Arabidopsis were dried and immediately sown to screen for T2 generation positive Arabidopsis. Arabidopsis thaliana is a diploid plant. The positive Arabidopsis thaliana plants selected in the first screening are heterozygotes. In the T2 generation, the ratio of positive to negative plants will be 3:1. Single-copy lines that meet the segregation ratio are retained, and leaf DNA is extracted for PCR detection. Positive plants are retained.The seeds of the harvested T2 generation line were further screened by spraying herbicides to obtain stable transgenic Arabidopsis thaliana T3 generation lines, and leaf DNA was extracted for PCR detection. Figure 7 B) Retain homozygous lines, collect seeds, and dry and store them.

[0034] 3.3 Identification of resistance to Verticillium wilt in Arabidopsis thaliana overexpressing GbABH132 Overexpression lines OE7, OE8, and wild-type Col-0 (WT) Arabidopsis thaliana were sown in nutrient soil. When the plants reached four weeks of age, Verticillium wilt pathogen strain V991 was activated, and the spore suspension concentration was adjusted to 1×10⁶ spores / mL. The inoculum was applied by watering the roots, with an inoculum dosage of 10 mL per plant. Fifteen days after inoculation, wild-type WT leaves showed obvious yellowing phenotype, while the overexpression line GbABH132 exhibited milder disease progression. Figure 7 A), after statistical analysis of the disease index, the disease index of wild-type WT reached 28.57%, while that of the overexpression lines OE7 and OE8 were 13.88% and 11.875%, respectively. The figure clearly shows that the disease index of the overexpression GbABH132 line was lower than that of wild-type WT. Figure 7 C). Therefore, overexpression of the GbABH132 gene significantly enhanced resistance to Verticillium wilt in Arabidopsis thaliana.

[0035] Example 4 4. Luciferase Complementation Reporter Gene Assay Using the PlantTFDB (https: / / planttfdb.gao-lab.org / ) transcription factor prediction website, the transcription factor binding to the upstream promoter of GbABH132 was predicted, and GB_A04G1261 was obtained. It is speculated that GB_A04G1261 (i.e., GbERF054, belonging to the ERF family) may be a potential transcription factor binding to the upstream promoter of GbABH132. To verify whether the transcription factor GB_A04G1261 has a regulatory effect on the upstream promoter of GbABH132, a 2000 bp promoter upstream of GbABH132 was cloned from the mixed DNA of cotton Z36, ligated into the pGreen-0800-LUC vector, and the pGreen-0800-GbABH132 recombinant plasmid was constructed. Simultaneously, a 969 bp full-length CDS of GbERF054 was cloned from the cDNA of mixed cotton Z36 plants and ligated into the pGreen-62-sk vector to construct the GbERF054-62-SK recombinant plasmid. GbERF054-62-SK and pGreen-0800-GbABH132 were used as the experimental group, and pGreen-0800-GbABH132 and pGreen-62-sk were used as the control group. Protein expression was quantified by detecting luciferase activity, and the results were observed using a fluorescence imaging system. It was found that co-transformation of tobacco with GbERF054-62-SK and pGreen-0800-GbABH132 produced a fluorescent signal. Figure 8 Therefore, it was hypothesized that GbERF054 could bind to the GbABH132 promoter, and the results showed that GbERF054 affects the transcription of GbABH132 by binding to the promoter.

[0036] This application analyzed the sequence structure, evolutionary relationship, and expression pattern of the cotton GbABH132 gene. It was found that after infection with Verticillium wilt, the GbABH132 gene responded to Verticillium wilt induction, with its expression level reaching a peak after 24 hours. Analysis revealed that this gene contains cis-acting elements and conserved motifs related to disease resistance. Furthermore, downregulating or silencing the GbABH132 gene expression in cotton significantly enhanced its susceptibility to Verticillium wilt. Simultaneously, using transgenic technology, homozygous Arabidopsis thaliana lines overexpressing the GbABH132 gene were obtained. After infection with Verticillium wilt, these lines showed significantly enhanced resistance to the pathogen. In summary, it is clear that the GbABH132 gene positively regulates cotton resistance to Verticillium wilt. Therefore, this invention holds promise for providing new ideas for breeding high-quality upland cotton lines with high disease resistance by creating transgenic cotton materials overexpressing GbABH132.

Claims

1. A cotton gene GbABH132, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

2. The application of the cotton gene GbABH132 as described in claim 1 in plant resistance to Verticillium wilt.

3. The application of the cotton gene GbABH132 as described in claim 2 in plant resistance to Verticillium wilt, characterized in that, The plant in question is cotton or Arabidopsis thaliana.

4. The application of the cotton gene GbABH132 as described in claim 2 in plant resistance to Verticillium wilt, characterized in that, Overexpression of the GbABH132 gene in plants enhances their resistance to Verticillium wilt.

5. A method for obtaining crops resistant to Verticillium wilt, characterized in that, Includes the following steps: (1) Obtain the GbABH132 gene as described in claim 1 and ligate it with the expression vector plasmid to construct a recombinant vector containing GbABH132; (2) The recombinant vector containing GbABH132 was transformed into competent Agrobacterium tumefaciens cells by freeze-thaw method, and positive transformants were obtained by screening. (3) Transfect wild-type crops with bacterial solutions containing positive transformants, harvest the T0 generation after the crops mature, and screen the T0 generation to obtain transgenic crops resistant to Verticillium wilt; (4) Self-pollinate the transgenic crop, screen and harvest transgenic plants or seeds with stable traits; select homozygotes of the T3 generation GbABH132 gene with stable traits in the self-pollination offspring and retain them to obtain crops resistant to Verticillium wilt.

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