Application of GhEPC1 gene in regulation and control of verticillium wilt resistance of cotton

Abstract

The invention relates to the technical field of biology, and discloses application of a GhEPC1 gene in regulation and control of cotton verticillium wilt resistance. How the GhEPC1 gene affects cell wall synthesis is analyzed by means of biochemistry, molecular biology, cell histology, genetics and the like so as to regulate and control a molecular mechanism of cotton resistance to verticillium wilt bacteria; the GhEPC1 gene disease-resistant mechanism can be revealed from the perspective of molecular level, and new theoretical basis and technical support are provided for cotton molecular breeding.

Description

[0001] The application relates to the field of biotechnology, and more particularly to application of a GhEPC1 gene in regulating cotton resistance to verticillium wilt. BACKGROUND

[0002] Cotton is an indispensable basic product and basic industry for the national economy and people's livelihood. The unique light and heat resources in Xinjiang provide a congenital advantage for the region to become the largest high-quality cotton base in China. However, cotton verticillium wilt is a major obstacle to the sustainable development of the cotton industry in Xinjiang. Plant glycosyltransferase genes play a key role in regulating biological stress response processes. At present, the research on glycosyltransferase genes in cotton is insufficient. Through analysis of cotton transcriptome data in response to verticillium wilt stress, a glycosyltransferase gene GhEPC1 related to cell wall synthesis and capable of responding to verticillium wilt stress is obtained. Time-space expression, gene silencing, tobacco heterologous expression experiments and cotton transgenic experiments show that the gene is dominantly expressed in the resistant variety, and the cotton plants with silenced genes are more sensitive to verticillium wilt fungus, but the tobacco with overexpressed genes improves the tolerance to verticillium wilt fungus, and the transgenic cotton improves the resistance to verticillium wilt. On this basis, biochemical, molecular biological, cytological and genetic methods are used to analyze how the GhEPC1 gene affects cell wall synthesis and further regulates the molecular mechanism of cotton resistance to verticillium wilt fungus, which helps to reveal the disease resistance mechanism of the GhEPC1 gene from the molecular level and provides new theoretical basis and technical support for cotton molecular breeding. SUMMARY

[0003] In order to overcome the above-mentioned defects in the prior art, the application provides application of a GhEPC1 gene in regulating cotton resistance to verticillium wilt. Biochemical, molecular biological, cytological and genetic methods are used to analyze how the GhEPC1 gene affects cell wall synthesis and further regulates the molecular mechanism of cotton resistance to verticillium wilt fungus, which helps to reveal the disease resistance mechanism of the GhEPC1 gene from the molecular level and provides new theoretical basis and technical support for cotton molecular breeding.

[0004] The above technical purpose of the application is achieved by the following technical scheme: application of a GhEPC1 gene in regulating cotton resistance to verticillium wilt.

[0005] Further, the GhEPC1 gene regulates the disease resistance of transgenic cotton to verticillium wilt by regulating cell wall synthesis related genes.

[0006] In summary, the present invention has the following beneficial effects: This application analyzes the molecular mechanism of how the GhEPC1 gene affects cell wall synthesis and thus regulates cotton resistance to Verticillium dahliae through biochemistry, molecular biology, cell histology, genetics and other means, which helps to reveal the disease resistance mechanism of the GhEPC1 gene from a molecular level perspective and provide new theoretical basis and technical support for cotton molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is a phylogenetic tree of the GMT1 family in Arabidopsis thaliana and four major cotton species ( G. hirsutum , G. barbadense , G. arboreum , and G. arboreum ) in the embodiments of the present invention;

[0008] Figure 2 This is a distribution diagram of the GMT1 gene on chromosomes in four major cotton species (G. hirsutum, G. barbadense, G. arboreum, and G. arboreum) according to the present invention. In the figure: a: G. arboreum, b: G. raimondii, c: G. hirsutum, d: G. barbadense;

[0009] Figure 3 is a cluster analysis diagram of GhEPC1 and other glycosyltransferases in the embodiment of the present invention;

[0010] Figure 4 1. It is a phylogenetic tree, conserved motif and gene structure analysis diagram of the GMT1 family protein sequence of upland cotton in the embodiment of the present invention;

[0011] Figure 5 is a linear analysis graph of GMT1 family members in four major cotton species (G. hirsutum, G. barbadense, G. arboreum, and G. arboreum) according to the embodiment of the present invention;

[0012] Figure 6 This is a diagram showing tissue-specific expression analysis of the GhEPC1 gene in an embodiment of the present invention;

[0013] Figure 7 2 is an analysis diagram of the expression pattern and subcellular localization of the GhEPC1 gene after treatment with Verticillium dahliae in an embodiment of the present invention, wherein: A: expression pattern of the GhEPC1 gene after treatment with Verticillium dahliae; B: subcellular localization of the GhEPC1 gene in tobacco cells, scale bar = 50 μm;

[0014] Figure 8: This is a diagram showing disease resistance identification after GhEPC1 gene silencing in cotton and expression analysis of disease resistance-related genes in silenced lines according to an embodiment of the present invention. In the figure: a: leaf albino phenotype of cotton seedlings injected with a reporter gene (GhCLA1); b: inoculation identification of cotton seedlings injected with TRV:GhEPC1; c: inoculation identification of cotton seedlings injected with TRV:0; d: statistics of inoculation disease index after GhEPC1 gene silencing (* indicates significant difference at the 0.05 level);

[0015] Figure 9 This is a graph showing the detection of disease resistance-related enzyme activities in susceptible and disease-resistant plants after GhEPC1 gene silencing in an embodiment of the present invention;

[0016] Figure 10 1 is a graph showing the disease resistance identification and expression characteristics of disease resistance-related genes in transgenic tobacco positive plants in an embodiment of the present invention, wherein: a is untreated wild-type tobacco, b is wild-type inoculated with Vd592, c is GhEPC1-overexpressing transgenic tobacco inoculated with Vd592, d to i: expression characteristics of NbPR1a, NbPR2, NbPR9, NbPR10, NbLox6, and NbERF1;

[0017] Figure 11 : This is a diagram of GhEPC1 gene genetic transformation of cotton and positive seedling detection in an embodiment of the present invention, in which: a to f: cotton transformation process; g: PCR detection of transgenic positive seedlings (DNA Marker III, 1-23 transgenic tobacco detection, WT is wild type);

[0018] Figure 12 This is a Southern hybridization analysis diagram of GhEPC1 gene-transgenic cotton in an embodiment of the present invention;

[0019] Figure 13 This is a diagram of GhEPC1 gene expression analysis of positive plants of different strains in the embodiment of the present invention;

[0020] Figure 14 This is a positive plant disease resistance identification diagram in the embodiment of the present invention, in which: A is a cotton phenotypic identification diagram, and B is a disease index statistical diagram;

[0021] Figure 15 This is a graph showing the activity of disease-resistant enzymes according to an embodiment of the present invention;

[0022] Figure 16 This is a differential gene analysis diagram of "Zhong 24" and "OE" without pathogen inoculation in the embodiment of the present invention;

[0023] Figure 17 is a heat map of the association between the gene co-expression network module and different samples in an embodiment of the present invention;

[0024] Figure 183 is an expression characteristic analysis diagram of cell wall-related genes in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following is combined with Figures 1-18 The present invention is described in further detail.

[0026] Example:

[0027] Glycosyltransferase family 64 gene family analysis:

[0028] Through sequence alignment, the gene GhEPC1 studied in this project belongs to the glycosyltransferase family 64. Based on the whole genome data of upland cotton (TM-1_V2.1), sea island cotton (Hai7124_V1.1), raimondii cotton (Graimondii_genome_HAU_v1.0), and cotton herb (G. arboreum_ICR), the glycosyltransferase family 64 gene members in different cotton genera and Arabidopsis were analyzed and phylogenetic tree analysis was performed. The results showed that 6 GMT1 family members were identified in upland cotton, 5 in sea island cotton, 3 in raimondii cotton, and 2 in cotton herb. Figure 1 shown.

[0029] Based on the whole genome data of upland cotton, sea island cotton, raimondii cotton and herbaceous cotton, the location information of the family genes on chromosomes was analyzed. The results showed that the gene family was distributed on chromosomes 4 and 12 of subgroup A and chromosomes 4, 5 and 12 of subgroup D. There was no GMT1 gene family on other chromosomes. Statistics showed that subgroups A and D contained 2 and 3 GMT1 family genes, respectively. Figure 2 shown.

[0030] This suggests that the evolution of GMT1 family genes on the A chromosome is symmetrical, while their evolution on the D chromosome is asymmetrical. Allotetraploid upland cotton (G. hirsutum) has one more GMT1 gene than G. barbadense (AD genome), and diploid cotton (G. raimondii) has one more GMT1 than G. arboreum. The GMT1 ratio between tetraploid and diploid cotton is greater than 2:1.

[0031] The protein structure of GhEPC1 contains a typical glycosyltransferase family 64 domain. The gene has a similarity of 65.34% with the Arabidopsis gene AT3G55830 (ECTOPICALLY PARTING CELLS, EPC1). Phylogenetic tree analysis shows that the gene is most closely related to the Glycosyltransferase family 64 protein C4-like isoform X2 of upland cotton (G. hirsutum). Figure 3 shown.

[0032] The amino acid sequences of six GMT1 family genes in upland cotton were used to construct a phylogenetic tree and visualize the family gene structure and conserved motifs. Figure 4 As shown, the six GMT1 family genes clustered together in pairs and were divided into three branches, clustering with the three genes of Arabidopsis thaliana respectively; MEME online software was used to analyze the conserved motifs of the GMT1 family protein sequences in upland cotton. The analysis found that the GH_A05G2929 and GH_D05G2953 gene members both contained motifs 1 to 10, four CDS regions, and three introns; the GH_A05G3441 and GH_D04G0669 group gene members both contained motifs 6, 2, 3, 4, 7, and 8, five CDS regions, and four introns; while the GH_A12G1345 and GH_A12G1366 gene members contained motifs 1, 2, 3, 4, 5, and 8, one CDS region, and no introns.

[0033] To further explore the evolutionary process of the GMT1 family genes in the four major cotton species, this application analyzed the collinearity between different genes in the GMT1 family of the four major cotton species, such as Figure 5 As shown, 61 gene pairs exhibited collinearity, including 27 pairs of segmental duplications. Eight pairs of GMT1 genes on chromosomes A and D exhibited collinearity, while five and six pairs of GMT1 genes on chromosomes A and D, respectively, also exhibited collinearity. Furthermore, the GMT1 gene family exhibited a single gene corresponding to multiple genes, suggesting that a chromosome segmental duplication event occurred during evolution. The GMT1 genes in these four species have corresponding homologous genes in almost every branch, indicating that the GMT1 genes in these species are closely related.

[0034] Tissue-specific expression analysis of the GhEPC1 gene:

[0035] The tissue expression specificity analysis of the GhEPC1 gene was performed using the database. The results showed that among the tissues, the highest expression level was in the stem. At flowering, the highest expression level was in the ovule. At 20 days after flowering, the highest expression level was in the fiber. The highest expression level was in the cotyledon at 96 hours of growth, in the root at 120 hours of growth, and in the seed at 5 hours of germination. This indicates that this gene plays a key role in the development of cotton. Figure 6 -A), and the expression level of the gene was further verified by qPCR technology in roots, stems, and leaves. The results showed that the expression level in the stems was the highest, and the difference was not significant compared with the data in roots and leaves ( Figure 6 -B), the results are consistent with those in the database.

[0036] Expression and subcellular localization analysis of GhEPC1 gene under Verticillium dahliae stress:

[0037] After infection with Verticillium dahliae, the relative expression level of the GhEPC1 gene in the resistant variety Zhongzhimian 2 was higher than that in the susceptible variety Xinluzao 36 at all treatment time points. At 120 hours after treatment, the relative expression level of the gene reached its maximum in the resistant variety Zhongzhimian 2, 3.2 times the expression level in the susceptible variety at the same time point, indicating that the gene can be induced to upregulate expression by Verticillium dahliae and is speculated to be involved in the cotton Verticillium wilt stress response ( Figure 7 A). To examine the subcellular localization of GhEPC1, we performed an expression experiment by transiently expressing GFP-GhEPC1 and GFP constructs into tobacco epidermal cells. In cells expressing GFP alone, GFP fluorescence was observed in the nucleus and cell membrane, while GFP fluorescence from GhEPC1 was detected in the endoplasmic reticulum and Golgi apparatus. Results ( Figure 7 B) shows that GhEPC1 is mainly localized in the endoplasmic reticulum and Golgi apparatus.

[0038] Preliminary study on the function of GhEPC1 gene in cotton resistance to Verticillium wilt:

[0039] After silencing the gene using VIGS technology, cotton growth and development were restricted, and resistance to Verticillium dahliae was significantly reduced. Further analysis of the expression characteristics of disease resistance-related genes in normal plants and silenced plants ( Figure 8 -b / c), the disease index statistics showed that the disease index in the silent plants was significantly higher than that in the control ( Figure 8 -d). Analysis of the expression characteristics of disease resistance-related genes in silenced strains ( Figure 8-e), the results showed that compared with the control plant pTRV:00, the key genes of the salicylic acid pathway PAL (phenylalanine ammonia lyase) and PR1 (pathogenesis-related protein 1) in the pTRV:GhEPC1 plant were significantly decreased, and EDS1 (Enhanced Disease Susceptibility 1) The expression level did not change significantly; the expression characteristic analysis results of key genes in the ethylene pathway showed that the expression level of ACO (ACC oxidase gene) increased; the expression characteristic analysis results of key genes in the jasmonic acid pathway showed that the expression level of AOC (allene oxide cyclase) decreased; the expression characteristic analysis results of key genes in the lignin synthesis pathway showed that the expression level of 4CL (4-coumaric acid: coenzyme A ligase) decreased; the expression characteristic analysis results of chitinase and antioxidant enzyme-related genes showed that the expression level of PPO (polyphenolase) decreased, the expression levels of CHI (chitinase), SOD (superoxide dismutase), and CAT (catalase) increased, and the expression level of POD (peroxidase) did not change significantly; combined with the above experimental results, it was shown that GhEPC1 positively regulated the expression of PAL, 4CL, PPO, PR1 and AOC genes, and negatively regulated the expression of CHI, SOD, CAT and ACO. Therefore, GhEPC1 mainly affects cotton resistance by affecting hormone signaling pathways and lignin synthesis.

[0040] Detection of disease resistance-related enzyme activities after GhEPC1 gene silencing:

[0041] The activities of CHI, PAL, POD and PPO in normal plants and silent plants were detected respectively. The results showed that before and after pathogen infection, the susceptible plants ( Figure 9 -B / D / F / H), there was no significant difference in the activities of CHI, PAL, POD, and PPO after silencing the GhEPC1 gene, but in the disease-resistant plants ( Figure 9 -A / C / E / G), after GhEPC1 gene silencing, the activities of CHI and PAL enzymes decreased in silenced plants, while there was no significant difference in the activities of POD and PPO enzymes.

[0042] Functional study of the GhEPC1 gene in tobacco against Verticillium wilt:

[0043] To further verify the function of the GhEPC1 gene after ectopic expression in tobacco, a plant overexpression vector was constructed. The gene was introduced into Agrobacterium GV3101 by electroporation, and positive strains were identified by PCR screening of the bacterial solution. The tobacco was transformed using the Agrobacterium-mediated method. T3 generation transformed plants were obtained through laboratory kanamycin screening and PCR detection. The resistance of these plants to Verticillium dahliae was identified. The results showed that compared with wild-type tobacco, the transgenic (GhEPC1) tobacco had improved resistance to Verticillium dahliae ( Figure 10-a / b / c), the disease index of the wild type was 76.94 and that of the overexpression plant was 26.02 20 days after inoculation. The wild type plant began to die on the 20th day after inoculation, while the overexpression plant did not die although its leaves turned yellow. As time went on, the wild type plant began to die (30 days), while the overexpression plant began to die 45 days after inoculation. After inoculation with Verticillium wilt, the expression characteristics of disease resistance-related genes (NbPR1a, NbPR2, NbPR9, NbPR10, NbLox6, NbERF1) in tobacco were analyzed by qRT-PCR technology ( Figure 10 -d / e / f / g / h / i). The results showed that, except for NbERF1 and NbLox6, the expression levels of the other genes in transgenic tobacco were higher than those in wild-type tobacco after 12h / 24h treatment, indicating that the genes can respond quickly to the initial stage of Verticillium dahliae stress. Studies have shown that PR (Pathogenesis-related proteins, PRs) are a class of genes that can encode pathogenesis-related proteins when plants are induced by stress. PR1a-like proteins inhibit the growth and development of fungal cells by decomposing fungal cell walls or causing plasmolysis of pathogen cells. PR2 proteins have β-1,3-glucanase activity, PR9 proteins have peroxidase activity, and resist pathogen invasion by thickening cell walls. PR10 proteins have ribonuclease-like activity and resist pathogen invasion by phosphorylation. These substances constitute an important component of the plant defense system and play an important role in plant disease resistance. The above results indicate that the target gene can be ectopically expressed in tobacco and can activate the expression of pathogenesis-related protein genes.

[0044] Genetic transformation of GhEPC1 gene in cotton and screening of positive plants:

[0045] The plant overexpression vector containing the GhEPC1 gene was introduced into cotton ( Figure 11 -a~f), the target gene sequence and the NOS end sequence of the plant expression vector pCAMBIA2300 were selected to design primers, and PCR positive seedlings were screened. The target fragment was 410bp. After testing, 8 transgenic cotton plants could amplify the target fragment ( Figure 11 -g), in order to identify the copy number of T2 generation positive plants, high-purity genomic DNA from leaves was extracted by CTAB method, and the cotton genome was digested with HindⅢ. A sequence amplified by KanR was used as a probe for Southern hybridization, and three single-copy lines were identified ( Figure 12 ), which can be used for the next functional analysis.

[0046] Analysis of GhEPC1 expression in positive plants:

[0047] In the early stage, three single-copy positive plants were obtained through Agrobacterium-mediated genetic transformation. Further, the expression level of the GhEPC1 gene in the positive plants and wild-type plants was detected by qPCR technology. The results showed that the expression level of the GhEPC1 gene in the positive plants was higher than that in the wild-type plants. Further analysis of the expression level of the GhEPC1 gene in the positive plants of different strains showed that there was no significant difference in the expression level of the GhEPC1 gene between different positive plants ( Figure 13 ).

[0048] Identification of disease resistance in overexpression plants

[0049] The transgenic cotton and wild-type cotton were inoculated with the Verticillium dahliae strain V592 by the root irrigation method. The results showed that about 15 days after the inoculation of the pathogen, the leaves of the transgenic plants and the wild-type plants began to wrinkle to varying degrees, and then slowly turned yellow near the veins. The transgenic cotton began to develop disease 25 days after inoculation and reached its peak at 35 days; the wild-type cotton began to develop disease about 20 days after inoculation and reached its peak at 28 days. The results showed that compared with the wild-type, the transgenic cotton was more resistant to the disease ( Figure 14 ).

[0050] Determination of physiological and biochemical indicators of positive plants overexpressing GhEPC1 gene and wild-type plants

[0051] The chitinase, phenylalanine ammonia lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) contents of wild-type and transgenic plants were detected. The results showed that when the pathogen was not infected, the chitinase, PAL, and POD enzyme activities in transgenic plants were higher than those in wild-type plants, but not significantly. There was no significant difference in PPO activity between wild-type and transgenic plants. Figure 15 A / B), and further determined the flavonoid content, lignin content, cellulose content, and hemicellulose content. The results showed that compared with Zhong 24, the flavonoid content and lignin content remained basically unchanged, but the cellulose content and hemicellulose content increased significantly ( Figure 15 C).

[0052] Transcriptome analysis of positive plants overexpressing the GhEPC1 gene and wild-type plants:

[0053] In the absence of pathogenic fungal infection, a total of 1180 differentially expressed genes were identified between the two cultivars, of which 531 genes were upregulated in the ‘Zhong 24’ material and 649 genes were upregulated in the ‘OE’ material ( Figure 16A and B). Functional annotation of these genes using GO-BP (biological process), GO-MF (molecular function), and GO-CC (cellular component) terms showed that the 649 up-regulated genes in the ‘OE’ material had a total of 41 GO-BP / CC / MF terms ( Figure 16 C), stress-related terms "antioxidant activity (6 genes), immune system process (17 genes), response to stimulus (121 genes), multi-organism process (83 genes), biological regulation (128 genes), detoxification (7 genes), signaling (37 genes) were significantly enriched. At the same time, 531 up-regulated genes in the 'Zhong 24' material were annotated with a total of 40 GO-BP / CC / MFterms ( Figure 16 D), among which "antioxidant activity (6 genes), immune system process (11 genes), response to stimulus (112 genes), multi-organism process (53 genes), biological regulation (87 genes), signaling (21 genes), detoxification (7 genes), by comparing the number of genes enriched in stress-related terms, compared with the '24' material, in the 'OE' material, the number of genes enriched in immune system process (17 genes), response to stimulus (121 genes), multi-organism process (83 genes), biological regulation (128 genes), and signaling (37 genes) were greater.

[0054] KEGG is a comprehensive database that integrates genomic, chemical, and system functional information, facilitating the study of genes and their expression in a holistic network. Analysis of 649 upregulated genes in the 'OE' material showed that they were enriched in plant stress-related pathways: cutin, suberin, and wax biosynthesis; phenylpropanoid biosynthesis; plant hormone signaling ( Figure 16 E). The 531 up-regulated genes in the ‘Zhong 24’ material were more enriched in photosynthesis-related terms, nitrogen metabolism, glyoxylate and dicarboxylic acid metabolism pathways ( Figure 16F) Given that the GO terms and pathway enrichment of upregulated genes in transgenic and wild-type materials were significantly different, the introduction of the GhEPC1 gene into cotton affected the response mechanism of biotic stress.

[0055] A total of 17,335 differentially expressed genes were obtained by further comparison of Z24_CKvsZ24_1d, Z24_CKvsZ24_3d, Z24_CKvsZ24_5d, OE_CKvsOE_1d, OE_CKvsOE_3d, OE_CKvsOE_5d, Z24_CKvsOE_CK, Z24_1dvsOE_1d, Z24_3dvsOE_3d, and Z24_5dvsOE_5d. Key gene mining, based on the correlation of gene expression, a cluster tree was constructed. The generated cluster tree was cut using the dynamic cutting method, and genes with similar expression patterns were merged on the same branch. Each branch represents a co-expression module. Modules with similar expression patterns were merged according to the module similarity (0.8) and then divided into modules. Finally, 12 co-expression modules were obtained. The obtained modules were associated with samples and 12 modules associated with different materials and processing times were obtained. Some modules were highly associated with varieties and processing times. For example, the lightyellow module was significantly positively correlated with the Z24_3d processing time, the black module was significantly positively correlated with the OE_1d processing time, and the red module was significantly positively correlated with the OE_3d processing time. Figure 17 As shown in the figure, the red and blue cells represent the positive and negative correlations between traits and modules, respectively. Therefore, these three modules are studied in depth as disease resistance-related specific modules to explore the core genes in the modules.

[0056] Differential expression analysis of genes related to cellulose and hemicellulose synthesis pathways in the lightyellow module, black module, and red module showed that there were 40 genes in the lightyellow module, 104 genes in the black module, and 111 genes in the red module. The genes obtained in the black and red modules were annotated, and 21 genes were directly related to cell wall synthesis. The 20 genes were caffeic acid 3-O-methyltransferase (COMT), pectin acetylesterase 9 (PAE9), β-1,4-xylosyltransferase (IRX9), cellulose synthase A catalytic subunit 8 (CESA8), laccase-22 (LAC22), β-D-xylosidase 1 (BXL1), UDP xylose transporter 1 (UXT1), cellulose synthase A catalytic subunit 4 (CESA4), cellulose synthase A catalytic subunit 7 (CESA7), xyloglucan endotransferase Glucosylase / hydrolase protein 32 (), UDP-glucuronic acid decarboxylase 6 (UXS6), xylan α-glucuronosyltransferase 1 / 2 (GUX1 / 2), GDSL esterase / lipase 1 (GLIP1), cell wall-associated receptor kinase-like 22 (WAKL22), UDP glycosyltransferase 13 (), cell wall-associated receptor kinase-like 8 / 9 / 2 (WAKL8 / 9 / 2), glucan endo-1,3-β-glucosidase (GNS1), but only one gene (fructokinase 4 (FRK4)) in the lightyellow module is directly related to cell wall synthesis.

[0057] Further expression analysis of the above 21 genes showed that the expression levels of GLIP1, PAE9, LAC22, WAKL22, WAKL2, IRX9, CESA8, WAKL9, GNS1, CESA7, BXL1, XTH32, and GUX2 in OE were higher than those in Zhong-24, and the expression levels of OMT, CGT, WAKL8, and UXS6 in Zhong-24 were higher than those in OE. The expression of FRK4 gene was upregulated in Zhong-24 under pathogen stress, and the expression of the remaining 20 genes was upregulated in OE under pathogen stress ( Figure 18 ).

[0058] In summary, it is speculated that the GhEPC1 gene regulates the disease resistance of transgenic cotton to Verticillium wilt by regulating cell wall synthesis-related genes.

[0059] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. Application of GhEPC1 gene in regulating cotton resistance to Verticillium wilt.

2. The use of the GhEPC1 gene in regulating cotton resistance to Verticillium wilt according to claim 1, characterized in that: The GhEPC1 gene regulates the disease resistance of transgenic cotton to Verticillium wilt by regulating cell wall synthesis-related genes.

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