GhHSL1 gene and application thereof in regulating verticillium wilt resistance of upland cotton

Abstract

The invention relates to the field of biotechnology application, and discloses a GhHSL1 gene and application thereof in regulation and control of upland cotton verticillium wilt resistance. The nucleotide sequence of the GhHSL1 gene upland cotton genetic standard line TM-1 is shown as SEQ ID NO. 1, and the amino acid sequence of the GhHSL1 gene upland cotton genetic standard line TM-1 is shown as SEQ ID NO. 2. The gene is induced by verticillium dahlia to realize obvious up-regulation expression. According to the invention, a virus-induced gene silencing technology and a transgenic overexpression technology are further utilized to preliminarily research the function of the cotton GhHSL1 gene in cotton verticillium wilt resistance, and the result shows that the verticillium wilt resistance of cotton plants is obviously reduced after the GhHSL1 gene in upland cotton is silenced; after the GhHSL1 gene is over-expressed in upland cotton, the verticillium wilt resistance of the cotton is remarkably improved, and it is determined that the GhHSL1 gene positively regulates the resistance of the upland cotton to verticillium dahliae; the invention discloses the application of the GhHSL1 gene in regulating and controlling the disease resistance of upland cotton verticillium wilt for the first time, and provides theoretical guidance and genetic resources for improving the disease resistance of cotton through molecular design breeding or genetic engineering.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications, specifically relating to a... GhHSL1 Genes and their application in regulating resistance to Verticillium wilt in upland cotton. Background Technology

[0002] Cotton (Gossypium spp.) is one of the world's most important economic crops, with wide applications in agriculture, textile industry, and medical and health fields. my country is the world's largest cotton producer, but it faces a serious disease known as "cotton cancer"—Verticillium wilt. This disease causes yellowing of cotton leaves and wilting, significantly reducing cotton yield and severely damaging the quality of cotton fibers, becoming a major factor affecting cotton production.

[0003] Verticillium wilt is a fungal soil-borne disease caused by Verticillium dahliae Kleb. The pathogen can infect the vascular bundles of cotton, causing systemic disease. It is characterized by its wide distribution, severe damage, and long survival time, and has become a major obstacle to cotton production worldwide.

[0004] The cotton genome is large and its genetic background is complex. Resources of Verticillium wilt resistance genes are scarce, and the pathogenic mechanism of Verticillium wilt pathogen is complex. These factors have consistently constrained research on cotton resistance to Verticillium wilt and the progress of molecular breeding for resistance. Cloning and functional identification of cotton Verticillium wilt resistance genes, and studying the molecular mechanisms of interaction between cotton and Verticillium wilt pathogen, are important topics in cotton Verticillium wilt resistance breeding research. However, because the pathogen causing this disease lives in the top 40 cm of soil and mutates frequently, traditional fungicides and chemical agents are ineffective. Moreover, it hosts more than 120 plant species, and traditional crop rotation patterns are not ideal in the short term.

[0005] Therefore, planting Verticillium wilt-resistant varieties is the most effective way to control this disease. However, the disease resistance of cotton varieties in my country is currently only at the level of resistance to tolerance, so breeding highly resistant Verticillium wilt-resistant superior varieties remains a bottleneck that urgently needs to be addressed in cotton variety breeding. Researching the pathogenic mechanism of Verticillium wilt pathogen and the disease resistance mechanism of cotton, and using molecular markers and genetic engineering techniques to transfer resistance traits into existing superior cultivars is the current direction of Verticillium wilt-resistant breeding. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies and provide a solution. GhHSL1 Genes and their potential and application in regulating Verticillium wilt resistance in upland cotton and in breeding new Verticillium wilt-resistant cotton germplasm.

[0007] To achieve the above objectives, the present invention proposes the following technical solution: In a first aspect, the present invention provides a substance isolated from upland cotton that is associated with regulating resistance to Verticillium wilt. GhHSL1 The gene, whose nucleotide sequence is shown in SEQ ID NO. 1, and whose encoded protein sequence is shown in SEQ ID NO. 2.

[0008] A cotton Verticillium wilt resistance-related GhHSL1 The nucleotide sequence of this gene in the upland cotton standard line TM-1 is shown in SEQ ID NO. 1. The expression level of this gene is positively correlated with cotton Verticillium wilt resistance, and overexpression in upland cotton is shown to be positive. GhHSL1 The gene can significantly reduce susceptibility to Verticillium wilt infection, lower the disease index, alleviate vascular bundle darkening, and increase gossypol content; in upland cotton, it reduces... GhHSL1 Gene expression can significantly enhance susceptibility to Verticillium wilt infection, increase disease index, increase vascular bundle darkening, and reduce gossypol content.

[0009] A second aspect of the present invention provides a method described above. GhHSL1 Application of genes in enhancing resistance to Verticillium wilt in upland cotton, improving the aforementioned resistance in upland cotton. GhHSL1 Gene expression enhances resistance to Verticillium wilt in upland cotton. For example, overexpression of the gene shown in SEQ ID NO. 1... GhHSL1 Application of genes in enhancing the resistance of upland cotton to Verticillium wilt or in breeding new cotton varieties resistant to Verticillium wilt.

[0010] A third aspect of the invention provides an improvement over the aforementioned GhHSL1 Application of gene-expressing biological materials in enhancing Verticillium wilt resistance in upland cotton and / or in breeding new Verticillium wilt-resistant cotton germplasm.

[0011] Furthermore, the biomaterial comprises at least one of the following: a) capable of enabling the... GhHSL1 a) an expression cassette with significantly upregulated genes; b) a recombinant vector having the function described in a); c) a recombinant bacterium having the function described in a) or a recombinant vector described in b).

[0012] A fourth aspect of the present invention provides a method for enhancing resistance to Verticillium wilt in upland cotton by upregulating or overexpressing the expression shown in SEQ ID NO. 1 in cotton. GhHSL1 Genes that enhance resistance to Verticillium wilt in upland cotton.

[0013] Furthermore, in accordance with the aforementioned GhHSL1 The gene is used as a template gene and is overexpressed in cotton through Agrobacterium-mediated plant transformation. GhHSL1 The gene can enhance cotton's resistance to Verticillium wilt or be used in the breeding of new cotton varieties with enhanced resistance to Verticillium wilt.

[0014] The beneficial effect of this invention is that it upregulates or overexpresses [certain substances] in cotton. GhHSL1 This invention can enhance cotton's resistance to Verticillium wilt by reducing its susceptibility to infection, lowering the disease index, mitigating the darkening of vascular bundles, and increasing gossypol content. This invention lays the foundation for establishing new Verticillium wilt-resistant upland cotton germplasm and has significant practical implications for both basic scientific research and future agricultural applications. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 This invention provides a gene silencing genome (TRV2: GhHSL1 ) and control group (TRV2: 00 Analysis of cotton plant phenotype and related genes; among which, Figure 1 A in the figure represents the positive control. Two weeks after cotton plants were injected with Agrobacterium, TRV2: CLA Leaf whitening indicates that the silencing efficiency of this experiment was good; Figure 1 B in the figure represents the overall phenotypic difference between the gene silencing group and the control group cotton plants injected with Agrobacterium tumefaciens two weeks after inoculation with Verticillium dahliae V991; Figure 1 C in the figure represents the disease index statistics of cotton plants in the gene silencing group and the control group after two weeks of injection with Agrobacterium tumefaciens and then inoculation with Verticillium dahliae V991. Figure 1 In the diagram, D represents the root, stem, and leaf tissues of cotton plants from the gene silencing group and the control group two weeks after injection of Agrobacterium. GhHSL1 The relative expression levels of genes were detected using RT-qPCR. Figure 1 In the figure, E represents the gossypol content in the root, stem, and leaf tissues of cotton plants in the gene silencing group and the control group two weeks after injection of Agrobacterium; where ns, P > 0.005 ***, P < 0.001; ****, P < 0.0001; t - test.

[0017] Figure 2 This invention is an overexpression GhHSL1 Phenotypic analysis of transgenic cotton following gene modification; among which... Figure 2 In the diagram, A represents the PCR identification of overexpressing transgenic lines; Figure 2 In the diagram, B represents the transgenic receptor TM-1 and one of the 14 selected transgenic genes. GhHSL1In gene-positive lines GhHSL1 The relative expression levels of genes were detected using RT-qPCR. Figure 2 In the figure, C represents the period from 12 to 144 hours after inoculation of *Verticillium dahliae* V991 into *Upland Cotton* TM-1. GhHSL1 Changes in gene expression levels; Figure 2 In this context, D represents the transgenic receptor TM-1. GhHSL1 Disease index statistics of transgenic line OE-9 with overexpression of gene and germplasm Hai7124 resistant to Verticillium wilt after inoculation with Verticillium dahliae V991 at the two-leaf-one-heart stage; Figure 2 In this context, E represents the transgenic receptor TM-1 and... GhHSL1 Determination of gossypol content in transgenic lines OE-5, OE-9, and OE-10 with overexpression of genes; Figure 2 In this context, F represents the transgenic receptor TM-1. GhHSL1 The vascular tissue lesions of the transgenic lines OE-9 (overexpressing gene) and Hai7124 (resistant to Verticillium dahliae) were observed 30 days after inoculation with Verticillium dahliae V991 at the two-leaf-one-heart stage. The oblique sections of the stem segments 1 cm above the cotyledonary nodes of each material were examined under a stereomicroscope. P < 0.01; ***, P < 0.001; ****, P < 0.0001; t - test. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In the following examples, the upland cotton standard series TM-1 is preserved in our laboratory.

[0020] In the following embodiments, the VIGS vectors pTRV2 and pTRV1 can be obtained commercially, and TRV2: CLA This vector was used to construct a pTRV2 vector that silences the CLA gene. It is a commonly used positive control group in this field. Silencing the CLA gene causes the new leaves of positive plants to turn white.

[0021] Example 1: GhHSL1 Obtaining gene sequences A gene that was significantly differentially expressed in the context of changes in secondary metabolites was identified through transcriptomics and metabolomics analysis. GhHSL1The nucleotide sequence of the gene is shown in SEQ ID NO.1. The gene encodes a leucine-rich repeat receptor protein kinase, which belongs to the LRR-RLK family. The encoded amino acid sequence is shown in SEQ ID NO.2.

[0022] right GhHSL1 Gene cloning is performed as follows: (1) Cotton planting Seeds of the standard upland cotton line TM-1 were sown in seedling trays with a nutrient substrate ratio of nutrient soil: vermiculite: perlite = 1:1:1. The seedlings were placed in a light-in-light / dark cultivation room with 16 hours of light and 8 hours of darkness, and watered as needed to maintain a certain level of soil moisture.

[0023] (2) RNA extraction Root tissue samples were taken as described above. A grinding rod was placed in a mortar, and 3–4 mL of anhydrous ethanol was added and ignited to inactivate RNase. The mortar was then rapidly cooled with liquid nitrogen. The root sample was then added and rapidly ground in liquid nitrogen until a powder without particles was formed. The powder was then quickly transferred to 1.5 mL RNase-free centrifuge tubes and stored again in liquid nitrogen or at -80°C for long-term preservation. Total RNA extraction from cotton was performed according to the instructions of the SpectrumPlant Plant Total RNA Extraction Kit (Sigma-Aldrich, DE). Total RNA was stored at -80°C for subsequent analysis.

[0024] (3) Obtaining cDNA via reverse transcription Reverse transcription was performed using ABclonal reverse transcriptase. RNA template denaturation facilitates the opening of secondary structures, significantly increasing the yield of first-strand cDNA. The reverse transcription reaction system is shown in Table 1 below. The reverse transcription conditions were: 37 °C for 2 min, 55 °C for 15 min, and 85 °C to terminate the reaction. The reverse transcription product can be used immediately for subsequent experiments or stored at -20 °C and used within six months, avoiding repeated freeze-thaw cycles.

[0025] Table 1 Reverse transcription reaction system

[0026] (4) GhHSL1 Amplification of the full length of the gene Based on the principle of homologous recombination, amplification primers were designed using Primer 5.0 to ligate the target gene into the vector pT2-shp53-GFP4. Xba Ⅰ~ BamHBetween the restriction enzyme sites, the 5' end of both upstream and downstream primers is pre-added with the restriction enzyme site and the corresponding 15-base vector fragment before or after that site in the respective vector to ensure the correct orientation when the target fragment is inserted into the vector. Primers are generally around 40 bp in length and have a GC content of 40%-60%. The primers designed in this example are as follows: Upstream primer (SEQ ID NO. 3): 5'-gaacgatagggtacccccgggATGTCTAAAACCCGGTTCACATTT -3'; Downstream primer (SEQ ID NO. 4): 5'- gcccttgctcaccatggatccAACTCTTTCCTCGGATCCCTCT -3'; Fragment PCR amplification was performed using diluted primers and cDNA obtained in step (3). The PCR amplification system is shown in Table 2.

[0027] Table 2 PCR amplification system

[0028] The PCR program was as follows: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 10 sec, 59 °C annealing for 5 sec, 68 °C extension for 1-10 sec / kb, 25-35 cycles; 68 °C final extension for 5 min.

[0029] (5) Recovery and purification of PCR amplification products PCR products were separated by 1% agarose gel electrophoresis. The product recovery procedure was performed according to the instructions of the TianSai Gel / PCR Purificationkit kit. The concentration and quality of the recovered products were determined using a NanoDrop One 2000 spectrophotometer.

[0030] (6) Homologous recombination reaction Using the ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., a homologous recombination reaction system was prepared by combining the linearized pT2-shp53-GFP4 vector with the recovered PCR product, as shown in Table 3. The reaction was carried out at 37 ℃ for 30 min. The recombination product was used immediately or stored at -20 ℃.

[0031] Table 3 Homologous recombination reaction system

[0032] (7) Transformation of the recombinant product into competent E. coli DH5α cells, sequencing and sequence analysis, including the following steps: (a) Add 5-10 μL of the recombinant product to be transformed into 50-100 μL of competent cells, gently tap to mix, and incubate on ice for 30 min.

[0033] (b) Place the centrifuge tubes in a 42 °C water bath for 90 sec for heat shock.

[0034] (c) Add 700 μL of antibiotic-free LB liquid medium to a centrifuge tube and incubate at 37°C and 200 rpm for 45 min / h.

[0035] (d) Centrifuge the incubated bacterial culture at 3500 rpm for 5 min, discard the supernatant, resuspend the bacterial pellet in 100 μL of non-resistant LB liquid medium, and transfer it to LB solid medium containing kanamycin (added at a ratio of 1:1000) and spread it evenly.

[0036] (e) After the bacterial culture on the surface of the plate has been absorbed, seal the culture dish and invert it at 37 ℃ for 12-16 h.

[0037] (f) Use a sterilized toothpick to pick a single colony and place it in 600 μL of antibiotic liquid LB medium. Incubate at 37 ℃ and shake at 200 rpm for 6-8 h. Then, take 1 μL of the bacterial solution for PCR reaction. Send the PCR-positive bacterial solution to Qingke Biotechnology for Sanger sequencing. GhHSL1 The reference nucleotide sequence of the gene is shown in SEQ ID NO. 1. The measured sequences are compared, and plasmids and recombinant bacterial cultures that are completely identical can be stored in an ultra-low temperature freezer for subsequent experiments.

[0038] Example 2: Silence GhHSL1 Application of genes in reducing resistance to cotton Verticillium wilt Virus-induced gene silencing (VIGS) is a technique for studying gene function, capable of inducing endogenous gene silencing in contemporary plants. This example utilizes VIGS technology to... GhHSL1 Functional studies of the genes were conducted to verify the role of the target genes in the response to cotton Verticillium wilt, providing a scientific basis for cotton breeding and genetic improvement, as detailed below: (1) GhHSL1 Construction of the VIGS gene vector Based on the target gene GhHSL1 Based on the CDS sequence information, a specific silencing target sequence was designed, and suitable restriction enzyme sites (EcoRI and BamHI) were added according to the target sequence and the information on the pTRV2 vector. TM-1 leaf tissue cDNA was used as the amplification template for specific amplification. The specific amplification system is shown in Table 4, and the primers used are as follows: Forward primer (SEQ ID NO. 5): 5'-gtgagtaaggttaccgaattcATGTCGCCATGATTGTACCACTT -3'; Reverse primer (SEQ ID NO. 6): 5'- cgtgagctcggtaccggatccAACACCAATGAGATTATCATCTACTAAATT -3'; Table 4 Specific amplification system

[0039] Following Example 1, the PCR product was recovered, the pTRV2 vector was linearized, recombinantly transformed into *E. coli*, and sequenced. The plasmid with correct sequencing was identified as TRV2. GhHSL1 Store in a refrigerator at -20 ℃ for later use.

[0040] (2) Transformation of Agrobacterium Plasmid transformation was performed using Agrobacterium competent cells GV3101 from Weidi Biotechnology. The specific steps are as follows: (a) Take the competent Agrobacterium cells stored at -80 °C and place them on ice to thaw.

[0041] (b) Add 0.01-1 μg TRV2 per 100 μL of Agrobacterium competent cells: GhHSL1 Mix the plasmid gently by stirring the bottom of the tube, let it stand on ice for 5 min, then flash freeze in liquid nitrogen for 5 min, then in a water bath at 37°C for 5 min, and finally in an ice bath for 5 min.

[0042] (c) Add 600 μL of antibiotic-free LB liquid medium and incubate at 28 °C with shaking for 2-4 h.

[0043] (d) Centrifuge at 3500 rpm for 5 min, discard the supernatant, resuspend the bacterial pellet in 100 μL of LB liquid medium without resistance, and spread it on LB solid medium containing Kan / Rif resistance. Incubate upside down at 28 ℃ for 2-3 days.

[0044] (3) Agrobacterium-mediated VIGS infection of cotton Will contain TRV2: GhHSL1 TRV2: CLA The recombinant bacteria containing pTRV2 and pTRV1 plasmids were cultured separately in liquid LB medium containing Kan / Rif at 28 ℃ and 220 rpm until OD. 600 Once the bacterial culture reaches 2.0, centrifuge at 4000 rpm for 10 min and discard the supernatant; resuspend the bacterial cells in an equal volume of suspension (10 mM MgCl2, 100 mM MES, 200 μM AS) until the OD value is reached.600 After standing at 28 ℃ for 3 hours, TRV2: (The value is 2.0). GhHSL1 TRV2: CLA The pTRV2 empty suspension and the pTRV1 suspension were mixed at a 1:1 volume ratio. The mixed bacteria were then injected into the cotyledons of cotton plants of two leaves and one bud, until the cotyledons were completely saturated. The injection contained TRV2: GhHSL1 The cotton boll containing the bacteria was a gene-silenced genome, and was injected with TRV2: CLA The cotton seedlings containing the pTRV2 empty vector served as the positive control group, while those injected with the pTRV2 empty vector served as the control group. After injection, the cotton seedlings were cultured overnight in the dark at 22 °C, followed by normal light / dark culture at 23 °C for 16 h / 8 h. Two weeks later, the new true leaves in the positive control group showed whitening, demonstrating the good silencing efficiency of this batch of VIGS injection and the effectiveness of this technical system.

[0045] (4) TRV2: GhHSL1 Gene silencing efficiency detection RNA was extracted from different tissues of cotton in the control and experimental groups according to Example 1, and cDNA was obtained by reverse transcription. Real-time quantitative PCR (RT-qPCR) was performed using quantitative upstream and downstream primers (SEQ ID NO. 7 and SEQ ID NO. 8) to detect TRV2. GhHSL1 Gene silencing.

[0046] Design specific primers: Forward primer (SEQ ID NO. 7): 5'-TCCTGTTTCCTTGGTTATTGTTG -3'; Reverse primer (SEQ ID NO. 8): 5'-GAGGTCAGCTTCCATGTTTCT-3'; RT-qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix kit purchased from Novizan Pharmaceuticals in Nanjing. The specific procedures are as follows: (a) Melt all reagents on ice and mix thoroughly, then prepare the following systems in a 96-well fluoropolymer plate as shown in Table 5: Table 5 qPCR reaction system

[0047] (b) When adding samples, please note: dilute the DNA obtained from reverse transcription to 200 ng / μL. The gene expression level of each cDNA sample needs to be performed in 3 technical replicates. At the same time, the internal reference gene (e.g., 8991) of the cDNA sample also needs to be performed in 3 technical replicates.

[0048] (c) The qRT-PCR reaction program was as follows: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s, 60 °C annealing for 15 s, 72 °C extension for 30 s, for 40 cycles, followed by a 72 °C extension for 10 min. Finally, starting from 60 °C, the temperature was increased to 95 °C in increments of 0.5 °C, with each temperature held for 1 s, to plot the melting curve.

[0049] (d) Analysis was performed on a real-time RT-PCR instrument (ABI StepOnePlus system). Based on the exported Ct values ​​for each well, technical replicates and data standardization were performed. First, the ΔCt for each sample technical replicate (experimental group and control group) was calculated as Ct = target gene - Ct internal reference gene. The average of the ΔCt values ​​from the three replicates was taken. Then, ΔΔCt was calculated as ΔΔCt = ΔCt experiment - ΔCt control. Finally, the relative expression level was obtained as 2^(-ΔΔCt).

[0050] RT-qPCR test results are as follows Figure 1 As shown in D, different tissues in the gene-silencing group of cotton plants GhHSL1 The gene expression level was significantly lower than that of the control group, indicating that the silencing effect was good.

[0051] (5) TRV2: GhHSL1 Determination of gossypol content in plants Confirmed in TRV2: GhHSL1 In the plant GhHSL1 After the genes were indeed silenced, samples were taken separately. GhHSL1 Root (after washing away soil from the roots with clean water), stem (about 3-4 cm from the bottom of the second stem segment), and leaf (second true leaf) tissues from gene-silenced plants were sent to Nanjing Cavens Testing Technology Co., Ltd. for gossypol content determination. The test results are as follows: Figure 1 As shown in E, the gossypol content in different tissues of cotton plants in the gene-silenced group was lower than that in the control group, indicating that gene silencing... GhHSL1 Genes can reduce the gossypol content in cotton.

[0052] (6) TRV2: GhHSL1 Verticillium wilt resistance identification in plants For analysis GhHSL1 To determine whether the gene is involved in the formation of resistance to Verticillium wilt in cotton, the silencing gene was followed by inoculation with Verticillium dahliae V991, and resistance performance was observed and statistically analyzed 20 days later. The specific methods for Verticillium wilt activation and inoculation, and for disease index statistical analysis, are as follows: (a) Activation of Verticillium dahliae spores: Frozen Verticillium dahliae V991 spore solution was removed from a -80 °C freezer, thawed, and 100 μL was spread onto PDA solid medium. After the medium was dried, it was inverted and placed in a 25 °C incubator. When white mycelia covered the medium, a mycelial block was cut off with a sterilized toothpick and placed in 200 mL of sterilized Czapek's medium. The medium was then incubated on a shaker at 25 °C and 200 rpm for 4 days. The spore concentration was then determined and diluted to 6 × 10⁻⁶. 7 per mL.

[0053] (b) Verticillium wilt inoculation: Cotton seedlings were subjected to gene silencing treatment. When the second true leaf of the control TRV2:CLA showed reticulate whitening of the leaf veins, inoculation with Verticillium wilt began. Seedlings with similar growth stages were selected, and the bottoms of disposable paper cups were removed. 20 mL of an appropriate concentration of spore solution was poured into an empty disposable paper cup, and the seedlings with the bottoms removed were slowly placed into the paper cup, allowing the spore solution to completely saturate the cotton roots. The seedlings were cultured in an artificial climate chamber, and watered uniformly on the 4th day.

[0054] (c) Disease Index Statistics: The severity of cotton disease can be divided into 5 levels based on the leaf growth status. Disease incidence is generally assessed 7 days after cotton seedlings are inoculated with Verticillium wilt spores. Level 0: Cotton leaves are intact, showing no disease characteristics. Level 1: 0-25% of leaves show yellowing or other symptoms. Level 2: 25%-50% of leaves show symptoms. Level 3: 50%-75% of leaves show symptoms. Level 4: 75%-100% of leaves show symptoms or the entire plant dies completely. The formula for calculating the disease index is as follows:

[0055] The results are as follows Figure 1 As shown in AC, where TRV2: CLA As a positive control group, carrying TRV2: 00 Plants with empty vectors exhibited typical symptoms of Verticillium wilt, including marked yellowing and wilting of leaves and stunted growth, and were associated with TRV2: 00 Compared to the leaf, TRV2: GhHSL1 The lesions formed on plants infected with the necrotic fungus *Verticillium dahliae* were significantly larger, and the disease index was also significantly higher than that of the control group, indicating that the silencing agent was present. GhHSL1 It can significantly reduce the resistance of cotton to Verticillium dahliae, the pathogen of Verticillium wilt.

[0056] Example 3 Overexpression GhHSL1 Application of genes in improving resistance to cotton Verticillium wilt (1) GhHSL1 Creation of overexpression plants and positive identification To create GhHSL1Gain-of-function materials, using upland cotton TM-1 as the acceptor, were constructed to carry AADA resistance markers driven by the 35S promoter. GhHSL1 The overexpression vector was used, and 20 independent T0 generation overexpression lines (OE-1 to OE-20) were obtained through genetic transformation.

[0057] To identify positive transformants, the vector plasmid and TM-1 genomic DNA were used as positive and negative controls, respectively. PCR detection of the AADA marker gene was performed on the leaf DNA of the strains, as shown in Example 1. Agarose gel electrophoresis results are as follows. Figure 2 As shown in A, some strains showed a band at the expected position (3000bp) consistent with the positive control, and were preliminarily identified as transgenic positive strains.

[0058] Further detection of positive strains by RT-qPCR GhHSL1 The transcriptional level was determined using the method described in Example 2. The results were as follows: Figure 2 As shown in B, compared with wild-type TM-1, most positive lines... GhHSL1 Expression levels were significantly increased, but there were differences among different strains, with the OE-5, OE-9 and OE-10 strains showing the most significant upregulation.

[0059] The above results confirm that the desired result was obtained. GhHSL1 Overexpressing plants can be screened to identify lines with high expression levels for subsequent disease resistance identification.

[0060] (2) GhHSL1 Determination of gossypol content in overexpressed plants Stable overexpression was confirmed. GhHSL1 After different cotton lines, samples were taken respectively. GhHSL1 Leaf tissues from three overexpression lines (OE-5, OE-9, and OE-10) with high expression levels, with three biological replicates for each line, were sent to Nanjing Cavens Testing Technology Co., Ltd. for gossypol content determination. The results are as follows: Figure 2 As shown in E, three GhHSL1 The gossypol content in the leaves of the overexpressing strains was significantly higher than that in the control group, indicating that... GhHSL1 Overexpression can increase the gossypol content in cotton.

[0061] (3) GhHSL1 Identification of Verticillium wilt resistance in overexpressed plants For in-depth identification GhHSL1 The function of genes in cotton Verticillium wilt resistance was studied by selecting upland cotton wild-type TM-1, GhHSL1Using the overexpression line OE-9 and the resistant strain Hai7124 as materials, Verticillium dahliae V991 was inoculated at the two-leaf-one-heart stage to systematically evaluate their disease resistance phenotype. The specific methods for Verticillium wilt activation and inoculation, and disease index statistics, are as shown in Example 2. Thirty days after inoculation, stem sections were dissected for identification. A slant section was made 1 cm above the cotyledon node, and the vascular tissue lesions were observed under a stereomicroscope.

[0062] The results are as follows Figure 2 As shown in D and F, GhHSL1 The disease index of the overexpressing plants was significantly lower than that of the control group, and the degree of vascular browning was also significantly milder, indicating a significant reduction in disease severity. GhHSL1 Overexpression can inhibit the spread of pathogens in the plant's vascular system and significantly improve cotton's resistance to Verticillium dahliae, the pathogen of Verticillium wilt.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. GhHSL1 The application of genes in regulating resistance to Verticillium wilt in upland cotton is characterized by, The GhHSL1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the protein sequence it encodes is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, overexpression GhHSL1 Genes can enhance resistance to Verticillium wilt in upland cotton; silencing GhHSL1 Genes can reduce resistance to Verticillium wilt in upland cotton.

3. The application according to claim 1, characterized in that, The overexpression GhHSL1 Genes can improve resistance to Verticillium wilt in upland cotton by: constructing a gene containing... GhHSL1 Gene expression cassettes, recombinant expression vectors, recombinant host cells or recombinant bacteria are transferred into upland cotton to improve its resistance to Verticillium wilt.

4. The application according to claim 1, characterized in that, The silence GhHSL1 The gene can reduce the resistance of upland cotton to Verticillium wilt by: using primers to amplify the VIGS fragment and then using it to induce viral gene silencing in upland cotton, thereby reducing the resistance of upland cotton to Verticillium wilt. GhHSL1 Gene expression was used to reduce the resistance of upland cotton to Verticillium wilt.

5. The application according to claim 1, characterized in that, The primers include an upstream primer VIGS-F and a downstream primer VIGS-R. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

6.

6. The application according to any one of claims 1-5, characterized in that, Verticillium wilt is a plant disease caused by Verticillium dahliae.

7. A method for improving plant resistance to Verticillium wilt, characterized in that, include: a) Constructing a structure containing GhHSL1 Gene overexpression recombinant plant expression vectors; b) containing GhHSL1 Gene overexpression recombinant plant expression vectors transform plants, enabling GhHSL1 Genes are overexpressed in plants, thereby enhancing the plant's resistance to Verticillium wilt; The plant in question is upland cotton; the Verticillium wilt is a plant disease caused by Verticillium dahliae.

8. A method for cultivating plant varieties resistant to Verticillium wilt, characterized in that, include: a) Constructing a structure containing GhHSL1 Gene overexpression recombinant plant expression vectors; b) containing GhHSL1 Transformation of plants using gene overexpression plant expression vectors enables... GhHSL1 Genes were overexpressed in plants to obtain transgenic positive plants, and plant varieties with improved resistance to Verticillium wilt were screened. The plant in question is upland cotton; the Verticillium wilt is a plant disease caused by Verticillium dahliae.

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