Application of GhWAKL37 gene in plant regulation of resistance to fusarium wilt

Abstract

The application provides a kind of GhWAKL37 The application of the gene in regulating the resistance of plants to verticillium wilt belongs to the technical field of genetic engineering. GhWAKL37 The gene plays a positive regulation role in the process of plant resistance to verticillium wilt, GhWAKL37 positively regulates the defense response of plants to verticillium wilt, GhWAKL37 The performance of plants to verticillium wilt is reduced after gene silencing or knockdown, GhWAKL37 The gene provides an important theoretical basis and gene resource for improving the resistance of plants to verticillium wilt and creating disease-resistant germplasm.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and particularly relates to... GhWAKL37 Application of genes in regulating plant resistance to Verticillium wilt. Background Technology

[0002] Cotton, as an important economic crop, directly impacts agricultural economic benefits and the development of the textile industry. Verticillium wilt, a significant soil-borne disease caused by Verticillium dahliae, is characterized by its wide spread and severe damage, significantly reducing cotton yield and fiber quality, and has become a major factor restricting the development of the cotton industry. Currently, production mainly relies on integrated management of this disease through chemical control, biological control, cultivation regulation, and the breeding of disease-resistant varieties. Among these, breeding disease-resistant varieties is widely considered the fundamental approach to controlling Verticillium wilt due to its economic advantages and sustainable development. However, research on the mechanisms of cotton disease resistance remains relatively weak, limiting related disease-resistant breeding efforts. Therefore, in-depth exploration of the molecular mechanisms of cotton disease resistance is of significant theoretical and practical value for improving the efficiency of disease-resistant breeding and cultivating superior disease-resistant varieties. The infection and resistance mechanisms of pathogens to plants have always been a research hotspot in the field of plant immunity.

[0003] Cell wall-associated receptor kinase-like proteins (WAKLs) are plant-specific transmembrane proteins, primarily located in the cell membrane. They sense extracellular signals and transmit information intracellularly, thus participating in plant growth, development, and responses to abiotic stress. Most WAKLs possess the typical structure of WAKs, including an EGF domain, a transmembrane domain, an intracellular serine / threonine kinase domain, and two introns with conserved insertion sites. Some genes lack certain sequences; for example, WAKL7 lacks both the transmembrane and kinase domains. Although the WAKL family has many members, their functions are highly differentiated, with different proteins within the same family exhibiting varying and unpredictable effects. For instance, at the mRNA level, WAKLs, such as WAKL1, WAKL3, and WAKL5, are highly expressed in Arabidopsis roots and flowers. WAKL expression also varies at different developmental stages; for example, WAKL2 and WAKL4 are expressed in developing Arabidopsis seeds but not in mature seeds, while WAKL5 is expressed only in young leaves. Of course, there are also opposite cases, such as WAKL6 expression being higher in mature leaves than in young leaves. These different gene expression patterns suggest that different WAKL members may have different functions in different parts of the plant or at different developmental stages. Therefore, providing a WAKL gene for plant resistance to Verticillium wilt, especially Verticillium wilt caused by Verticillium dahliae infection, is an urgent technical problem to be solved. However, to date, GhWAKL37 No studies have been reported on the role of genes in disease resistance in plants. Summary of the Invention

[0004] Therefore, the object of the present invention is to provide a GhWAKL37 Application of genes or related materials in regulating plant resistance to Verticillium wilt.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides GhWAKL37 The application of genes in (1) and / or (2), (1) Regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing... GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

[0006] This invention provides GhWAKL37 Application of gene-related biomaterials in (1) and / or (2), (1) regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing... GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

[0007] Preferably, the Verticillium wilt is caused by Verticillium dahliae.

[0008] Preferably, the GhWAKL37 gene-related biological material is a GhWAKL37 gene overexpression biological material or a GhWAKL37 gene knockdown or silencing material; The GhWAKL37 gene knockdown or silencing material is a biological material containing CRISPR / Cas9 or a VIGS vector. The CRISPR / Cas9-containing biological material includes sgRNA1 and sgRNA2, the sequence of which is shown in SEQ ID NO.13; the sequence of which is shown in SEQ ID NO.14. The biomaterials are carriers, host bacteria, plant tissues, plant cells, or plant organs.

[0009] This invention provides an overexpression GhWAKL37 The application of the gene in cotton or Arabidopsis thaliana in resistance to Verticillium dahliae infection, wherein the amino acid sequence of the protein encoded by the GhWAKL37 gene is shown in SEQ ID NO.8.

[0010] Preferred, GhWAKL37 The coding region sequence of the gene is shown in SEQ ID NO.7.

[0011] This invention provides a cotton or Arabidopsis thaliana resistant to Verticillium wilt, wherein the cotton or Arabidopsis thaliana overexpresses the above-mentioned... GhWAKL37 Gene.

[0012] This invention provides a method for preparing plants resistant to Verticillium wilt, comprising the following steps to improve the quality of recipient plants. GhWAKL37 Gene expression levels and activity were obtained. GhWAKL37 Genetically modified plants; The recipient plants are cotton and / or Arabidopsis thaliana.

[0013] Preferably, the Verticillium wilt is caused by Verticillium dahliae.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides GhWAKL37 Application of genes or related materials in regulating plant resistance to Verticillium wilt. The study found that... GhWAKL37 Genes play a positive regulatory role in plant resistance to Verticillium wilt. GhWAKL37 Positive regulation of plant defense responses to Verticillium wilt, GhWAKL37 Gene silencing or knockdown reduces the plant's resistance to Verticillium wilt. GhWAKL37 Genes provide important theoretical basis and genetic resources for improving plant resistance to Verticillium wilt and creating new disease-resistant germplasm. In addition, breeding plants with reduced resistance to Verticillium wilt can be used as disease-susceptible plant models for mechanistic studies and disease efficacy testing. Attached Figure Description

[0015] Figure 1 for GhWAKL37 Gene structure analysis.

[0016] Figure 2 for GhWAKL37 Distribution of Arabidopsis protoplasts.

[0017] Figure 3 After *Verticillium dahliae* infects cotton, at different infection times... GhWAKL37 The results of the expression level analysis are shown, with blue representing the result after treatment and yellow representing the result before treatment.

[0018] Figure 4 for GhWAKL37The results of VIGS silencing efficiency analysis are shown in Figure A, where A represents the phenotype of cotton seedlings infected with TRV::CLA strain, and B represents the qRT-PCR detection. GhWAKL37 Genes in control and GhWAKL37 Relative expression levels in silent plants. Data are the mean ± standard deviation of three biological replicates. This indicates that P < 0.01 (t-test).

[0019] Figure 5 Results of the effect of GhWAKL37 silencing on the susceptibility of cotton to Verticillium dahliae.

[0020] Figure 6 The results of the analysis of cotton disease resistance-related indicators are shown. A represents the TRV::00 control group and TRV:: GhWAKL37 Phenotypic characteristics of stem vascular tissue in the silent group after inoculation with *Verticillium dahliae*; B represents the TRV::00 control group and TRV:: GhWAKL37 Biomass of *Verticillium dahliae* on PDA medium under a silent group background; C represents the TRV::00 control group and TRV:: GhWAKL37 Statistical analysis of the disease index in the silent group; D represents TRV::00 control group and TRV:: GhWAKL37 Quantitative analysis of relative fungal biomass in cotton plants. This indicates a highly significant difference at the P<0.01 level (t-test); error bars represent the standard deviation (SD) of three biological replicates.

[0021] Figure 7 For heterologous expression GhWAKL37 Molecular identification of Arabidopsis thaliana lines, A represents the detection of wild-type and heterologous expression by qRT-PCR. GhWAKL37 Arabidopsis plants GhWAKL37 B represents the expression level of wild-type and heterologous expression, analyzed using semi-quantitative RT-PCR. GhWAKL37 Arabidopsis plants GhWAKL37 The transcription status was monitored, with AtHis3 serving as an internal reference gene.

[0022] Figure 8 To enhance Arabidopsis resistance to Verticillium dahliae through heterologous expression of GhWAKL37, A shows the plant phenotypes of wild-type (WT) and GhWAKL37 heterologous expression lines (OE1, OE2, OE3) before inoculation with Verticillium dahliae (0 dpi) and 14 days after inoculation (14 dpi); B shows the statistical analysis of disease index between wild-type and heterologous expression lines 14 days after inoculation; C shows the quantitative analysis of relative fungal biomass between wild-type and heterologous expression lines 14 days after inoculation. This indicates a highly significant difference at the P<0.01 level (t-test); error bars represent the standard deviation of three biological replicates.

[0023] Figure 9 for GhWAKL37 Molecular identification of cotton plants overexpressing the vector, A represents the detection of wild-type (WT), empty vector control (NULL), and [other plants] using semi-quantitative RT-PCR. GhWAKL37 Overexpression lines (OE-1, OE-2, OE-3) GhWAKL37 The expression situation, GhHis3 B is used as an internal reference gene; B is used for quantitative detection of the gene in each line by qRT-PCR. GhWAKL37 The relative expression level. Three biological replicates were set up for each sample. The p-value represents a highly significant difference at the 0.01 level (t-test), and the error bars represent the standard deviation (SD) of biological replicates.

[0024] Figure 10 for GhWAKL37 Overexpression enhances cotton resistance to Verticillium wilt. A shows the disease phenotype of wild-type (WT), empty vector control (Null), and GhWAKL37 overexpressing cotton plants at different time points (0 dpi, 7 dpi, 14 dpi, 21 dpi, 28 dpi) after inoculation with Verticillium dahliae, scale bar = 1 cm; B shows the symptoms of vascular tissue in the stems of cotton plants of various lines after inoculation with Verticillium dahliae, scale bar = 5 mm. Figure 11 for GhWAKL37 Analysis of disease resistance-related indicators in cotton overexpression: A shows the disease index statistics at different time points (0 dpi, 7 dpi, 14 dpi, 21 dpi, 28 dpi) after inoculation with *Verticillium dahliae* in wild-type (WT), empty vector control (Null), and GhWAKL37 overexpressing cotton (OE-1, OE-2, OE-3); B shows the relative fungal biomass of each strain at different time points after inoculation, quantitatively detected by qRT-PCR. Data are obtained from three biological replicates, each containing 20 cotton plants; error bars represent the standard deviation (SD) of the three biological replicates. This indicates a highly significant difference at the P<0.01 level. This indicates a significant difference at the P<0.05 level.

[0025] Figure 12 for GhWAKL37 Identification of gene knockout cotton, A is GhWAKL37 A schematic diagram of gene knockout vector construction, with the sgRNA target site and PAM sequence labeled; B represents wild-type (WT) upland cotton HM-1 and... GhWAKL37The sequencing results of the gene knockout lines showed the base deletion mutations in the target region.

[0026] Figure 13 for GhWAKL37 Gene knockout cotton exhibits decreased disease resistance; A represents wild-type (WT), empty vector control (Null), and... GhWAKL37 The results of phenotypic identification of disease in gene-edited mutant lines at different time points (0 dpi, 7 dpi, 14 dpi, 21 dpi, 28 dpi) after inoculation with Verticillium dahliae, scale bar = 1 cm; B shows the symptomatic manifestations of the vascular tissue of cotton stems after inoculation, scale bar = 5 mm.

[0027] Figure 14 for GhWAKL37 Analysis of disease resistance-related indicators in gene knockout cotton, where A represents wild-type (WT), empty vector control (Null), and... GhWAKL37 Disease index statistics at different time points (0 dpi, 7 dpi, 14 dpi, 21 dpi, 28 dpi) after inoculation of gene knockout mutant lines with *Verticillium dahliae*; B represents the relative fungal biomass of each line at different time points after inoculation, quantitatively detected by qRT-PCR. Data are obtained from three biological replicates, with each replicate containing 20 cotton plants; error bars represent the standard deviation (SD) of three biological replicates. This indicates a highly significant difference at the P<0.05 level (t-test). Detailed Implementation

[0028] This invention provides GhWAKL37 The application of genes in (1) and / or (2), (1) Regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing... GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

[0029] This invention provides GhWAKL37 Application of gene-related biomaterials in (1) and / or (2), (1) regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing... GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

[0030] This invention provides an overexpression GhWAKL37 The application of the gene in cotton or Arabidopsis thaliana in resistance to Verticillium dahliae infection, wherein the amino acid sequence of the protein encoded by the GhWAKL37 gene is shown in SEQ ID NO.8.

[0031] In this invention, the GhWAKL37 The genome sequence of the gene is accessed in the CottonFGD database under accession number Gohir.D02G17766.1. The genome sequence of GhWAKL37 is 4035 bp in length, with a coding region of 2919 bp. GhWAKL37 The coding region sequence of the gene is shown in SEQ ID NO.7; GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.8. The GhWAKL37 protein contains two important conserved domains: the GUB_WAK_bind domain and a protein kinase domain.

[0032] This invention has found that subcellular localization experiments show that... GhWAKL37 The fluorescence signal corresponding to the protein is mainly concentrated in the plasma membrane region. Real-time quantitative PCR results show that the transcription level of this gene is significantly upregulated after infection with Verticillium dahliae, indicating that its expression is induced by pathogen infection and may be involved in related defense processes. GhWAKL37 Silent plants inoculated with *Verticillium dahliae* exhibited a stronger susceptible phenotype, with significantly decreased resistance, increased disease index, and more severe browning of vascular tissues. Simultaneously, the fungal colonization and relative fungal biomass in the stem segments also significantly increased. This indicates that... GhWAKL37 It participates in the plant's defense response to Verticillium wilt and plays a positive regulatory role in it. Compared with transgenic materials, wild-type plants showed more pronounced leaf yellowing and abscission, indicating that overexpression... GhWAKL37 It can enhance the resistance of Arabidopsis thaliana or cotton to Verticillium wilt.

[0033] In this invention, the regulation is to increase or decrease, or to overexpress... GhWAKL37 When the gene is knocked down, the plant's resistance to Verticillium wilt is increased; knockdown or silencing... GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

[0034] In this invention, the GhWAKL37 Gene-related biomaterials GhWAKL37 Gene overexpression biomaterials or GhWAKL37 Gene knockdown or silencing materials; The GhWAKL37Gene knockdown or silencing materials are biological materials containing CRISPR / Cas9 or VIGS vectors; The CRISPR / Cas9-containing biological material includes sgRNA1 and sgRNA2, the sequence of which is shown in SEQ ID NO.13; the sequence of which is shown in SEQ ID NO.14. The biomaterials are carriers, host bacteria, plant tissues, plant cells, or plant organs.

[0035] In a preferred embodiment, the Verticillium wilt is caused by Verticillium dahliae.

[0036] This invention cultivates plants with reduced resistance to Verticillium wilt, which can be used as susceptible plant models for mechanism research and disease efficacy detection.

[0037] This invention provides a cotton or Arabidopsis thaliana resistant to Verticillium wilt, wherein the cotton or Arabidopsis thaliana overexpresses the above-mentioned... GhWAKL37 Gene.

[0038] This invention provides a method for preparing plants resistant to Verticillium wilt, comprising the following steps to improve the quality of recipient plants. GhWAKL37 Gene expression levels and activity were obtained. GhWAKL37 Genetically modified plants; The recipient plant is cotton or Arabidopsis thaliana.

[0039] Preferably, the Verticillium wilt is caused by Verticillium dahliae.

[0040] In this invention, the VIGS vector is a gene silencing vector induced by tobacco brittle virus (TRV) (TRV-VIGS). The VIGS vector is named TRV::GhWAKL37, and the construction method of TRV::GhWAKL37 includes the following steps: extracting total RNA from cotton root tissue, reverse transcribing to synthesize cDNA, performing PCR amplification using TRV::GhWAKL37-F and TRV::GhWAKL37-R as shown in SEQ ID NO.9~SEQ ID NO.10 respectively, obtaining amplification products, and then processing the TRV::RNA2 plasmid. EcoR I and Sac After double digestion with enzyme I, the enzyme-treated vector was obtained. The amplification product was then ligated with the enzyme-treated vector to obtain TRV::GhWAKL37.

[0041] In this invention, the GhWAKL37 In the gene overexpression biological material, the empty vector of the vector includes pCambia2300-GFP; the host bacteria include Escherichia coli or Agrobacterium, such as Escherichia coli Match1-T1 or Agrobacterium GV3101.

[0042] In this invention, the empty vector of the expression vector in the CRISPR / Cas9-containing biological material is pRGEB32-GhU6.9. The method for preparing the CRISPR / Cas9 system includes digesting the empty vector pRGEB32-GhU6.9 with Bsa I to obtain a linearized vector; fusing sgRNA1 and sgRNA2 via overlap extension PCR to obtain the sgRNA1-sgRNA2 fusion sequence, which is then introduced into the linearized vector to obtain the CRISPR / Cas9 system. This invention utilizes CRISPR / Cas9-containing biological materials or VIGS vectors to significantly reduce the concentration of CRISPR / Cas9 in plant materials. GhWAKL37 Gene expression levels.

[0043] In this invention, the Verticillium wilt is caused by Verticillium dahliae. The Verticillium dahliae is Verticillium dahliae V592.

[0044] This invention provides a cotton or Arabidopsis thaliana resistant to Verticillium wilt, wherein the cotton or Arabidopsis thaliana overexpresses the above-mentioned... GhWAKL37 Gene.

[0045] This invention provides a method for preparing plants resistant to Verticillium wilt, comprising the following steps to improve the quality of recipient plants. GhWAKL37 Gene expression levels and activity were obtained. GhWAKL37 Transgenic plants; the recipient plant is cotton or Arabidopsis thaliana.

[0046] In the above preparation method, as a preferred embodiment, the above... GhWAKL37 Gene or GhWAKL37 Gene overexpression material was introduced into recipient plants to obtain overexpressed genes. GhWAKL37 Plants containing the above-mentioned genes. For example, this can be achieved by introducing the above-mentioned genes into recipient plants. GhWAKL37 This can be achieved through gene expression, by introducing the aforementioned expression vector into the recipient plant, or by transforming the recipient plant with recombinant bacteria.

[0047] In this invention, the Verticillium wilt is caused by Verticillium dahliae, and the Verticillium dahliae is Verticillium dahliae V592.

[0048] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] In the following embodiments, (1) Experimental material varieties: The plant materials used in the study included the Verticillium wilt-resistant upland cotton variety "Zhongzhimian No. 2" (Gossypium hirsutum Cotton seedlings bred from L., and upland cotton variety Huakangmian 1 (HM-1). Among them, the negative control (NULL) is HM-1 plants that have undergone genetic transformation but have not been introduced with foreign genes; GhWAKL37 overexpression and knockout cotton lines all use upland cotton HM-1 as recipient material and have been successfully transformed and stably inherited to the T3 generation.

[0051] Colombian wild type ( Columbia Arabidopsis thaliana (WT) was used as wild-type material, while Nicotiana benthamiana (WT) was used as wild-type material. Nicotiana benthamiana () is used for subcellular localization experiments.

[0052] (2) Planting conditions and treatment sampling of experimental materials: Cotton Cultivation: Plump, high-quality cotton seeds were selected and washed with purified water. The seeds were then soaked overnight in a 37℃ incubator to promote germination, yielding the selected seeds. The next day, the selected seeds were sown in culture pots containing sterilized substrate, a mixture of vermiculite and nutrient soil (purchased from Zhengzhou Weizhan Biotechnology Co., Ltd., DH-018) at a 1:1 (v / v) ratio. After sowing, the seeds were covered with plastic film to maintain suitable humidity. The film was gradually removed to harden the seedlings after emergence. During the growth period, the plants were cultivated in a greenhouse at 28℃, using a natural light environment with alternating 16 hours of light and 8 hours of darkness. Regular watering was provided, and nutrient solution was added as needed based on the plant growth.

[0053] Verticillium wilt treatment experiment: When the plants developed to the two-leaf-one-heart stage, they were inoculated with Verticillium wilt. The control group consisted of plants that had not undergone Verticillium wilt treatment. In the experimental group, the roots of the plants were damaged, and 10 mL of *Verticillium dahliae* spore suspension was inoculated into each culture pot. Samples were collected from seedlings in both the experimental and control groups at 0h, 1h, 3h, 6h, 12h, 24h, 48h, 72h, and 96h after inoculation, and were rapidly flash-frozen in liquid nitrogen to ensure the integrity of the biological samples. The treated samples were then stored in an ultra-low temperature incubator at -80℃ for subsequent experimental analysis.

[0054] (3) Information on the carrier and strain is shown in Table 1.

[0055] Table 1. Carriers and strains

[0056] (4) The main reagents used in the experiment are shown in Table 2.

[0057] Table 2 Main reagents used in the experiment

[0058] (5) Preparation of main solutions, see Table 3 for details.

[0059] Table 3 Preparation of the main solutions

[0060] Example 1 1.1 Experimental Methods 1.1.1 GhWAKL37 Bioinformatics analysis of genes This study first retrieved and obtained data from the CottonFGD database (https: / / cottonfgd.net). GhWAKL37 The gene sequence (database accession number Gohir.D02G17766.1) was obtained. To clarify the structural features and family attributes of the protein encoded by this gene, its protein sequence was imported into the HMMER online analysis platform (https: / / www.ebi.ac.uk / Tools / hmmer / search / hmmscan) for conserved domain prediction.

[0061] Figure 1 The results show that, through the CottonFGD database, it is known that GhWAKL37 The genome sequence is 4035 base pairs (bp) long, with a coding region of 2919 bp, predicted to translate into 973 amino acid residues. Furthermore, gene structure analysis revealed... GhWAKL37 Composed of four exons and three introns, it provides a possible structural basis for its complex gene expression regulation. Scanning results using the HMMER tool indicate that... GhWAKL37 The protein contains two important conserved domains: the GUB_WAK_bind domain and the protein kinase domain.

[0062] 1.1.2 Extraction of total RNA from cotton The RNA extraction kit used was the RN53-EASYspin Plus Complex Polysaccharide and Polyphenol Plant RNA Rapid Extraction Kit (see Table 2). Total RNA from tissues was extracted using a combination of liquid nitrogen grinding and column purification. The specific steps are as follows: (1) After thoroughly cleaning the mortar, pestle and weighing tools, wipe them with a 95% ethanol solution and sterilize them by flame. Let them cool naturally before use.

[0063] (2) Take an appropriate amount of root tissue from the disease-resistant upland cotton variety No. 2 and place it in a pre-cooled mortar. Add liquid nitrogen and grind it thoroughly until it becomes a fine powder. Then, quickly weigh about 0.1g of the tissue powder and transfer it to a pre-cooled 2.0mL enzyme-free centrifuge tube to obtain a cotton tissue sample.

[0064] (3) The lysis buffer CLB needs to be preheated to 65°C and added to mercaptoethanol at a volume percentage of 5% before use to obtain the lysis buffer. Take 1 mL of the lysis buffer and add it to the cotton tissue sample. After thorough shaking and mixing, promote the full lysis of the tissue to obtain a well mixed sample.

[0065] (4) After mixing, the sample was placed in a 65°C water bath for 15 min, and the mixture was inverted intermittently during the incubation to promote nucleic acid release. After incubation, the sample was centrifuged at 13,000 rpm for 10 min, and the supernatant was used for subsequent RNA purification.

[0066] (5) Transfer 700µL of supernatant into a brand new 1.5mL centrifuge tube, add 350µL of anhydrous ethanol, and immediately invert to mix well to obtain mixture 1, so that the nucleic acid can be bound to the subsequent purification medium under suitable conditions.

[0067] (6) Add the mixture 1 to the genome removal column and centrifuge at 13000 rpm for 2 min to remove the genomic DNA from the sample. Discard the waste liquid (if the single loading volume exceeds the column capacity, it can be added in multiple times and centrifuged repeatedly) to obtain a removal column containing genomic DNA.

[0068] (7) Place the genomic DNA removal column into a clean 2.0 mL centrifuge tube, add 500 µL of lysis buffer RLT Plus to the column, centrifuge at 13000 rpm for 1 min, collect the filtrate, add about 250 µL of anhydrous ethanol to the filtrate, and use a pipette to thoroughly mix to ensure uniform mixing, to obtain mixture 2.

[0069] (8) Transfer the above mixture 2 to a new RNA adsorption column, let stand for 1 min, centrifuge at 13000 rpm for 2 min to allow the RNA to bind to the adsorption membrane, and discard the lower layer of filtrate.

[0070] (9) Then add 700µL of RW1 protein removal solution, let stand at room temperature for 2 min, centrifuge at 13000rpm for 3 min, and discard the lower layer filtrate.

[0071] (10) Add 500µL of RW washing solution to the adsorption column of step (9) above, centrifuge at 13000rpm for 2min, discard the lower layer filtrate and repeat the washing once (in the washing step, add an appropriate amount of anhydrous ethanol to the RW washing solution before use according to the instructions).

[0072] (11) To remove residual ethanol as much as possible, the empty column (adsorption column) was centrifuged again at 13000 rpm for 4 min.

[0073] (12) The adsorption column needs to be transferred into a fresh RNase-free centrifuge tube, and then 40µL of elution solution preheated to 65℃ is injected. The tube is placed at room temperature and incubated for 2 minutes to ensure sufficient elution. Then, the tube is centrifuged at 12000rpm for 2 minutes to complete the elution and obtain total RNA from cotton.

[0074] (13) The obtained RNA should be stored at -80℃ immediately.

[0075] 1.1.3 Reverse transcription to synthesize cDNA (suitable for qRT-PCR) Total RNA extracted from cotton (as described in section 1.1.2) was used as a template for reverse transcription to synthesize cDNA for qRT-PCR. The reagents used are listed in Table 2, and the specific steps are as follows: (1) Remove genomic DNA: Prepare the reaction system in the PCR tube according to the formula shown in Table 4.

[0076] (2) After thoroughly mixing the reaction system of step (1), briefly centrifuge and place it in a PCR instrument. React at 42℃ for 2 min to complete the removal of gDNA.

[0077] (3) Place the reacted eight-tube bundle on ice, add 4µL of 5×HiScriptII qRT SuperMixII, and mix gently.

[0078] (4) The reverse transcription program was set as follows: 50℃ for 15 min, 85℃ for 5 s.

[0079] (5) After the reaction is complete, the obtained cDNA product is stored at -20℃ for subsequent qRT-PCR analysis.

[0080] Table 4 Genomic DNA Removal Reaction System

[0081] 1.1.4 Design of primers for real-time quantitative PCR (qRT-PCR) Using the SGN VIGS Tool online platform (https: / / vigs.solgenomics.net / ) to... GhWAKL37 Sequences were screened for specific regions, and a fragment of approximately 200 bp was selected as a primer design template. Subsequently, qRT-PCR specific primers were designed using SnapGene software. GhUBQ7 As an internal reference gene, its primers were used for subsequent quantitative fluorescence analysis. The final quantitative fluorescence primer sequences are shown in Table 5.

[0082] Table 5 Primer sequences for quantitative fluorescence detection

[0083] 1.1.5 Real-time quantitative PCR (qRT-PCR) Real-time quantitative PCR (qRT-PCR) was used for detection. The reagents and consumables used in the experiment are shown in Table 2. GhUBQ7 As an internal reference gene, the amplification reaction of each sample was completed in a 96-well qRT-PCR plate. The qRT-PCR reaction system is shown in Table 6. The amplification program was performed according to the conditions shown in Table 7. Three replicates were set up for each sample to ensure the accuracy and reproducibility of the results.

[0084] Table 6. GhWAKL37 Real-Time PCR Reaction System

[0085] Table 7 qRT-PCR reaction procedure

[0086] 1.1.6 Reverse transcription to synthesize cDNA (suitable for gene cloning) The main reagents used for reverse transcription to synthesize cDNA are shown in Table 2. The main steps are as follows: (1) RNA template pretreatment: Prepare the reaction system as shown in Table 8 in RNase-free centrifuge tubes. After heating the prepared mixture at 65°C for 5 min, quickly place it on ice to cool it down and let it stand on ice for 2 min.

[0087] (2) Genomic DNA removal: Add 4µL of 4×gDNA wiper Mix to the reaction system obtained in step (1). After gently mixing with a pipette, react at 42℃ for 2min to obtain the treated system, thereby eliminating the interference of residual genomic DNA.

[0088] (3) Preparation of reverse transcription reaction system: Add 2µL of 10×RT Mix and 2µL of HiScript IIEnzyme Mix to the treated system, gently mix by pipetting to obtain the first-strand cDNA synthesis reaction system.

[0089] (4) First-strand cDNA synthesis: The first-strand cDNA synthesis reaction system was placed in a PCR instrument and the reverse transcription reaction was completed at 25℃ for 5 min, 50℃ for 45 min, and 85℃ for 2 min.

[0090] (5) Product preservation and use: After the reaction is completed, the obtained cDNA product is diluted 5 times for subsequent experiments, or stored at -20℃ for later use.

[0091] Table 8 RNA template pretreatment reaction system

[0092] 1.1.7 Extraction of DNA from Cotton Plant DNA extraction was performed using appropriate kits, the reagents of which are listed in Table 2. The specific operating procedure is as follows: (1) Preparation of pretreatment buffer: Before use, add β-mercaptoethanol to P1 buffer to make the final concentration reach 0.2%. At the same time, preheat P1 buffer and elution buffer at 65℃ for later use. Add 500 μL of equilibration solution to DNA adsorption column, centrifuge at 12000 rpm for 1 min, discard the lower layer filtrate, and return adsorption column AC to the original collection tube.

[0093] (2) Tissue grinding: Take 5mg of root sample of upland cotton No. 2 and place it in a mortar. Add liquid nitrogen and grind it thoroughly until it becomes fine powder to obtain the ground sample.

[0094] (3) Lysis and RNA removal: Transfer the ground sample to a 1.5 mL centrifuge tube, taking care not to thaw the sample. Add 4 μL RNase A and 550 μL P1 buffer (preheated to 65 °C), vortex vigorously for 1 min, and let stand at room temperature for 10 min to obtain the processed sample.

[0095] (4) Protein and impurity precipitation: Add 130 μL of P2 buffer to the treated sample, vortex thoroughly for 1 min, and then centrifuge at 12000 rpm for 3 min.

[0096] (5) Transfer of supernatant: Carefully aspirate the supernatant into the separation column A, avoiding aspirating the interfacial precipitate. Centrifuge at 12000 rpm for 1 min, collect the lower layer filtrate, and obtain the centrifuged filtrate.

[0097] (6) Condition adjustment: Transfer the obtained centrifuged filtrate to a new 2.0 mL centrifuge tube, add 1.5 times the volume of Buffer P3 according to the filtrate volume, and immediately vortex gently after adding to make the system uniformly mixed to obtain the processed mixture.

[0098] (7) DNA adsorption: Add the above-treated mixture to the adsorption column AC placed in the collection tube, and then centrifuge at 12000 rpm for 1 min to adsorb DNA onto the column membrane. After centrifugation, discard the lower layer of filtrate.

[0099] (8) First cleaning of the column membrane: Add 700 μL of washing solution WB to the adsorption column of step (7), centrifuge at 12000 rpm for 1 min, and discard the lower layer filtrate.

[0100] (9) Second wash: Add 500 μL of WB washing solution to the adsorption column of step (8) again, and treat at 12000 rpm for 1 min. After treatment, remove the lower layer filtrate.

[0101] (10) Remove residual washing liquid: Place the adsorption column AC from step (9) into a new empty collection tube and centrifuge at 13000 rpm for 3-5 min to remove residual washing liquid in the column as much as possible to avoid ethanol inhibiting the subsequent reaction.

[0102] (11) DNA elution: Transfer the adsorption column to a clean centrifuge tube, add 50 μL of elution buffer EB to the center of the membrane, let it stand at room temperature for 4 min, centrifuge at 12000 rpm for 1 min, collect the lower layer filtrate, and obtain the DNA sample.

[0103] (12) Preservation: The obtained DNA sample is stored at -20℃ for later use.

[0104] 1.1.8 GhWAKL37 Gene cloning and recycling First, search and download from the CottonGen database (https: / / www.cottongen.org). GhWAKL37 The CDS sequence information of the gene was obtained, and then specific amplification primers for the coding region of the gene were designed using SnapGene software. The final CDS primer sequences are shown in Table 9.

[0105] The PCR reaction system was prepared according to Table 10. All components were added sequentially on ice and then placed in a PCR instrument for amplification. The PCR amplification reaction system was performed according to the following program: first, pre-denaturation was carried out at 95℃ for 3 min; then 30 cycles were performed, with each cycle set as follows: denaturation at 95℃ for 15 s, annealing at 57℃ for 15 s, and extension at 72℃ for 3 min; after the cycle, final extension was carried out at 72℃ for 5 min to obtain the target DNA fragment (PCR product), and finally stored at 4℃.

[0106] Table 9 GhWAKL37 Primer sequences for amplifying the CDS region of the gene

[0107] Table 10 GhWAKL37 Gene PCR amplification reaction system

[0108] After the PCR products were separated by agarose gel electrophoresis, the gel containing the target band was excised and purified using the appropriate recovery kit. The specific procedures are as follows: (1) Place the gel block of the target DNA fragment in a 2.0 mL centrifuge tube and add 500 µL of gel dissolution buffer; (2) Heat in a 65°C water bath for 5 minutes, inverting and mixing every 2.5 minutes until the gel is completely dissolved and the solution turns pale yellow, thus obtaining the dissolved liquid; (3) Transfer the dissolved liquid to the adsorption column EC, centrifuge at 12000 rpm for 1 min, discard the lower layer of liquid, and then put the adsorption column back into the original centrifuge tube.

[0109] (4) Add 600µL of washing buffer W1 to the adsorption column EC, centrifuge at 12000rpm for 1min, and then discard the lower layer filtrate. (5) Perform the washing operation again according to step (4) above; (6) Then put the adsorption column back into a new empty collection tube, centrifuge at 12,000 rpm for 2 min, and discard the lower layer of filtrate to minimize the residual water inside. (7) Then place the column into a clean 1.5 mL centrifuge tube, open the cap and let it stand at room temperature for 2 min. Add 50 µL of elution buffer EB preheated at 60~65℃ to the center of the adsorption membrane, let it stand at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to elute the DNA. The lower layer filtrate yields the PCR gel recovery product (also known as...). GhWAKL37 PCR product).

[0110] 1.1.9 Cloning Vector Construction The vectors used were digested with restriction endonucleases to obtain linearized vectors. The vectors involved and their restriction sites are shown in Table 11.

[0111] Table 11 Vectors and Enzyme Restriction Sites

[0112] The linearized vector obtained from enzyme digestion was ligated with the target fragment purified by gel extraction. The vector used was then cleaved; the main vectors and cleavage sites are shown in Table 11. Specific reagents and parameters are shown in Table 2. The reaction system was prepared on ice, with 5 µL of 2×ClonExpress Mix, 3 µL of linearized vector, and 2 µL of PCR gel-extracted product added in the specified proportions. After mixing, the mixture was briefly centrifuged to collect the reaction solution at the bottom of the tube. Subsequently, it was incubated at 50 °C for 10 min to obtain pCambia2300-GFP- GhWAKL37 The recombinant plasmid was stored at 4°C after the reaction was completed, and will be used for subsequent transformations.

[0113] In addition, the T-load connection adopts GhWAKL37 The PCR amplification product was then used to construct the pCE2 TA / Blunt-Zero Vector cloning vector. Specifically, 4 µL of the amplified product was taken. GhWAKL37The PCR product was added to 1 µL of Blunt Zero vector in a reaction tube, gently mixed, and incubated at 25 °C for 15 min to obtain pCE2TA / Blunt-Zero Vector- GhWAKL37 The recombinant plasmid was used to generate the ligation product for subsequent transformation of competent E. coli cells.

[0114] 1.1.10 Transformation of Escherichia coli (1) Place Match1-T1 Escherichia coli competent cells on ice and thaw slowly.

[0115] (2) Add recombinant product (pCambia2300-GFP-) GhWAKL37 Recombinant plasmid or pCE2 TA / Blunt-ZeroVector- GhWAKL37 After mixing the recombinant plasmid, gently touch the tube wall to mix, avoiding vigorous shaking, and continue incubation in an ice bath for 30 minutes.

[0116] (3) Heat shock at 42℃ for 45s, then immediately transfer to ice to cool for 2min.

[0117] (4) Add 700 μL of antibiotic-free LB liquid medium and revive the culture at 37°C and 175 rpm for 75 min.

[0118] (5) After the culture is completed, centrifuge at 4000 rpm for 5 min, discard most of the supernatant, retain 50 μL of culture medium to resuspend the bacterial cells, and spread the bacterial solution on the corresponding resistance plate.

[0119] (6) Invert the plate and incubate it in a 37°C incubator for 14 hours.

[0120] (7) The next day, select a number of single clones for colony PCR identification and send positive clones for sequencing verification to obtain recombinant plasmids with correct sequences.

[0121] 1.1.11 Colony PCR Detection Single, normally developing colonies were selected from the plate and transferred to 600 μL of LB medium containing kanamycin. The culture was incubated at 37°C and 200 rpm for 7 hours with shaking until the bacterial cells became turbid. The cultured bacterial solution was then used directly as a template for amplification, and PCR was performed using 2×Taq Plus Master Mix and M13F / R universal primers. The reaction conditions are shown in Table 12. Several clones from the recombinant reaction transformation plate were selected for colony PCR identification and sent for assay to obtain plasmids with correct sequences.

[0122] Table 12 PCR reaction procedure for colony PCR detection

[0123] Sequencing analysis confirmed that the obtained pCambia2300-GFP- GhWAKL37 Recombinant plasmid or pCE2 TA / Blunt-ZeroVector- GhWAKL37 All recombinant plasmids were successfully ligated. GhWAKL37 Genes, the ones mentioned GhWAKL37 The nucleotide sequence of the gene is as follows:

[0124] The GhWAKL37 The amino acid sequence of the gene is as follows: MGIRMVFYFILQLPQLIQSASTFEAGEAGCKETCENVSIPYPFGIKRGCYQNSWFRVTCNKTINGTKPFISRINMELLPSYWLVEDNRVTVNNPVTYLNCDDKGNNGTTSSSSVNLQGSPFFLSEQNIFGSVGCGYLAIIFRNNQTDPIAACLQQRCEDHISSKLPGCLTMVPENLTSYTTALRPMTEIISPGEKESSKRCTSTFVGDSNEFSEISIDMTHVPATLEWNPVKCDLEASLCSMVRPNYALPYKTSCNERCGNVDIPFPFGIKVGCYKSEWFRVTCNKTADGEKPFISSINMQLLNVSFYEGTVLVNNSVIYSYCPGKDRENNEGSVNLTGTPFFFSHIFDRFMSIGCGNLATFLDSPTNDHRIGGCKLPPCENNMTSIVRCAVNIPPGLSSFVTNIRRIYPNNGSKSSCISSFIVDTRFLDSLEANSDHNATTTNRSGTYVPTTLQWGIPKRGLCELGEESGTLCSPDGRYCWTSLSQMHLCVCTPDTYNDYDYLSTDVCQEIGKCVDMKYRNCFIHCLNADGNDCSSSCPDRYKYLGHMCRPLNVLDSSEVPTKKSKRSQNLPVIIGCSTSIGTIVVLIGTWHMHKLIERRNNIKLKQKYFKRNGGLLLQQQLSNNKGNFEKIKLFASKELEKATDYYNENRILGRGGQGTVFKGMLTDGSIVAIKKSKMTEDKKLDENELKQFITEVMILSQINHRNVVKLLRCCLETKVPLLVYEFVPNGTLSQLLHVPNEEFPLTWEMRLRIAIEIANSLSYLHSAASVPIYHRDIKSSNILLDDKYRAKVSDFGTSRSVALEQTHVTTRVQGTFGYLDPEYFRSSQFTEKSDVYSFGVVLIELITGQKPVSSCQSEEVVRSLANFFLHSMKENSLLNIVDPLVMNDNAEEEIVAVAKLAKRCLNLNGKRRPTMKQVALELERIRSSEEANGMQQSADEDSDTDAMIEALGVDSFSTSGSILKDSVTLK(SEQ IDNO.8)。

[0125] 1.1.12 Transformation of Agrobacterium The recombinant plasmid pCambia2300:GFP-GhWAKL37, which was verified by sequencing, was transformed into Agrobacterium GV3101. The procedure is as follows: (1) Place Agrobacterium GV3101 competent cells on ice and thaw slowly.

[0126] (2) Take 1µL of recombinant plasmid and add it to Agrobacterium GV3101 competent cells. Incubate on ice for 5 min, then transfer to liquid nitrogen for rapid freezing for 5 min, then transfer to a 37℃ constant temperature water bath for 5 min, and finally cool and retain for 5 min.

[0127] (3) Then add 600µL of antibiotic-free LB medium to the above system in step (2) and shake and culture at 28℃ and 200rpm for 3h to obtain bacterial solution.

[0128] (4) Centrifuge the bacterial culture at 5000 rpm for 1 min, discard the supernatant, resuspend the culture, spread it on the corresponding screening medium, and incubate it in the dark at 28℃ for 48 h.

[0129] (5) Select single colonies for PCR identification. After confirming a positive result, continue amplification culture and preserve the strain to obtain 35S- GhWAKL37 Agrobacterium.

[0130] 1.1.13 Subcellular localization of GhWAKL37 Arabidopsis protoplasts were prepared using an Arabidopsis thaliana protoplast preparation and transformation kit following standard operating procedures.

[0131] (1) First, prepare the enzyme hydrolysate according to the formula in Table 13, and then use deionized water to make up to 10 mL.

[0132] Table 13 Composition of the enzymatic hydrolysis system

[0133] (2) After removing the main vein, the young leaves of Arabidopsis thaliana in the non-bolting stage cultivated in the greenhouse are cut into thin strips along the direction of the secondary veins. This operation helps to efficiently release and extract the protoplasts in the subsequent process.

[0134] (3) Immerse the treated leaf strips in the enzymatic hydrolysate and shake at a low speed of 40 rpm for 4 hours in a dark environment, then adjust to 80 rpm and shake vigorously for 5 minutes to promote the full release of protoplasts.

[0135] (4) After the enzymatic hydrolysis process is completed, the enzymatic hydrolysis products are separated by cell filtration sieve, the inner wall of the container and residual tissue are rinsed with 10 mL W5 buffer, and the filtrate is combined into a 50 mL centrifuge tube.

[0136] (5) Set the centrifugation parameters (increase speed parameter 3, decrease speed parameter 3), centrifuge at 100×g for 2min, and remove the supernatant.

[0137] (6) Add 5 mL of W5 solution pre-cooled at 4°C to resuspend the precipitate, and centrifuge again under the same conditions for 60 s. Discard the supernatant.

[0138] (7) Take 5 mL of pre-cooled II-W5 mixture to resuspend the protoplast precipitate, and then place it in an ice box for 30 min of low-temperature static culture.

[0139] (8) After incubation, centrifuge at 100×g for 1 min (acceleration parameter 3, deceleration parameter 3), remove the supernatant, and resuspend in 1 mL MMg solution to finally obtain Arabidopsis protoplast suspension.

[0140] (9) The prepared protoplast suspension was quantitatively analyzed by hemocytometer and observed by microscopic morphology. Once the quality was confirmed to be qualified, it could be used for subsequent transformation experiments.

[0141] The specific steps of protoplast transformation are as follows: (1) Take two centrifuge tubes and put 10 μg of purified pCambia2300-GFP plasmid and pCambia2300-GFP plasmid into each tube respectively. GhWAKL37 Recombinant plasmid.

[0142] (2) Slowly add 100 μL of protoplast suspension to each centrifuge tube, then inject 110 μL of polyethylene glycol (PEG), mix gently, and place in a room temperature environment for 15 min to react.

[0143] (3) Finally, add 0.44 mL of W5 buffer to stop the conversion process.

[0144] (4) Place the treated protoplasts horizontally and incubate them under weak light conditions at 25°C for 18 hours.

[0145] (5) After cultivation, the GFP fluorescence signal was observed using a confocal microscope. GFP was detected using 488nm excitation light, while chloroplast autofluorescence was observed at 633nm. 10 μg (>10 μL) of pCambia2300-GFP plasmid (referred to as GFP) and pCambia2300-GFP- were added to two centrifuge tubes, respectively. GhWAKL37 Recombinant plasmid (abbreviated as) GhWAKL37 -GFP).

[0146] To clarify GhWAKL37 This study constructed a subcellular localization model for proteins. GhWAKL37The pCambia2300-GFP fusion expression vector was used as a control, and the localization analysis was performed using a transient transformation system of Arabidopsis protoplasts.

[0147] Figure 2 The results showed that the GFP fluorescence signal in the control was diffusely distributed within the cells, while... GhWAKL37 The fluorescence signal of the -GFP fusion protein is mainly localized at the edge of the protoplast and highly coincides with the cell membrane outline, exhibiting obvious membrane localization characteristics. These results indicate that... GhWAKL37 Proteins are located in the cell membrane.

[0148] 1.1.14 Infection and Phenotypic Identification of Verticillium dahliae (1) Preparation of bacterial culture: The preserved highly pathogenic Verticillium dahliae strain V592 was inoculated into a suitable liquid culture medium and cultured with shaking at 27°C. After the mycelia or spores grew to the appropriate stage, they were collected and prepared into a spore concentration of 10. 7 A bacterial suspension of *Verticillium dahliae* was prepared by obtaining a bacterial suspension of *Verticillium dahliae* per mL for later use.

[0149] (2) Preparation of cotton materials: Select cotton seedlings with uniform growth and cultivate them to the two-leaf-one-heart stage as subsequent infection materials. Stop watering or control watering appropriately before infection to improve the consistency of materials after root damage treatment.

[0150] (3) Root damage treatment: Gently remove the cotton seedling, wash the substrate attached to the roots with sterile water, and then perform appropriate root damage treatment on the root system or the base of the main root. Cutting, scratching or shortening the lateral roots can be used to create uniform wounds on the roots, so that the roots of the cotton seedling after root damage are easier for pathogens to infect.

[0151] (4) Inoculation with bacterial solution: Immerse or drench the roots of cotton seedlings after root injury in a suspension of Verticillium dahliae to ensure that the bacterial solution fully contacts the wound area; use cotton root samples under normal growth conditions as control materials, and treat the control materials with sterile culture medium in the same way.

[0152] (5) Post-inoculation culture: After treatment, the plants were replanted in the substrate and cultured under suitable temperature and humidity conditions. Root tissues were collected at 0h, 1h, 3h, 6h, 9h, 12h, 24h, 48h, and 72h after infection, and the relative expression level of GhWAKL37 was detected by real-time quantitative PCR (qRT-PCR) (the relative expression level of GhWAKL37 was detected according to the real-time quantitative PCR method described in 1.1.4~1.1.5). High humidity was maintained after inoculation to facilitate pathogen infection and disease development.

[0153] (6) Disease observation and phenotypic statistics: After inoculation, observe the symptoms of wilting, yellowing and growth inhibition of the plants regularly, and record the disease time, disease severity and mortality rate, and compare the disease resistance differences among different materials.

[0154] Figure 3 The results showed that, compared with the control, GhWAKL37 The expression of *Verticillium dahliae* was significantly upregulated 3 hours after infection, and then gradually increased, reaching a peak at 24 hours, followed by a downward trend. These results indicate that *Verticillium dahliae* treatment of cotton... GhWAKL37 The expression level of this gene changed significantly, suggesting that it may play a role in cotton's response to Verticillium wilt stress and may also play a role in disease resistance regulation.

[0155] 1.1.15 GhWAKL37 Obtaining and phenotyping cotton with gene silencing expression 1.1.15.1 Construction of VIGS Vector (1) For analysis GhWAKL37 Based on the biological functions of [the virus], this study constructed a virus-induced gene silencing (VIGS) vector. GhWAKL37 Sequence information was used to design specific amplification primers, as shown in Table 14. The reverse transcription product cDNA obtained in 1.1.3 was used as a template, and TRV:: GhWAKL37-F / TRV:: GhWAKL37-R PCR amplification was performed. The reaction conditions were set as follows: pre-denaturation at 95℃ for 1 min; 35 amplification cycles were performed at 95℃ for 20 s, 56℃ for 20 s, and 72℃ for 30 s; a final extension at 72℃ for 5 min was performed to obtain the amplified product, which was then stored at 4℃. The amplified fragments were recovered from the gel and sequenced to confirm their specificity.

[0156] (2) At the same time, select EcoR I and Sac I was used as the restriction site for double digestion of the TRV::RNA2 plasmid. The digestion reaction system is shown in Table 15 below. The target vector fragment was recovered to obtain the digested vector.

[0157] (3) The above amplification product is ligated with the enzyme-digested vector to finally obtain TRV:: GhWAKL37 Recombinant plasmid. This recombinant plasmid was subsequently transformed into Agrobacterium GV3101 competent cells, providing a material basis for subsequent experimental research.

[0158] Table 14 GhWKAL37 Primer sequence information for VIGS vector construction

[0159] In Table 14, lowercase letter sequences represent vector homologous recombination adapters and restriction enzyme sites; uppercase letter sequences represent specific sequences of the GhWAKL37 gene.

[0160] Table 15 Enzyme digestion reaction system

[0161] 1.1.15.2 Obtaining cotton with silenced GhWAKL37 gene expression (1) Cultivate the disease-resistant upland cotton variety Zhongzhimian No. 2 until the cotyledons are fully expanded.

[0162] (2) In a sterile laminar flow hood, the pre-constructed TRV::CLA, TRV::GhWAKL37, TRV::RNA1 and TRV::RNA2 Agrobacterium were inoculated into LB medium containing kanamycin and rifampicin at a final concentration of 50 μg / mL and a final concentration of 50 μg / mL respectively. The culture was shaken at 28℃, 180 rpm and in the dark. The culture was first activated in small quantities and then expanded.

[0163] Among them, TRV::CLA, TRV::RNA1 and TRV::RNA2 are the pTRV2-GhCLA1 vector, pTRV1 (also known as pTRV1 empty vector) and pTRV2 (also known as pTRV2 empty vector) disclosed in the literature Wang Xinyu, Lü Kun, Cai Caiping, Xu Jun, Guo Wangzhen. Establishment and application of TRV virus-mediated gene silencing system in cotton. Acta Agronomica Sinica, 2014, 40(8):1356-1363.

[0164] (3) After the culture is completed, centrifuge at 5000 rpm for 10 min and collect the bacterial precipitate.

[0165] (4) Prepare resuspension buffer using deionized water as solvent, adding MgCl2, AS, and MES to achieve final concentrations of 10 mmol / L, 200 μmol / L, and 10 mmol / L, respectively. Measure OD after resuspending the bacteria. 600 The concentration was adjusted to 1.0. The mixture was then incubated at 28°C in the dark for 3 hours. Finally, the bacterial culture containing TRV::RNA1 was mixed with equal volumes of TRV::CLA, TRV::GhWAKL37, and TRV::RNA2 for later use.

[0166] (5) Ninety seedlings with fully expanded cotyledons and emerging true leaves were selected and divided into three groups. Bacterial culture was injected into the underside of the cotyledons using a sterile syringe, ensuring that both cotyledons were injected. The seedlings were inoculated with TRV::RNA2 as an empty vector control, TRV::GhWAKL37 as a silencing treatment group, and TRV::CLA as a positive control group. After inoculation, the seedlings were placed in a high-humidity, dark environment for 12 hours, and then cultured normally.

[0167] (6) When true leaves turn white in plants treated with TRV::CLA, it indicates that the VIGS system has been successfully constructed. After the seedlings have grown to the two-leaf-one-heart stage, root damage is performed and they are inoculated with Verticillium dahliae (see section 1.1.14 for the infection mode of Verticillium dahliae). After inoculation, the plants are cultured in a greenhouse for 14 days, the phenotype is observed and recorded, and finally the disease index is calculated.

[0168] 1.1.16 Disease Index Statistics Seedling diseases were classified into four levels (0-4) based on the severity of leaf disease and wilting, with the specific grading standards as follows: Level 0: Healthy plants, no lesions or diseased leaves; Level 1: Mildly diseased plants, with approximately 1%–33% of leaves showing symptoms; Level 2: Moderately diseased plants, with approximately 34%–66% of leaves showing symptoms or falling off; Level 3: Severely diseased plants, with approximately 67%–99% of leaves showing symptoms or falling off; Level 4: Extremely severe diseased plants, with 100% of leaves showing symptoms and the terminal bud dying. The number of diseased plants at each level was then counted, and the disease index was calculated using the disease index formula. DI = [Σ(number of disease grades × number of plants at each disease grade) / (total number of plants investigated × most severe disease grade)] × 100.

[0169] 4 represents the highest disease severity. Each treatment was performed in three biological replicates, and the severity of the disease in each treatment was ultimately represented by a disease index.

[0170] 1.1.17 Detection of fungal content To extract fungal DNA from plant tissues, a column chromatography method was used for purification, and the fungal content was determined. (1) Take 100 mg of cotton root sample that has been thoroughly ground and crushed by liquid nitrogen, quickly transfer it to a 1.5 mL centrifuge tube, then add 500 µL of CSPL buffer that has been preheated to 65 °C, and vortex thoroughly to mix the sample and buffer solution evenly to obtain a mixture.

[0171] (2) Place the above mixture into a constant temperature water bath and keep it at 65°C for 15 minutes. During the incubation process, gently turn the centrifuge tube every once in a while to ensure that the plant tissue can be fully lysed and to ensure the subsequent DNA extraction effect.

[0172] (3) Add 800µL of chloroform-isoamyl alcohol mixture (chloroform to isoamyl alcohol volume ratio of 24:1) to the centrifuge tube above, shake the centrifuge tube vigorously by hand to mix the mixture thoroughly, and then put the centrifuge tube into a high-speed centrifuge and centrifuge at 13000rpm for 5min.

[0173] (4) After centrifugation, carefully aspirate the upper aqueous phase liquid (about 300µL) with a pipette and transfer it to a new 1.5mL centrifuge tube; then add 150µL of sample binding buffer and 300µL of anhydrous ethanol in sequence, gently invert the centrifuge tube to mix, and then transfer all the liquid in the tube to the DNA adsorption column.

[0174] (5) Place the DNA adsorption column containing liquid into the matching collection tube, place it in a centrifuge, and centrifuge at 13000 rpm for 1 min. After centrifugation, discard the lower layer of filtrate in the collection tube, retain the adsorption column, and obtain the DNA adsorption column.

[0175] (6) Add 650µL of DNA washing buffer to the DNA adsorption column and centrifuge again at 13000rpm for 1min to complete the washing operation of the adsorption column. This washing step needs to be repeated once to completely remove impurities.

[0176] (7) Transfer the washed DNA adsorption column to a new matching collection tube and centrifuge at 13,000 rpm for 2 minutes. This step thoroughly removes the residual washing buffer from the adsorption column to avoid affecting subsequent DNA elution.

[0177] (8) Transfer the adsorption column to a new centrifuge tube, add 100µL of elution buffer preheated at 65℃, let stand, and centrifuge at 13000rpm for 1min to complete the elution.

[0178] (9) Collect the eluted DNA and store it at -20℃ for subsequent fungal content determination.

[0179] GhUBQ7 was used as an internal reference gene, and fungal genomic DNA extracted from the sample was used as an amplification template. qRT-PCR amplification experiments were carried out using the universal fungal forward primer ITS1-F and the Verticillium dahliae-specific reverse primer STVe1-R (see Table 16) to detect the content level of Verticillium dahliae in plant tissues.

[0180] Table 16 Specific primer sequences for fungal detection

[0181] To explore GhWAKL37The function of the TRV::GhWAKL37 silencing vector in cotton's resistance to Verticillium dahliae infection was investigated by silencing the vector using virus-induced gene silencing (VIGS) technology. The constructed TRV::GhWAKL37 silencing vector was then used to infect cotton seedlings with Agrobacterium-mediated infection (denoted as TRV::GhWAKL37). GhWAKL37 Phenotypic identification was performed using the silent group (TRV::CLA as a positive control and TRV::RNA2 as a negative control, denoted as TRV::00 control group).

[0182] Figure 4 The results of the study showed that 10 days after infection, the new true leaves of the positive control plants showed obvious whitening phenotype, indicating that the experimental operation of the VIGS system was correct.

[0183] Figure 4 The results of B showed that, compared with the TRV::00 control, TRV:: GhWAKL37 Treatment of newly emerging true leaves of plants GhWAKL37 The relative expression level of the gene was significantly reduced, indicating that the gene was effectively silenced in cotton, which shows that the GhWAKL37 gene-silenced cotton was successfully constructed and can be used for subsequent disease resistance function analysis.

[0184] For system analysis GhWAKL37 To investigate the specific function of cotton Verticillium wilt resistance regulation, this study used the TRV::00 empty vector control group (TRV::RNA2) and the TRV::GhWAKL37 silent group, which were grown to the two-leaf stage and had consistent growth, for the Verticillium dahliae inoculation experiment.

[0185] Figure 5 The results showed that, observed 14 days post-inoculation, compared with the TRV::00 negative control, GhWAKL37 The cotton plants with gene silencing showed more severe symptoms, with widespread yellowing and wilting of true leaves, and some leaves further drying out and falling off, resulting in a significant decline in overall plant growth. In contrast, the TRV::00 control plants only showed a few localized lesions on their true leaves, with relatively milder overall symptoms. These results indicate that gene silencing... GhWAKL37 The resistance of cotton to Verticillium dahliae decreased significantly afterward, suggesting that this... GhWAKL37 Genes may play a positive role in the regulation of resistance to Verticillium wilt in cotton.

[0186] To further confirm the phenotypic differences among plants under different treatments, this study observed and analyzed the disease incidence in the vascular tissues of their stems.

[0187] Take TRV::00 control group and TRV:: GhWAKL37 Longitudinal sections of stems from the silent group were prepared and observed under an optical microscope. Compared to the TRV::00 control group, TRV:: GhWAKL37 The vascular tissue damage was more pronounced in the silent group, with obvious browning of the vascular bundles (see...).Figure 6 (A). Quantitative analysis of the disease index was performed on the two groups of plants, and the results showed TRV:: GhWAKL37 The severity of disease in the silent group was significantly higher than that in the TRV::00 control group (see...) Figure 6 (C)

[0188] To confirm that the phenotypic differences in silent GhWAKL37 plants were caused by Verticillium dahliae infection, a fungal recovery experiment was conducted. Six inoculated plants were randomly selected from each group for subsequent pathogen isolation and colonization testing. The results showed that the pathogen biomass in the TRV::00 control group was significantly lower than that in the TRV::00 control group. GhWAKL37 Silent plants (see) Figure 6 (B) Further analysis of root samples from both groups of plants revealed the presence of TRV:: GhWAKL37 The root system of the silent group had a higher content of Verticillium dahliae (see Figure 6 (D).

[0189] 1.1.18 GhWAKL37 Obtaining gene knockout cotton To obtain GhWAKL37 First, the corresponding recombinant vector is constructed using the knockout material. The gene knockout portion utilizes the CRISPR / Cas9 system, targeting... GhWAKL37 Two sgRNAs (sgRNA1 and sgRNA2) were designed, and the primer information is shown in Table 17. Finally, the CRISPR / Cas9-WAKL37 vector was constructed.

[0190] Construction of the CRISPR / Cas9-WAKL37 vector: The sgRNA1 and sgRNA2 were fused by overlap extension PCR to obtain the sgRNA1-sgRNA2 fusion sequence. Then, the sgRNA1-sgRNA2 fusion sequence was ligated into the pRGEB32-GhU6.9 vector digested with Bsa I using the ClonExpress® II one-step cloning kit (Vazyme) to obtain the CRISPR / Cas9-WAKL37 vector. The CRISPR / Cas9-WAKL37 vector was then transformed into Agrobacterium LBA4404 to obtain the transformed Agrobacterium.

[0191] The transformation of the CRISPR / Cas9-WAKL37 vector into Agrobacterium LBA4404 can be found in: Optimization of the cotton genetic transformation system and creation of mutants, Doctoral dissertation, Jin Shuangxia, 2006, Chapter 4, Section 4.1, "Agrobacterium-mediated genetic transformation of cotton embryogenic callus".

[0192] (1) After the outer seed coat of the seed kernel of the upland cotton variety HM-1 was peeled off, it was first disinfected with 75% ethanol solution for 1 min, then treated with 0.1% mercuric chloride solution for 15 min, then rinsed repeatedly with sterile distilled water 6 times, and finally inoculated into germination medium and cultured in a dark environment at 28℃ for 72 h.

[0193] (2) Agrobacterium was inoculated into LB liquid medium and placed in a constant temperature shaker at 28°C and shaken at 200 rpm until the absorbance of the culture medium at a wavelength of 600 nm reached the range of 0.6 to 0.8. After the culture was completed, the bacterial cells were separated by centrifugation, and the precipitated bacterial cells were resuspended in co-culture medium.

[0194] (3) Cut the hypocotyl of the cotton seedling into segments of about 0.5 cm (aseptic treatment is required), immerse them in the bacterial solution prepared in step (2) for infection treatment for 1 hour; after treatment, remove the residual bacterial solution, transfer the infected tissue block to a culture dish containing solid culture medium, and place it in a constant temperature incubator at 28℃ for dark culture for 2 days.

[0195] (4) After the co-culture stage is completed, the explants are inoculated into the selection medium and cultured for 15 days. Then they are transferred to the subculture medium for further culture, and the medium is changed every 14 days. Once kanamycin-resistant plants are obtained, hardening and transplanting can be carried out.

[0196] (5) DNA and RNA samples were extracted from plant leaf tissues, and PCR and qRT-PCR techniques were used to detect and analyze transgenic positive plants. For gene knockout lines, Cas9- GhWAKL37 -F / R specific primers amplify the target region containing sgRNA1 and sgRNA2 target sites. The amplified products are sequenced and compared with the control wild-type sequence to determine the gene mutation type.

[0197] Table 17 Primer sequences used for identification.

[0198] 1.1.19 GhWAKL37 Overexpression of cotton acquisition 1.1.19.1 Cloning and purification of the target fragment Based on the laboratory-preserved overexpression vector pCambia2300-GFP, the following were selected BamH I and EcoRI was used as the restriction enzyme site for the target fragment, and specific primers containing the corresponding restriction endonuclease recognition sites were designed based on the target fragment using an online website (https: / / crm.vazyme.com / cetool / singlefragment.html). The relevant sequence information is shown in Table 18. Subsequently, PCR amplification was performed using the extracted plasmid DNA as a template. After amplification, the target product was recovered and purified.

[0199] Table 18 GhWAKL37 overexpression vector primers

[0200] In Table 18, lowercase letters represent vector homologous recombination linkers, and uppercase letters represent... GhWAKL37 The specific sequence of a gene.

[0201] 1.1.19.2 Enzyme digestion and recovery of overexpression vectors use BamH I and EcoR I. Prepare the reaction system according to Table 19 and incubate at 37°C for 6 hours. After the reaction, perform agarose gel electrophoresis to recover the target carrier fragment and store it at -20°C.

[0202] Table 19 Restriction Enzyme Digestion Reaction System

[0203] 1.1.19.3 GhWAKL37 Construction of overexpression vectors In this study, the target fragment obtained by PCR amplification and the linearized vector were mixed in the proportions shown in Table 20 to prepare a ligation system. The mixture was incubated at 50°C for 5 min in a PCR instrument, followed immediately by an ice bath to obtain the ligation product. This product was then introduced into competent *E. coli* cells, with the specific transformation steps described in 1.1.10. After colony growth, single clones were selected, and colony PCR was performed using the universal primer HP158 for the pCambia2300-GFP vector and the gene-specific primer GhWAKL37-R, as described in 1.1.11. Sequencing-positive single clones were selected to obtain the GhWAKL37 overexpression vector.

[0204] Table 20 Homologous recombination reaction system

[0205] Refer to section 1.1.12 for the method. GhWAKL37 The overexpression vector was transformed into Agrobacterium GV3101 to obtain a strain containing... GhWAKL37 Agrobacterium.

[0206] Referring to the method in section 1.1.18, it will contain GhWAKL37Agrobacterium infection of upland cotton variety HM-1 yielded a product containing... GhWAKL37 Genetically modified cotton.

[0207] 1.1.20 GhWAKL37 Acquisition of heterologous expression of Arabidopsis thaliana 1.1.20.1 Genetic transformation and identification of Arabidopsis thaliana heterologous expression of GhWAKL37 This experiment uses the 35S- constructed in 1.1.12 GhWAKL37 The specific steps for Agrobacterium-mediated transformation of transgenic Arabidopsis thaliana are as follows: (1) In a clean bench, Arabidopsis seeds were sterilized by immersing them in 4% sodium hypochlorite solution and 75% ethanol solution for 5 min each. After that, they were rinsed repeatedly with sterile distilled water 8 to 10 times to ensure that residual ethanol was completely removed. Finally, the seeds were resuspended in sterile water and evenly sown onto the surface of antibiotic-free MS solid medium using a pipette.

[0208] (2) Set the airflow of the clean bench to the maximum setting to quickly evaporate the residual moisture on the surface of the MS medium. After the medium is completely dry, immediately seal the culture dish with sealing film, and then place the sealed culture dish upside down in a 4°C refrigerator for a 2-day low-temperature vernalization treatment.

[0209] (3) The vernalization-treated seeds were transferred to a light incubator. When the Arabidopsis thaliana developed to the stage of four true leaves, they were transplanted into small culture pots for soil cultivation. The plants were cultivated under the following conditions: temperature 22 ℃, light 16 h, dark 8 h alternating, and relative humidity 60% until they entered the flowering period. Then, the genetic transformation of Arabidopsis thaliana was carried out.

[0210] (4) 35S- GhWAKL37 Agrobacterium was transferred to an Erlenmeyer flask containing 50 mL of LB liquid medium supplemented with kanamycin and rifampin. The culture was then placed in a 28°C shaker at 200 rpm for 17 h of continuous shaking culture.

[0211] (5) Prepare the resuspension according to the method shown in Table 21. Transfer the bacterial culture to a 50 mL centrifuge tube and centrifuge at 3000 rpm at room temperature for 10 min. Collect the precipitate to enrich the bacterial precipitate.

[0212] Table 21 Resuspension Formulation

[0213] The recombinant vector was introduced into Arabidopsis thaliana using the Agrobacterium inflorescence inoculation method, and positive transgenic plants were obtained through resistance selection and PCR detection. The specific procedures are as follows: (1) Inoculate Agrobacterium with the corresponding recombinant vector into a suitable culture medium and culture it overnight under suitable conditions so that the bacteria are in the logarithmic growth phase.

[0214] (2) Prepare Arabidopsis inflorescence staining solution with the following composition: 1 / 2 MS, 5% (w / v) sucrose, 0.05% (v / v) Silwet L-77, pH 5.7.

[0215] (3) Collect the bacteria by centrifuging the overnight cultured Agrobacterium at 6000 rpm for 5 min, discard the supernatant, resuspend in the infiltration solution, and adjust the concentration of the bacterial suspension to OD. 600 =0.8.

[0216] (4) The Arabidopsis inflorescences were immersed in the bacterial suspension for infection treatment, and the immersion time was controlled within 1 minute. After the infection was completed, the plants were placed in a dark and high-humidity environment for overnight cultivation. The next day, the plants were restored to normal growth conditions and continued to be cultivated until the seeds matured. Depending on the development of the inflorescences, the infection could be repeated 2 to 3 times to improve the transformation efficiency.

[0217] (5) Screening of T0 generation seeds: T0 generation seeds are harvested after the seeds mature. After surface disinfection, the seeds are sown on a culture medium containing the corresponding resistance screening agent. After 1 to 2 weeks of culture, green resistant seedlings are selected as positive candidate plants and transplanted into nutrient pots for further culture.

[0218] (6) Identification of positive plants: After transplanting, the plants were cultured for about 3 weeks, and genomic DNA was extracted from their leaves. The DNA extraction method was as described in 1.1.7. Then, PCR detection was performed using HP158 as the upstream primer and GhWAKL37-R as the downstream primer to screen for positive plants.

[0219] (7) Harvesting and subsequent use of T1 generation seeds: Plants that tested positive by PCR were cultured until maturity and T1 generation transgenic seeds were harvested. Subsequent experiments all used T1 generation seeds for culture to obtain heterologous expression. GhWAKL37 Arabidopsis strains.

[0220] To detect heterologous expression GhWAKL37 The expression level of the target gene in Arabidopsis thaliana lines was determined by qRT-PCR as described above, and the expression level in rosette leaves of transgenic lines was analyzed. GhWAKL37 The expression levels were analyzed.

[0221] Figure 7 The results showed that, compared with the wild type (WT), GhWAKL37 The expression level was significantly increased in the overexpression lines ( Figure 7 (A). Further semi-quantitative RT-PCR results also showed that in the transgenic lines... GhWAKL37 The expression was significantly enhanced ( Figure 7(B). The above results indicate that, GhWAKL37 Heterologous expression has been successfully achieved in Arabidopsis thaliana, and the obtained transgenic lines can be used for subsequent phenotypic analysis.

[0222] 1.1.20.2 GhWAKL37 Phenotypic observation of transgenic Arabidopsis To verify GhWAKL37 To determine whether heterologous expression participates in the plant's defense response against pathogens, this study further examined its role. GhWAKL37 Resistance of Arabidopsis thaliana to Verticillium dahliae was investigated. The T3 generation homozygous overexpressing lines and wild-type controls were inoculated using the root-dip method, and phenotypic observations were performed 14 days post-inoculation.

[0223] The T3 generation will be obtained according to section 1.1.19. GhWAKL37 After transgenic Arabidopsis plants reached a growth stage of 4 weeks (before bolting), they were treated with Verticillium dahliae V592 infection. Before inoculation, the plants were carefully dug out of the culture soil along with their roots, and the soil particles attached to the root surface were thoroughly washed with clean water. Residual moisture was then absorbed with absorbent paper. Afterward, the plant roots were immersed in a solution of 1×10⁻⁶ ppm. 7 Inoculation was completed by soaking the plants in a suspension of *Verticillium dahliae* conidia at a concentration of 1 / mL for 90 seconds. After inoculation, excess bacterial solution was gently blotted off the surface of the plants with absorbent paper, and the plants were then transplanted into new small pots for continued soil cultivation. To promote pathogen infection, the treated plants were placed in the dark for 24 hours under moist conditions, and then transferred to normal light conditions for 14 days of further cultivation. During this period, the plants were observed for disease symptoms and disease resistance-related phenotypes were recorded.

[0224] Disease surveys were conducted according to pre-defined grading standards: Grade 0 indicated healthy plants with no diseased leaves; Grade 1 indicated ≤25% of the plant's leaves showed disease symptoms; Grade 2 indicated 25%–50% of the plant's leaves were diseased or had fallen off; Grade 3 indicated 51%–75% of the plant's leaves showed disease symptoms; and Grade 4 indicated ≥76% of the plant's leaves were diseased. Finally, the Arabidopsis disease index was calculated to evaluate its disease resistance.

[0225] Figure 8 The results showed that wild-type plants exhibited obvious symptoms of disease, such as yellowing, wilting, and leaf drop, while the disease progression of the overexpressing lines was significantly delayed, and the leaves maintained a higher degree of greenness overall. Figure 8 (A). Disease Index (DI) statistical results indicate that T3 generation... GhWAKL37 The disease severity in transgenic Arabidopsis was significantly lower than that in wild-type controls. Figure 8 (B)

[0226] Real-time quantitative PCR was used to detect the biomass of the pathogen in the plant.

[0227] The results show that the T3 generationGhWAKL37 The relative content of fungal DNA in transgenic Arabidopsis thaliana was significantly lower than that in wild-type plants. Figure 8 (C). Phenotypic observation and pathogen load analysis results indicate that GhWAKL37 Heterologous expression significantly improved Arabidopsis thaliana resistance to Verticillium dahliae, suggesting that this... GhWAKL37 Genes may play a positive role in the regulation of Verticillium wilt resistance.

[0228] To study overexpression GhWAKL37 In this study, a GhWAKL37 overexpression vector was constructed using the upland cotton standard line HM-1 (WT) as the recipient material. HM-1 plants that had undergone genetic transformation but had not integrated the target gene fragment were used as a negative control (Null). The obtained T3 generation overexpression vector was analyzed. GhWAKL37 Cotton samples were analyzed using semi-quantitative PCR and quantitative real-time PCR (qRT-PCR) to detect the transcriptional level of the target gene. All materials were cultured indoors at a constant temperature of 28°C, with 16 hours of light / day and 8 hours of darkness / day, and the relative humidity was maintained at approximately 60%.

[0229] Figure 9 The results showed that the internal reference gene GhHis3 The expression levels were relatively stable in WT, Null, and three independent overexpression lines, making them suitable for subsequent analysis. Compared to WT and Null, GhWAKL37 The relative expression level was significantly increased in the overexpression cotton lines, indicating that GhWAKL37 The overexpression of cotton has been successfully constructed.

[0230] Subsequently, this study used an inoculation experiment with *Verticillium dahliae* (see Section 1.1.14 for the infection pathway of *Verticillium dahliae*) to investigate the effects of *Verticillium dahliae* on... GhWAKL37 The disease resistance of cotton overexpressed was further analyzed.

[0231] Figure 10 Phenotypic observation results showed that 14 days after inoculation (dpi), obvious lesions appeared on the cotyledons of the control plants (WT and Null), while GhWAKL37 The overexpressing cotton plants maintained good growth, and no obvious symptoms were observed. By 21 days post-infection (dpi), the control plants (WT and Null) showed significantly worse symptoms, with extensive yellowing and wilting of true leaves; in contrast, GhWAKL37 Overexpressing the drug only resulted in a few lesions on the cotyledons of the cotton plants. By 28 days post-infection (dpi), the control plants (WT and Null) had essentially withered and died, while... GhWAKL37 The true leaves of cotton overexpressing the gene only showed localized yellowing and necrotic spots (see...) Figure 10 (A)

[0232] Further examination of the stem vascular bundles revealed that, compared to the control plants (WT and Null), the vascular bundles showed severe browning and rot. GhWAKL37 The browning of vascular bundles in cotton overexpressing the gene was significantly reduced, and the lesion area was smaller (see...). Figure 10 (B) The statistical results of the disease index were consistent with the above phenotypic observations.

[0233] Figure 11 The results show that GhWAKL37 The disease severity in cotton plants overexpressing the gene was significantly lower than in control plants (WT and Null). Compared to control plants (WT and Null), cotton plants showed significantly lower disease incidence. GhWAKL37 Overexpression significantly improves the resistance of cotton to Verticillium dahliae.

[0234] In conclusion, GhWAKL37 Overexpression of cotton significantly enhances cotton's resistance to Verticillium wilt.

[0235] For in-depth research GhWAKL37 This study investigated the function of genes during cotton growth and development using CRISPR / Cas9 gene knockout technology. GhWAKL37 Gene knockout cotton. First, a gene knockout cotton was designed and synthesized. GhWAKL37 A specific single guide RNA (sgRNA) for the gene was cloned and inserted into a CRISPR / Cas9 vector. Subsequently, the vector was introduced into cotton callus tissue via Agrobacterium-mediated transformation, and successfully transformed plants were screened. PCR amplification and sequencing analysis of these transformed plants identified knockout lines with frameshift mutations caused by base deletions in the sgRNA1 and sgRNA2 regions. Figure 12 (A). Three stable knockout lines were selected and self-crossed to obtain stable T3 generation lines ( ). Figure 12 (B), which laid the foundation for subsequent functional research.

[0236] The already obtained GhWAKL37 Transgenic knockout cotton lines were compared with control groups (WT and Null). Fourteen days after inoculation with *Verticillium dahliae*, obvious lesions were observed on the cotyledons of the control group plants, while the knockout lines showed significant cotyledon wilting and abscission, with a significantly increased proportion of withered leaves. Twenty-one days after inoculation, although the true leaves of the control group began to yellow and wilt, GhWAKL37 The knockout strain has completely died. Figure 13 (A)

[0237] To assess disease resistance in more detail, this study compared the control materials with... GhWAKL37Disease resistance-related indicators of the knockout lines were measured, and longitudinal sections of the plant stems were observed at different treatment stages. The GhWAKL37 knockout line showed more severe vascular bundle discoloration and earlier browning after infection with *Verticillium dahliae* compared to the control group (see...). Figure 13 (B)

[0238] This phenotypic difference was further confirmed by statistical analysis of disease index and relative fungal biomass.

[0239] Figure 14 The results proved GhWAKL37 Gene knockout significantly weakened cotton's resistance to Verticillium dahliae. These results reveal... GhWAKL37 Its key role in the disease resistance response of cotton.

[0240] In summary, compared with wild-type controls (WT and Null), the GhWAKL37 knockout lines showed a significant decrease in resistance after inoculation, manifested as early damage to vascular bundle structure, early yellowing and necrosis, and a deterioration of the overall disease phenotype.

[0241] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. GhWAKL37 The application of genes in (1) and / or (2) is characterized by, (1) Regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing of the gene increases resistance. GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

2. GhWAKL37 The application of gene-related biomaterials in (1) and / or (2), characterized in that, (1) Regulating plant resistance to Verticillium wilt; (2) Cultivate plants with increased or decreased resistance to Verticillium wilt; The GhWAKL37 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO. 8; The plant in question is Arabidopsis thaliana or cotton. overexpression GhWAKL37 When the gene is knocked down, plant resistance to Verticillium wilt is enhanced; knockdown or silencing of the gene increases resistance. GhWAKL37 When the gene is modified, the plant's resistance to Verticillium wilt decreases.

3. The application according to claim 1 or 2, characterized in that, The Verticillium wilt mentioned is caused by Verticillium dahliae.

4. The application according to claim 2, characterized in that, The GhWAKL37 Gene-related biomaterials GhWAKL37 Gene overexpression biological materials or GhWAKL37 Gene knockdown or silencing materials; The GhWAKL37 Gene knockdown or silencing materials are biological materials containing CRISPR / Cas9 or VIGS vectors; The CRISPR / Cas9-containing biological material includes sgRNA1 and sgRNA2, the sequence of which is shown in SEQ ID NO.13; the sequence of which is shown in SEQ ID NO.

14. The biomaterials are carriers, host bacteria, plant tissues, plant cells, or plant organs.

5. Overexpression GhWAKL37 The application of genes from cotton or Arabidopsis thaliana in resistance to Verticillium dahliae infection is characterized by, The amino acid sequence of the protein encoded by the GhWAKL37 gene is shown in SEQ ID NO.

8.

6. The application according to claim 1 or 2 or claim 5, characterized in that, GhWAKL37 The coding region sequence of the gene is shown in SEQ ID NO.

7.

7. A cotton or Arabidopsis thaliana resistant to Verticillium wilt, characterized in that, The cotton or Arabidopsis thaliana overexpression of any one of claims 1 to 6 GhWAKL37 Gene.

8. A method for preparing a plant resistant to Verticillium wilt, characterized in that, The following steps are included to improve the quality of recipient plants. GhWAKL37 Gene expression levels and activity were obtained. GhWAKL37 Genetically modified plants; The recipient plants are cotton and / or Arabidopsis thaliana.

9. The preparation method according to claim 8, characterized in that, The Verticillium wilt mentioned is caused by Verticillium dahliae.

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