Application of cotton gene GhVDRK1 in regulating resistance to verticillium wilt in cotton

Abstract

The present application relates to cotton gene GhVDRK1 In the application of regulating cotton verticillium wilt resistance, the cotton gene GhVDRK1 The encoded amino acid sequence is shown in sequence 2 in the sequence table. The present application obtains GhVDRK1 Gene silencing plants, the disease index of the silencing plants under the stress of verticillium wilt is increased, the resistance to verticillium wilt is reduced, and the expression amount of the gene in the leaves of cotton varieties with verticillium wilt resistance GhVDRK1 The expression amount of the gene in the leaves of cotton varieties susceptible to verticillium wilt GhVDRK1 The results show that GhVDRK1 GhVDRK1 The gene plays a role in regulating cotton verticillium wilt resistance, and the present application provides a good gene resource for the research of cotton verticillium wilt resistance and the cultivation of cotton varieties resistant to verticillium wilt.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a cotton gene. GhVDRK1 Application in regulating resistance to Verticillium wilt in cotton. Background Technology

[0002] Cotton, as one of the world's most important natural fiber and economic crops, has long been severely threatened by Verticillium dahliae, a soil-borne vascular disease. Infection with this pathogen causes plant wilting, leaf yellowing, vascular browning, and even death, leading to a significant decline in cotton yield and quality, earning it the nickname "cotton cancer." Current control methods are limited; breeding disease-resistant varieties is the most economical and effective approach. Plants rely on a multi-layered immune system to resist pathogen infection, with pattern recognition receptors located on the cell membrane surface playing a crucial role in sensing pathogen signals and activating defense responses.

[0003] The cell wall-associated protein kinase (CLP) gene family is a class of receptor kinases located on the plasma membrane and interacting with cell wall components. Serving as a bridge between extracellular stimuli and intracellular signal transduction, they play crucial roles in plant responses to biotic stress, maintaining cell wall integrity, and activating immune pathways. Currently, the CLP gene family in some crops has been systematically studied, such as Sea Island cotton, where approximately 70 genes have been identified. Among them, CLP 5 positively regulates salt tolerance by modulating ion homeostasis, thus controlling the salt stress resistance of Sea Island cotton. However, the function of the CLP gene family in cotton in responding to pathogens, especially devastating fungal diseases like Verticillium wilt, remains unreported, and related gene resources await further exploration.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide cotton genes. GhVDRK1 Application in regulating resistance to Verticillium wilt in cotton.

[0006] The second objective of this invention is to provide cotton genes. GhVDRK1 Application in the breeding of cotton varieties resistant to Verticillium wilt.

[0007] The third objective of this invention is to provide a biomaterial that improves resistance to Verticillium wilt in cotton.

[0008] The fourth objective of this invention is to provide a method for improving resistance to Verticillium wilt in cotton.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides cotton genes. GhVDRK1 Application in regulating cotton Verticillium wilt resistance, the cotton gene GhVDRK1 The encoded amino acid sequence is shown in Sequence 2 of the sequence listing. The regulation is to reduce the concentration of amino acids in cotton. GhVDRK1 The expression level of this gene will reduce resistance to Verticillium wilt in cotton, while increasing the expression level of the gene in cotton. GhVDRK1 The expression level of the gene will increase resistance to Verticillium wilt in cotton.

[0010] cotton genes GhVDRK1 The gene ID is Gh_A02G133400, and its encoded amino acid sequence is shown in Sequence 2 of the sequence listing, totaling 745 amino acids. Silencing of this gene using VIGS technology verified that reducing its expression level leads to an increase in the disease index of plants under Verticillium wilt stress, i.e., weakened resistance to Verticillium wilt. Furthermore, the expression level of this gene was significantly higher in highly resistant Verticillium wilt cotton varieties than in susceptible varieties. Therefore, it can be determined that increasing the expression level of this gene in cotton... GhVDRK1 The expression level of the gene increases resistance to Verticillium wilt in cotton.

[0011] Furthermore, the nucleotide sequence encoding the amino acid sequence shown in Sequence 2 of the sequence listing preferably includes the nucleotide sequence shown in Sequence 1 of the sequence listing, i.e., preferably the cotton gene. GhVDRK1 The CDS sequence is shown in Sequence 1 of the sequence listing. The nucleotide sequence shown in Sequence 1 consists of 2238 nucleotides.

[0012] Furthermore, the pathogen of the cotton Verticillium wilt is Verticillium dahliae.

[0013] The second aspect of the present invention provides cotton genes. GhVDRK1 Application in breeding cotton varieties resistant to Verticillium wilt, the cotton gene GhVDRK1 The encoded amino acid sequence is shown in Sequence 2 of the sequence listing, which increases the concentration of amino acids in cotton. GhVDRK1 Gene expression levels are used to breed cotton varieties resistant to Verticillium wilt.

[0014] Furthermore, the cotton gene GhVDRK1 The CDS sequence is shown in Sequence 1 of the sequence list.

[0015] Furthermore, the pathogen of cotton Verticillium wilt is preferably Verticillium dahliae.

[0016] A third aspect of the present invention provides a biomaterial for improving resistance to Verticillium wilt in cotton, said biomaterial comprising any one of the following (1) to (4): (1) DNA molecules encoding the amino acid sequence shown in Sequence 2 of the sequence listing; (2) An expression cassette containing the DNA molecule described in (1); (3) A recombinant vector comprising the DNA molecule of (1) or the expression cassette of (2); (4) A host cell or host bacterium containing the DNA molecule of (1) or the expression cassette of (2) or the recombinant vector of (3).

[0017] Furthermore, the nucleotide sequence of the DNA molecule is shown in Sequence 1 of the sequence listing.

[0018] In the aforementioned biological materials, the expression cassette includes at least a promoter, a target gene (a DNA molecule encoding the amino acid sequence shown in Sequence 2 of the sequence listing), and a terminator. The promoter in the expression cassette can be a general constitutive promoter suitable for plant genetic transformation and genetic engineering, such as the 35S or maize ubiquitin-1 promoter, or a tissue-specific promoter, such as the AtSUC2 (sucrose transporter) promoter.

[0019] In the aforementioned biological materials, the basic vector backbone used to construct the recombinant vector can be an Agrobacterium binary vector system, such as the pCAMBIA series, pBI series, etc. The basic vector backbone includes necessary selection marker genes and reporter genes for screening transformants or transgenic plants.

[0020] In the above-mentioned biological materials, the host cell or host bacterium can be Agrobacterium tumefaciens used for cotton genetic transformation or Escherichia coli used as a propagation vector, such as Agrobacterium tumefaciens EHA105.

[0021] A fourth aspect of the present invention provides a method for improving resistance to Verticillium wilt in cotton, the method comprising: increasing the concentration of... GhVDRK1 Gene expression levels.

[0022] Furthermore, the improvement in cotton material GhVDRK1 Specific methods for measuring gene expression levels include: S1: Introduce the biomaterial described in the third aspect above into cotton material; S2: Screening from the regenerated plants obtained in S1 GhVDRK1 Transgenic cotton lines with increased gene expression levels.

[0023] The biological materials described in the third aspect above can be introduced into cotton materials using conventional genetic transformation methods, such as Agrobacterium-mediated transformation.

[0024] Real-time quantitative PCR can be used to screen regenerated plants obtained from S1 to select GhVDRK1 Transgenic cotton lines with increased gene expression levels.

[0025] The advantages and positive effects of this invention are as follows: This invention provides cotton genes GhVDRK1 In regulating cotton resistance to Verticillium wilt or breeding cotton varieties resistant to Verticillium wilt, this invention uses VIGS technology to silence the gene, obtaining gene-silenced plants. Silent plants exhibit increased disease index under Verticillium wilt stress and decreased resistance to the disease. Simultaneously, the inventors discovered that in the leaves of cotton varieties resistant to Verticillium wilt... GhVDRK1 The gene expression level was significantly higher in the leaves of cotton varieties susceptible to Verticillium wilt than in those of cotton varieties susceptible to Verticillium wilt. GhVDRK1 Gene expression levels. The results showed that... GhVDRK1 Genes play a role in regulating cotton resistance to Verticillium wilt, and this invention provides excellent gene resources for research on cotton resistance to Verticillium wilt and for breeding cotton varieties resistant to Verticillium wilt. Attached Figure Description

[0026] To facilitate a more intuitive demonstration of the specific embodiments of the present invention, the accompanying drawings will be briefly described below. It should be understood that the accompanying drawings are merely illustrative of some embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art can make reasonable extensions or adjustments based on the essential content of the present invention without inventive effort, and thereby obtain the relevant drawings and embodiments.

[0027] Figure 1 The TRV:: provided in the embodiments of the present invention GhVDRK1 Plot of relative expression levels of the target gene in the stems and leaves of silent plants and cotton plants transformed with the TRV::00 empty vector; Figure 2 The TRV2:: provided in the embodiments of the present invention VDRK1 Phenotypic characteristics of silent cotton plants and cotton plants transformed with the empty TRV2::00 vector after infection with Verticillium dahliae, where A represents TRV2:: VDRK1 Overall phenotypes of silent cotton plants and cotton plants transformed with TRV2::00 empty vector after infection with Verticillium dahliae, B being TRV2:: VDRK1 Longitudinal sections of stem segments from silent cotton plants and cotton plants transformed with TRV2::00 empty vector 21 days after infection with Verticillium dahliae. Figure 3 The phenotype of TRV2::CLA1 albino plants; Figure 4 Wild-type upland cotton material AS98 Disease index graph of WT 21 days after inoculation with Verticillium wilt pathogen; Figure 5 For TRV:: VDRK1 Disease index of silent cotton plants and cotton plants transformed with TRV::00 empty vector 21 days after inoculation with Verticillium wilt pathogen. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof.

[0029] Unless otherwise specified, the experimental methods used in the following examples are generally performed under conventional conditions. For example, you can refer to the following books or manuals, such as Molecular Cloning: A Laboratory Manual (2nd Edition, by J. Sambrook et al., translated by Huang Peitang et al., Science Press, 2002), Plant Gene Silencing: Methods and Protocols in Molecular Biology, or other professional books on virus-induced gene silencing, or follow the conditions recommended by the manufacturer.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0032] Example 1: Validation using VIGS GhVDRK1 The role of cotton in resisting Verticillium wilt stress The cotton materials used in this embodiment are the natural dwarf mutant AS98 of the upland cotton variety LFH10 and its wild-type control WT (LFH10). Both cotton materials were provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0033] 1. GhVDRK1 Obtaining gene sequences (1) Extract separately AS98 Total RNA from young leaves of WT cotton plants was digested with DNase I to remove genomic DNA, and then reverse transcribed into first-strand cDNA using reverse transcriptase. (2) Using cDNA as a template, PCR amplification was performed using the following primer pairs. A virus-induced gene silencing system based on tobacco brittle virus (TRV) was used to design and synthesize specific amplification molecules. GhVDRK1 Primers for the gene fragment. The primer pair sequences are as follows: GhVDRK1- Forward primer: 5'-TAAGGTTACCGAATTCATGAAATTCCCTCCATTGCTTTCGC-3' (sequence 3). GhVDRK1- Reverse primer: 5'-GCTCGGTACCGGATCCATTCCTGCCGCCAAGACC-3' (Sequence 4).

[0034] The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2.

[0035] Table 1 Table 2 (3) Purification and recovery of gene products: The PCR products from the previous step were subjected to 1.2% agarose gel electrophoresis, i.e., electrophoresis at a constant voltage of 130 V for 25 min. The gel was then placed under a UV imager and the contents of the gel were cut off. GhVDRK1 Gel containing the target gene.

[0036] (4) The recovered fragments were sent to Beijing Nuosheng Genome Research Center Co., Ltd. for sequencing using the Novizan FastPure Gel DNA Extraction Mini Kit. AS98 Similar to the amplified product of WT cotton plants, the nucleotide sequence is shown in Sequence 1 of the sequence listing, totaling 2238 nucleotides. The encoded amino acid sequence is shown in Sequence 2 of the sequence listing, totaling 745 amino acids.

[0037] 2. Recombinant vector TRV:: GhVDRK1 Construction (1) The pTRV2 plasmid was linearized with EcoRI and BamHI restriction endonucleases, and the recovered target fragment was collected according to the instructions of the ClonExpress II One Step Cloning Kit. GhVDRK1 Homologous recombination with the linearized pTRV2 plasmid yielded the recombinant product.

[0038] (2) Genetic transformation and bacterial culture detection of Escherichia coli a) 100 μL of DH5α Escherichia coli competent cells were aliquoted into sterile 1.5 mL centrifuge tubes; b) Add the recombinant product, incubate on ice for 30 min, heat shock at 42 ℃ for 90 s, then incubate on ice for 5 min; c) Add 700 μL of antibiotic-free LB solution to the clean bench; d) Incubate at 37 ℃ with shaking for approximately 90 min; e) Centrifuge at 5000 rpm for 5 min, discard part of the supernatant, mix the remaining supernatant with the bacterial culture at the bottom, and then spread it evenly onto the solid LB containing kanamycin. f) Incubate upside down for approximately 16 hours under sterile conditions at 37 °C; g) Use a sterile toothpick to pick up a portion of the monoclonal antibodies from the plate and transfer them to 700 μL of LB liquid medium containing kanamycin for propagation; h) Take 2 μL of bacterial culture as a PCR template, and detect positive results by PCR amplification. Send the positive clones to Beijing Nuosai Genome Research Center Co., Ltd. for sequencing; plasmid extraction was performed using the Plasmid Miniprep Kit (PD1213, Biomiga).

[0039] (3) Agrobacterium transformation and bacterial culture detection a) Dispense 100 μL of GV3101 competent cells into two sterile 1.5 mL centrifuge tubes. Add 5 μL (approximately 800 ng) of the plasmid to be transformed, gently tap to mix, and incubate on ice for 30 min. Treat with liquid nitrogen for 5 min, heat shock at 37°C for 5 min, immediately place on ice, and ice bath for 5 min. In a clean bench, add 700 μL of antibiotic-free liquid LB to a centrifuge tube; Place in a shaker at 28℃ and shake for 4-6 hours; Take about 200 μL of bacterial suspension and spread it evenly on an LB agar plate containing kanamycin and rifampin antibiotics; Incubate upside down in an incubator at 28 ℃ for about 2 days; g) In a clean bench, pick a single colony into 700 μL of liquid LB containing kanamycin and rifampin, place it on a shaker at 28 ℃ and 180 rpm for about 24 h. h) PCR was used to detect positive results in the bacterial culture. The successfully transformed Agrobacterium culture was stored at -70°C for later use, following a bacterial culture to glycerol ratio of 6:4.

[0040] In this invention, the recombinant vector TRV:: GhVDRK1, Also known as TRV:: VDRK1 Or TRV2:: VDRK1 All of these refer to the same recombinant vector. When WT or AS98 is added before the vector, it means that the upland cotton variety LFH10 or AS98 has been transferred with the recombinant vector TRV:: GhVDRK1 .

[0041] In the preparation of recombinant vector TRV:: GhVDRK1 When processing Agrobacterium, the same method was also used to prepare vectors containing empty vector pTRV2, helper plasmid pTRV1, and positive control pTRV2-. GhCLA1 (Cotton is silent) CLA1After gene silencing, leaves and stems will exhibit chlorotic whitening symptoms (used to detect gene silencing effects). The empty vector pTRV2 is also known as TRV::00 or TRV2::00, pTRV2- GhCLA1 Also known as TRV2- CLA1 Add WT or before the carrier AS98 This indicates that the corresponding carrier has been introduced into the upland cotton varieties LFH10 or AS98.

[0042] 3. Agrobacterium-mediated transformation (1) Preparation of seedlings Upland cotton AS98 After soaking plump cotton seeds (WT seeds) in a 1% potassium dihydrogen phosphate solution for one day, they were wrapped in damp paper towels and placed in a 30°C incubator for suffocation. Well-germinating seeds were then sown in moist nutrient soil (vermiculite: humus: nutrient soil = 1:1:4) and cultured in a plant culture room at 25°C with a photoperiod of 16 h (light) / 8 h (dark). When the cotton seedlings had grown to 7-10 days and their cotyledons were fully expanded but the true leaves had not yet emerged, plants with uniform growth and good development were selected for the VIGS test, at which point the silencing effect was optimal.

[0043] (2) Activation of the strain Separately containing pTRV2- GhVDRK1 Agrobacterium, including empty vector pTRV2, helper plasmid pTRV1, and positive control pTRV2-GhCLA1, was inoculated into LB liquid medium (containing 50 µg / mL Kan) and activated in a shaker at 28 °C and 200 rpm for 12 h.

[0044] Activation and culture of Verticillium dahliae, the pathogen of Verticillium dahliae: Preserved Verticillium dahliae V991 (provided by Professor Cheng Hongmei's research group at the Institute of Biological Research, Chinese Academy of Agricultural Sciences) was activated and cultured for 5-7 days. Vigorous hyphae were selected, and mycelial blocks were taken using a sterile punch and placed into prepared PDA culture medium. The mixture was then cultured at 28 ℃ and 220 r / min in a constant temperature shaker for 3-5 days. The spore suspension was filtered through four layers of sterile gauze, and the spore concentration was recorded on a hemocytometer. The spore suspension was then diluted to a concentration of 1×10⁻⁶. 7 CFU / mL was used as the inoculum solution.

[0045] Preparation of VIGS resuspension Take 500 µL of the activated bacterial suspension containing the recombinant vector at a 1% volume ratio and inoculate it into 50 mL of LB liquid medium (containing 50 µg / mL Kan and 50 µg / mL Rif). Stop incubation when the OD600 of the bacterial suspension reaches 1 using a UV spectrophotometer. Dispense 50 mL of the bacterial suspension into sterile centrifuge tubes, centrifuge at 5000 rpm for 5 min, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells using VIGS resuspension (VIGS resuspension formula is shown in Table 3) and adjust the OD600 of the bacterial suspension to 0.8. Add the pTRV2-... GhVDRK1 The pTRV2-GhCLA1 and pTRV2 empty vector resuspensions were mixed with the pTRV1 resuspension containing the auxiliary plasmid vector at a ratio of 1:1 and allowed to stand at room temperature for 3 hours before injection.

[0046] Table 3 VIGS resuspension formulation (1 L) (4) Leaflet injection Select cotton seedlings with uniform growth and smooth, intact leaves. Using the needle of a 1 mL sterile syringe, make 2-3 small incisions on the underside of the cotyledons (avoiding the main vein to prevent puncture). Then, using a 1 mL syringe without the needle, draw up the bacterial solution and inject it into the incisions, ensuring that the leaf surface is injected to cover at least 80% of the leaf surface while minimizing the number of incisions. This will yield TRV:: GhVDRK1 TRV:: GhCLA1 TRV::00 and control plants were included. After injection, all plants were watered, covered with cotton in black plastic bags, and cultured in the dark for 24 hours. The next day, they were moved to normal light conditions for further culture.

[0047] 4. GhVDRK1 Gene silencing efficiency test Approximately 14 days after VIGS vector transformation of cotton plants, the positive control pTRV2- GhCLA1 The cotton plants exhibited chlorosis and whitishness in their leaves (e.g.) Figure 3 If the VIGS silencing system successfully silences the gene, it indicates that the gene has been successfully silenced. This serves as an indicator of viral vector infection efficiency and gene silencing effectiveness. VIGS vectors were used to transform cotton plants (TRV:: CLA1True leaf and stem tissues from *T. 10* (TRV::00) and control plants (LFH10 and AS98) were flash-frozen in liquid nitrogen, and total RNA was extracted and reverse transcribed to generate cDNA. Expression levels were analyzed using a CFX Connect Real-time PCR system (BIO-RAD, USA). The cotton GhHistone gene was used as an internal control. Three replicates were set up for each gene amplification. The relative expression level of the genes was calculated using the Comparative Ct method. See [link to results] for details. Figure 1 .

[0048] The forward and reverse primers used in quantitative real-time PCR are as follows: F: 5'-ATGAAATTCCCTCCATTGCTTTCGC-3' (Sequence 5) R: 5'-GGGCTGAAAGTGGTGTTACAAGTAA-3' (sequence 6) The real-time quantitative PCR amplification system is shown in Table 4, and the real-time quantitative PCR amplification program is shown in Table 5.

[0049] Table 4 Real-time quantitative PCR amplification system Table 5 Real-time quantitative PCR amplification program This example compares the results of transformation plants of upland cotton AS98 and LFH10. GhVDRK1 The relative expression level. From Figure 1 It can be seen that (WT)TRV:: VDRK1 (AS98)TRV:: VDRK1 In the silent plants, the level of silent expression of the target gene, whether in leaves or stems, was significantly lower than that in plants transformed with the empty vector TRV::00.

[0050] 5. GhVDRK1 Silent cotton plants inoculated with Verticillium wilt Approximately 14 days after the VIGS experiment, the silencing efficiency of the target gene was detected using qPCR. For successfully silencing transformed plants, TRV:: GhVDRK1 Cotton plants transformed with the TRV2::00 empty vector and wild-type control plants were inoculated with Verticillium wilt. Verticillium dahliae was inoculated using the root wound irrigation method: V991 spore suspension (1×10⁻⁶) was used. 7 Inject CFU / mL into the roots of the plants, and place the inoculated plants in the dark for 24 hours. Then, transfer the cotton to normal culture conditions for further cultivation. TRV2:: VDRK1The overall phenotypes of silent plants and cotton plants transformed with TRV2::00 empty vector after infection with Verticillium dahliae are as follows: Figure 2 As shown in A. From Figure 2 As shown in section A, the virus was successfully silenced after inoculation with Verticillium wilt pathogens. GhVDRK1 Plants with the silenced gene showed reduced resistance to Verticillium wilt, significantly lower than plants without the silenced gene.

[0051] 6. Analyzing the stem profile of inoculated cotton plants Twenty-one days after inoculating the plants with Verticillium wilt, collect TRV:: GhVDRK1 The stem segments, approximately 1 cm in length, were taken from the upper cotyledon nodes of silent plants, TRV: 00 plants, and control wild-type plants. The stem segments were longitudinally cut with a blade, and the browning of the vascular bundles was observed and photographed under a stereomicroscope. See [reference needed]. Figure 2 B. From Figure 2 B indicates that the virus was successfully silenced after inoculation with Verticillium wilt pathogens. GhVDRK1 Longitudinal section analysis of plant stems showed that the gene was successfully silenced after treatment. GhVDRK1 The secondary xylem of plants mutated with the gene turned black, while the secondary xylem of plants transformed with the empty vector TRV2::00 did not turn black, further demonstrating the effect of silencing. GhVDRK1 Subsequently, the plant's resistance to Verticillium wilt weakened.

[0052] 7. Disease Index 21 days after inoculating the plants with Verticillium wilt, the TRV:: GhVDRK1 Disease index was evaluated in silent plants, TRV::00 plants, and wild-type control plants. Resistance was assessed according to the cotton Verticillium wilt resistance level classification standard. The disease index was calculated as follows: DI = [Σ(disease grade × number of diseased plants at each grade) / (total number of plants surveyed × 4)] × 100. Disease grades were determined according to Table 6, and the disease index and resistance type were determined according to Table 7.

[0053] Based on the assessment of the disease index, the results showed that silence... GhVDRK1 Plants transformed with the TRV::00 empty vector showed significantly higher disease severity and lower resistance levels compared to plants transformed with this gene, demonstrating that this gene is an essential positive regulator of cotton resistance to Verticillium wilt. (See [link to relevant documentation]). Figure 5 .

[0054] Through wild type AS98 Disease index analysis with LFH10 (WT) revealed that the wild-type LFH10 had a relative disease index of 40, indicating a disease resistance level of susceptible. AS98 The relative disease index is 19.5, and the disease resistance is resistant. See [link to relevant documentation]. Figure 4 After successful gene silencing, the virus was inoculated with Verticillium wilt pathogen. AS98 And the WT disease resistance index increased significantly, see Figure 5 .

[0055] Table 6. Methods for determining disease severity (Source: Zhang Xinghua et al., Research progress on cotton resistance to blight and Verticillium wilt: Methods for identifying machine resistance, Jiangxi Journal of Agricultural Sciences) Table 7 Correspondence between Disease Index and Resistance Type (Source: Zhang Xinghua et al., Research progress on cotton resistance to blight and Verticillium wilt: Methods for identifying machine resistance, Jiangxi Journal of Agricultural Sciences) This invention compares upland cotton AS98 and LFH10 (WT) for GhVDRK1 Resistance of gene-silenced plants to Verticillium wilt after inoculation. The results showed that the leaves of gene-silenced plants under Verticillium wilt stress turned yellow and began to wilt, and their resistance to Verticillium wilt was significantly weakened, indicating that the gene played a positive role in the plant's resistance to Verticillium wilt pathogen.

Claims

1. Cotton genes GhVDRK1 Application in regulating cotton Verticillium wilt resistance, the cotton gene GhVDRK1 The encoded amino acid sequence is shown in Sequence 2 of the sequence listing. The regulation is to reduce the concentration of amino acids in cotton. GhVDRK1 The expression level of this gene will reduce resistance to Verticillium wilt in cotton, while increasing the expression level of the gene in cotton. GhVDRK1 The expression level of the gene will increase resistance to Verticillium wilt in cotton.

2. The application of the cotton gene GhVDRK1 according to claim 1 in regulating cotton Verticillium wilt resistance, characterized in that, The cotton gene GhVDRK1 The CDS sequence is shown in Sequence 1 of the sequence list.

3. The cotton gene according to claim 1 GhVDRK1 Its application in regulating resistance to Verticillium wilt in cotton is characterized by, The pathogen causing the cotton Verticillium wilt is Verticillium dahliae.

4. Cotton genes GhVDRK1 Application in breeding cotton varieties resistant to Verticillium wilt, the cotton gene GhVDRK1 The encoded amino acid sequence is shown in Sequence 2 of the sequence listing, which increases the concentration of amino acids in cotton. GhVDRK1 Gene expression levels are used to breed cotton varieties resistant to Verticillium wilt.

5. The cotton gene according to claim 4 GhVDRK1 Its application in breeding cotton varieties resistant to Verticillium wilt is characterized by, The cotton gene GhVDRK1 The CDS sequence is shown in Sequence 1 of the sequence list.

6. The cotton gene according to claim 4 GhVDRK1 Its application in breeding cotton varieties resistant to Verticillium wilt is characterized by, The pathogen causing the cotton Verticillium wilt is Verticillium dahliae.

7. A biomaterial for improving resistance to Verticillium wilt in cotton, characterized in that, The biomaterials include any one of the following (1) to (4): (1) DNA molecules encoding the amino acid sequence shown in Sequence 2 of the sequence listing; (2) An expression cassette containing the DNA molecule described in (1); (3) A recombinant vector comprising the DNA molecule of (1) or the expression cassette of (2); (4) A host cell or host bacterium containing the DNA molecule of (1) or the expression cassette of (2) or the recombinant vector of (3).

8. The biomaterial according to claim 7, characterized in that, The nucleotide sequence of the DNA molecule is shown in Sequence 1 of the sequence listing.

9. A method for improving resistance to Verticillium wilt in cotton, characterized in that, The method includes: improving the content of cotton materials GhVDRK1 Gene expression levels.

10. The method according to claim 9, characterized in that, The improvement of cotton materials GhVDRK1 Specific methods for measuring gene expression levels include: S1: Introducing the biomaterial of claim 7 or 8 into cotton material; S2: Screening out from the regenerated plants obtained from S1 GhVDRK1 Transgenic cotton lines with increased gene expression levels.

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