GhICDH3 gene and application thereof in regulation and control of verticillium wilt resistance of plants

Abstract

The invention relates to the field of plants, in particular to a GhICDH3 gene and application of the GhICDH3 gene in regulation and control of verticillium wilt resistance of plants. The invention provides an application of a GhICDH3 gene or a GhICDH3 protein coded by the GhICDH3 gene in any of the following items: an application in improvement of verticillium wilt resistance of plants; or the application in cultivating new germplasm with high verticillium wilt resistance. According to the invention, sequence characteristics, protein subcellular localization and ubiquitination modification site functions of the cotton GhICDH3 gene are systematically analyzed, and a disease-resistant mechanism of the gene for regulating reactive oxygen species outbreak, callose accumulation and lignin synthesis of cotton and an action relationship between the gene and interaction protein GhHSP70 are determined; a novel target gene capable of being used for genetic improvement of cotton verticillium wilt resistance is excavated, and the core technical problems that in cotton verticillium wilt resistance research, high-quality anti-disease genes are deficient, a key regulation mechanism is unknown, and efficient molecular breeding is difficult to achieve are solved.

Description

Technical Field

[0001] This invention relates to the field of plants, and more particularly to the GhICDH3 gene and its use in regulating plant resistance to Verticillium wilt. Background Technology

[0002] Cotton is a globally important economic crop, and its fiber is a core raw material for the textile industry. Verticillium wilt, a soil-borne vascular disease caused by Verticillium dahliae, leads to wilting of cotton plants, yellowing and drying of leaves, and in severe cases, plant death. It has become the primary biological stress factor restricting cotton yield and quality improvement. Currently, chemical control methods not only easily cause environmental pollution but also fail to completely eliminate pathogens in the soil; traditional disease-resistant breeding has drawbacks such as long cycles, scarcity of resistance sources, and the easy loss of resistance due to pathogen mutation.

[0003] In the study of the molecular mechanisms of plant-pathogen interactions, reactive oxygen species bursts, callose deposition, and lignin synthesis are key defense responses in plants against pathogen infection. Ubiquitination, as an important post-translational modification of proteins, can participate in regulating plant immune responses. The succinate signaling pathway is a core pathway for plant energy metabolism and biosynthesis, and its key enzyme, isocitrate dehydrogenase (ICDH), plays a crucial role in regulating carbon metabolism and redox homeostasis. However, the ubiquitination characteristics of ICDH family genes in cotton and their specific mechanisms of action in regulating Verticillium wilt resistance have not yet been systematically elucidated. Summary of the Invention

[0004] In view of this, the present invention provides the GhICDH3 gene and its use in regulating plant resistance to Verticillium wilt. This invention addresses the shortcomings in existing cotton Verticillium wilt research, such as the lack of a core resistance gene that can be efficiently utilized, the easy loss of resistance in resistant varieties bred using traditional breeding methods due to pathogen mutations, and the insufficient understanding of the molecular regulatory mechanisms of cotton's resistance to Verticillium wilt infection, particularly the inadequate research on ubiquitination modification of key proteins in the succinic acid signaling pathway and its mediated resistance pathways. By systematically analyzing the sequence characteristics, subcellular protein localization, and function of ubiquitination modification sites of the cotton GhICDH3 gene, this invention clarifies the disease resistance mechanism of this gene in regulating reactive oxygen species bursts, callose accumulation, and lignin synthesis in cotton, as well as its interaction with the protein GhHSP. 70 By studying the interaction relationships, we can discover novel target genes that can be used for genetic improvement of cotton resistance to Verticillium wilt, and solve the core technical problems of the lack of high-quality disease-resistant genes and the unclear key regulatory mechanisms in cotton research on Verticillium wilt, which make it difficult to achieve efficient molecular breeding.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of the GhICDH3 gene or the GhICDH3 protein it encodes in any of the following:

[0007] (a) Application in improving plant resistance to Verticillium wilt;

[0008] (b) Application in the cultivation of new germplasm with high resistance to Verticillium wilt;

[0009] The nucleotide sequence of the GhICDH3 gene is shown in SEQ ID NO:1;

[0010] The amino acid sequence of the GhICDH3 protein is shown in SEQ ID NO:2.

[0011] In some embodiments of the present invention, the above-described application of improving plant resistance to Verticillium wilt employs the step of overexpressing the GhICDH3 gene in plants.

[0012] In some embodiments of the present invention, the plant described above includes cotton.

[0013] In some embodiments of the present invention, the plant described above is cotton.

[0014] In some embodiments of the present invention, in the above applications, the GhICDH3 gene and GhHSP 70 Protein-protein interactions.

[0015] The present invention also provides a method for improving plant resistance to Verticillium wilt, comprising: overexpressing the GhICDH3 gene in the above-mentioned applications in plants.

[0016] The present invention also provides a method for cultivating plants with high resistance to Verticillium wilt, comprising: overexpressing the GhICDH3 gene used in the above-mentioned applications in the plant.

[0017] This invention also provides a GhICDH3 gene mutant, which has the following characteristics:

[0018] (1) An amino acid sequence as shown in SEQ ID NO:3; or

[0019] (2) An amino acid sequence obtained by substituting, deleting or adding one or more amino groups as shown in (1), and which has the same or similar function as the amino acid sequence shown in (1).

[0020] In some embodiments of the present invention, the sequence of the above-mentioned GhICDH3 gene mutant is shown in SEQ ID NO:3.

[0021] The present invention also provides a nucleic acid molecule encoding the above-mentioned GhICDH3 gene mutant, which has the following characteristics:

[0022] (3) A nucleotide sequence as shown in SEQ ID NO:4; or

[0023] (4) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (3); or

[0024] (5) A sequence having at least 80% homology to the nucleotide sequence described in (3) or (4); or

[0025] (6) The complementary sequence of the nucleotide sequence described in (3), (4) or (5).

[0026] In some embodiments of the present invention, the sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO:4.

[0027] This invention also provides the use of the above-mentioned GhICDH3 gene mutant and / or the above-mentioned nucleic acid molecule in any of the following:

[0028] (c) Application in improving plant resistance to Verticillium wilt;

[0029] (d) Application in cultivating new germplasm with high resistance to Verticillium wilt; the step of improving plant resistance to Verticillium wilt is to overexpress the nucleic acid molecule in the plant; the plant includes: cotton.

[0030] In some embodiments of the present invention, the plant described above is cotton.

[0031] The present invention also provides a method for improving plant resistance to Verticillium wilt, comprising: overexpressing the above-mentioned nucleic acid molecules in plants.

[0032] The present invention also provides a method for cultivating plants with high resistance to Verticillium wilt, comprising: overexpressing the above-mentioned nucleic acid molecules in the plant.

[0033] In some embodiments of the present invention, GhICDH3 K407RG AGCAAGACTTTGTTAA (as shown in SEQ ID NO:4).

[0034] GhICDH3 K407R Mutant protein sequence: MGFEKIKVANPIVEMDGDEMTRVIWKSIKDKLILPFVELDIKYFDLGLPHRDATDDKVTIESAEATLKYNVAIKCATITPDEARVKEFDLKQMWK SPNGTIRNILNGTVFREPIICKNVPRLVPGWTKPICIGRHAFGDQYRATDAVIKGAGKLKLVFVPEGQGEKTEYEVFNFTGEGGVSLAMYNTDESIRAFAEASM NTAYQKKWPLYLSTKNTILKKYDGRFKDIFQEVYEANWKSKYEAAGIWYEHRLIDDMVAYALKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLVCPDG KTIEAEAAHGTVTRHFRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNPKLLDFIEKLEAACIATVESGKMTKDLALIIHGSKLARDKYLNTEEFIDAVAADL R ARLC (as shown in SEQ ID NO:3).

[0035] This invention systematically analyzes the sequence characteristics, ubiquitination modification site function, and subcellular localization of the cotton GhICDH3 gene, clarifying the molecular mechanism by which this gene enhances cotton's resistance to Verticillium wilt by regulating reactive oxygen species bursts, callose deposition, and lignin synthesis; and verifies the relationship between GhICDH3 and GhHSP. 70 The study aims to investigate protein-protein interactions and key functional regions, and to identify novel target genes that can be used for genetic improvement of cotton resistance to Verticillium wilt, providing a theoretical basis and technical support for breeding cotton varieties with stable and durable disease resistance. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0037] Figure 1 This shows the K407 ubiquitination site of cotton isocitrate dehydrogenase GhICDH3;

[0038] Figure 2 Indicates the expression level of the GhICDH3 gene;

[0039] Figure 3This shows the subcellular localization results of GhICDH3 protein;

[0040] Figure 4 The phenotype and disease index of cotton after transient silencing of the GhICDH3 gene are shown; where: A shows that the positive control gene was effectively silenced; B shows the susceptibility phenotype of cotton; C shows the silencing efficiency of the GhICDH3 gene; and D shows the statistical cotton disease index.

[0041] Figure 5 The results of the test for cotton-related disease resistance indicators are shown below; A shows the level of reactive oxygen species in leaves; B shows the H2O2 content in leaves; C shows the callose deposition in cotton leaves; D shows the callose content in leaves; E shows the color of the xylem in cotton; F shows the lignin content in the stem; G shows the fungal residue in the cotton stem; H shows the degree of browning in the cotton stem.

[0042] Figure 6 The results of disease resistance identification of transgenic and mutant Arabidopsis thaliana with ICDH3 are shown; where: A shows the identification results of positive Arabidopsis thaliana plants; B shows the expression level of GhICDH3 gene in positive plants; C shows the disease phenotype of Arabidopsis thaliana; and D shows the statistical Arabidopsis thaliana disease index.

[0043] Figure 7 Show GhICDH3 and GhHSP 70 The results of protein-protein interaction verification; where: A shows the yeast two-hybrid assay results; B shows the bimolecular fluorescence complementation assay results; C shows the luciferase complementation assay results;

[0044] Figure 8 GhHSP 70 Gene expression levels after infection with Verticillium dahliae. Detailed Implementation

[0045] This invention discloses the GhICDH3 gene and its use in regulating plant resistance to Verticillium wilt.

[0046] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0047] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0048] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0049] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0050] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0051] The test materials involved in this invention include:

[0052] (1) Plant materials

[0053] The plant materials used in this embodiment were upland cotton and Arabidopsis thaliana. The upland cotton material was JM-11, and the tobacco material was Nicotiana benthamiana. The transgenic Arabidopsis thaliana recipient genotype was Colombia wild type (Colombia wild type, Col-0). The mutant Arabidopsis thaliana (T-DNA insertion mutant: through Agrobacterium-mediated T-DNA insertion technology, foreign DNA fragments are randomly inserted into the Arabidopsis thaliana genome, resulting in loss or alteration of gene function. Common T-DNA insertion mutant libraries include SALK, SAIL, GABI-Kat, WiscDsLox, etc. These mutant libraries cover most of the genes in the Arabidopsis thaliana genome, providing rich resources for functional genomics research) was purchased from the AraShare website. Cotton and tobacco were sown in a 1:1 mixture of nutrient soil and vermiculite and cultured in a constant temperature and humidity incubator at 28°C with 16 hours of light / 8 hours of darkness. Arabidopsis thaliana was sown in a 1:1 mixture of potting soil and vermiculite and cultured in a constant temperature and humidity incubator at 22°C with 16 hours of light and 8 hours of darkness.

[0054] (2) Vector and strain

[0055] Escherichia coli DH5α and Agrobacterium GV3101 were purchased from Beijing Coollab Technology Co., Ltd. The cotton deciduous wilt strain was the highly pathogenic Verticillium dahliae strain Vd991 (Vd991), which was preserved in our laboratory. Vectors used included pTRV1, pTRV2, pTRV2:CLA1, pCAMBIA2300, pGADT7 AD, pGBKT7 BD, pXY106-nYFP, pXY104-cYFP, pCAMBIA1300-cLuc, and pCAMBIA1300-nLuc, all of which were common vectors preserved in our laboratory.

[0056] (3) Main reagents

[0057] Plant total RNA mini-extraction kit, reverse transcription kit, and quantitative fluorescence kit were purchased from Beijing TransGen Biotechnology Co., Ltd.; plasmid mini-extraction kit was purchased from Beijing Jinsha Biotechnology Co., Ltd.; DNA Marker was purchased from Monad Biotechnology Co., Ltd.; high-fidelity PCR Mix was purchased from Nanjing Novizan Biotechnology Co., Ltd.; cotton protoplast preparation and transformation kit was purchased from Beijing Coolerbott Biotechnology Co., Ltd.; plant tissue reactive oxygen species detection kit (NBT), plant callose staining solution (aniline blue), lignin staining kit (phloroglucinol), reactive oxygen species content assay kit, and lignin content assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd.; callose content assay kit was purchased from Shanghai Yubo Biotechnology Co., Ltd.; restriction endonucleases EcoRI, BamHI, KpnI, and SalI were purchased from NEB (Beijing) Co., Ltd.

[0058] In Examples 1 to 12 and Effect Examples 1 to 8 of the present invention, the raw materials and reagents used can all be purchased from the market.

[0059] The present invention will be further illustrated below with reference to the embodiments:

[0060] Example 1 Planting of Plant Materials

[0061] Select plump, disease-free cotton seeds, delint them with concentrated sulfuric acid, rinse thoroughly with running water until neutral, and air dry in a cool, ventilated place. Soak the treated seeds in sterile distilled water for 24 hours, then sow them in a sterilized cultivation substrate (a mixture of peat moss, vermiculite, and perlite in a 3:1:1 volume ratio), and transfer them to an artificial climate chamber for cultivation. The cultivation conditions are controlled as follows: temperature 25±2℃, photoperiod 16 hours light / 8 hours dark, relative humidity 60%-70%. The same conditions apply to tobacco seeds. Arabidopsis seeds are disinfected by soaking in a 75% ethanol solution for 3 minutes, then rinsed three times with sterile distilled water for 2 minutes each time. Next, they are disinfected by soaking in a 5% sodium hypochlorite solution for 30 seconds and rinsing three times with sterile distilled water for 2 minutes each time to thoroughly remove residual disinfectant. Finally, the disinfected seeds are sown on the surface of 1 / 2 MS solid medium, vernalized at 4℃ for 48 hours, and then transferred to an artificial climate chamber for cultivation.

[0062] Example 2 Activation and culture of Verticillium wilt

[0063] The Vd991 strain, stored at -80℃, was revived and first streaked onto PDA plates and then incubated in a dark incubator at 28℃ for 24-48 hours to restore its activity. An appropriate amount of colonies was then inoculated into Czapek's agar and cultured at 28℃ with shaking at 200 rpm for 12-18 hours until the logarithmic growth phase. The culture medium was analyzed by optical density (OD) measurement. 600 The culture medium and equipment were determined and diluted to the required inoculum concentration for subsequent experiments. All operations were performed under aseptic conditions, and the culture medium and equipment were autoclaved at 121°C for 30 minutes. The incubation time and growth phase were recorded to ensure reproducibility.

[0064] Example 3: Inoculation treatment of cotton seedlings with Verticillium wilt

[0065] Healthy cotton seedlings at the two-leaf-one-heart stage were selected as inoculation material. Verticillium wilt pathogen Vd991 cultured on Czapek's medium was harvested during the logarithmic growth phase, and the spore concentration was adjusted to 1×10⁻⁶. 7 The concentration of bacteria per milliliter was 5 ml. Before inoculation, the seedling substrate was kept moderately moist. The root wound inoculation method was used for infection. After making a slight cut on the root, 5 ml of bacterial suspension was drawn and poured into the bottom of the soil for the seedlings to absorb naturally. After inoculation, the symptoms and disease progress were recorded regularly, and a sterile water treatment was set up as a negative control.

[0066] Example 4: Cotton Protein Ubiquitination Modification Omics Determination

[0067] The main steps include sample preparation, protein extraction and quantification, protein reduction, alkylation and enzymatic digestion, ubiquitinated peptide enrichment, LC-MS detection, and data analysis. Proteomics assays were performed by Beijing Novogene Technology Co., Ltd.

[0068] Example 5: Observation of cotton disease index

[0069] A control group (treated with sterile water) and an inoculated group (inoculated with Verticillium wilt according to the method in Example 3) were set up at the two-leaf-one-heart stage of cotton. Sampling began two days after inoculation and was recorded as hour 0. Subsequently, plant tissues were collected at 3, 6, 12, 24, and 48 hours for physiological, biochemical, and molecular index determination. Disease assessment was conducted on day 15 after inoculation. Based on the degree of leaf damage and overall plant symptoms, the disease was classified into four grades: Grade 0, no symptoms; Grade 1, 10%-20% of leaves slightly wilted or yellowed; Grade 2, 20%-50% of leaves moderately wilted or yellowed; Grade 3, 50%-75% of leaves significantly wilted or yellowed; Grade 4, more than 75% of leaves wilted, severely yellowed, or the plant died. The Disease Index (DI) is calculated using the following formula: DI = [Σ(Disease grade × Number of corresponding diseased plants) / (Total number of plants surveyed × 4)] × 100. At least three replicates should be set up for statistical analysis, with no fewer than 30 plants per replicate, and the disease grade of each plant should be recorded.

[0070] Example 6: RNA extraction from plant leaves and qRT-PCR experiment

[0071] 1. RNA extraction (EasyPure® RNA Kit)

[0072] (1) Take 20 mg of plant tissue ground with liquid nitrogen and immediately add 600 μL of BB4 and 30 μL of Proteinase K. After mixing, treat at 56°C for 10-20 minutes, centrifuge at 12,000 rpm for 5 minutes, and aspirate the supernatant into an RNase-free centrifuge tube.

[0073] (2) Add 1 volume of 70% ethanol to the clear liquid.

[0074] (3) Vortex thoroughly to disperse the precipitate. Add the resulting solution and precipitate together to a centrifuge column, centrifuge at 12,000 rpm for 30 seconds, and discard the eluent.

[0075] (4) Add 500 μL of CB4 to the centrifuge column, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the eluent. If genome removal is required, add 80 μL of DNase I working solution to the center of the centrifuge column, incubate at room temperature for 15 minutes, and repeat step 3 once.

[0076] (5) Add 500 μL of WB4, centrifuge at 12,000 rpm for 30 seconds at room temperature, and discard the effluent.

[0077] (6) Repeat step (4) once.

[0078] (7) Centrifuge at 12,000 rpm for 2 minutes at room temperature to completely remove residual ethanol.

[0079] (8) Transfer the centrifuge column into a new 1.5 mL RNase-free centrifuge tube and add 30-100 μL of RNase-free water to the center of the centrifuge column. Let it stand at room temperature for 1 minute.

[0080] (9) Centrifuge at 12,000 rpm for 2 minutes at room temperature to elute RNA.

[0081] 2. RNA reverse transcription (using the EasyScript® All-in-One First-Strand cDNASynthesis SuperMix for qPCR kit)

[0082] Table 1 Reverse transcription reaction system

[0083]

[0084] Table 2 Reverse transcription reaction procedure

[0085]

[0086] 3. qRT-PCR experiment (using PerfectStart® Green qPCR SuperMix dye premix for real-time PCR)

[0087] qRT-PCR was performed using a Light Cycler™ 480 System II (Roche, Switzerland). Expression levels of all target genes were determined by 2... -ΔΔCT Methods were used to calculate and select the cotton GhUBQ7 gene as the internal reference gene for qRT-PCR. All quantitative PCR analyses were performed at least three biological replicates. The qRT-PCR reaction system is shown in Table 3, and the qRT-PCR amplification program is shown in Table 4.

[0088] Table 3 qRT-PCR reaction system

[0089]

[0090] Table 4 qRT-PCR amplification program

[0091]

[0092] Example 7 Subcellular co-localization of GhICDH3 protein

[0093] 1. Source of GhICDH3 gene sequence

[0094] To achieve subcellular localization of the GhICDH3 protein, the complete CDS sequence and corresponding protein sequence of GhICDH3 were downloaded from the online website cotton MD. The CDS sequence of the GhICDH3 gene was amplified using GFP-GhICDH3-F / R primers (Table 5). The amplified CDS fragment was inserted into the expression vector pCAMBIA2300 containing the green fluorescent protein (GFP) gene. The successfully constructed recombinant plasmid was then transformed into cotton protoplasts and observed using a laser scanning confocal microscope (Olympus FV3000).

[0095]

[0096] GhICDH3 protein sequence: MGFEKIKVANPIVEMDGDEMTRVIWKSIKDKLILPFVELDIKYFDLGLPHRDATDDKVTIESAEATLKYNVAIKCATITPDEARVKEFDLKQMWKSPNGTIRNILNGTVFREPIICKNVPRLVPGWTKPICIGRHAFGDQYRATDAVIKGAGKLKLVFVPEGQGEKTEYEVFNFTGEGGVSLAMYNTDESIRAFAEASMNTA YQKKWPLYLSTKNTILKKYDGRFKDIFQEVYEANWKSKYEAAGIWYEHRLIDDMVAYALKSEGGYVWACKNYDGDVQSDFLAQGFGSLGLMTSVLVCPDGKTIEAEA AHGTVTRHFRVHQKGGETSTNSIASIFAWTRGLAHRAKLDDNPKLLDFIEKLEAACIATVESGKMTKDLALIIHGSKLARDKYLNTEEFIDAVAADLKARLC (as SEQ ID NO:2).

[0097] 2. Preparation and transformation of cotton protoplasts (Coollabo cotton protoplast preparation and transformation kit)

[0098] Isolation and preparation of protoplasts:

[0099] (1) Prepare cotton materials, preferably using cotyledons of seedlings 14 days after they are grown.

[0100] (2) Take 3-5 leaves and cut them into thin strips of 0.5-1.0 mm with a sharp blade parallel to the main vein.

[0101] (3) Place the cut strips (with the main vein removed) into a small beaker with a volume of 50 ml containing 20 ml of enzymatic hydrolysate, and wrap it with aluminum foil.

[0102] (4) Incubate at 25°C and 45 RPM for 5 hours in the dark.

[0103] (5) Rinse the cell sieve with 1 ml of culture medium CM to break the surface tension of the filter and facilitate smooth filtration in the future.

[0104] (6) Filter the enzymatically digested product through a cell sieve, and gently squeeze the digest with tweezers or the end of a pipette tip to help it fully release the protoplasts. Rinse the digestion vessel and undigested leaves twice with 5 ml of culture medium CM, and squeeze again to collect all the liquid into a 50 ml round-bottom centrifuge tube.

[0105] (7) Using a pointed pipette tip, take a drop of the enzyme digest for microscopic examination. If round protoplasts can be observed in a 100× field of view, the experiment can continue. If a large number of protoplasts are broken, shrunken, or blackened in the field of view, discard them.

[0106] (8) Select a horizontal rotor, centrifuge at 150 rpm for 3 minutes at room temperature, increase speed by 3, decrease speed by 3, and remove supernatant.

[0107] (9) Add 2 ml of culture medium CM to the above precipitate and gently resuspend the protoplasts at the bottom. Transfer them to a 2 ml round-bottom centrifuge tube using a pipette tip.

[0108] (10) Centrifuge at 150 rpm for 3 minutes at room temperature, then increase the speed by 3 and decrease it by 3, discarding the supernatant. Resuspend the protoplasts in an appropriate amount of RS resuspension solution and adjust the protoplast density to 2 × 10⁻⁶. 5 per milliliter.

[0109] Transformation of protoplasts:

[0110] (1) Add 5-20 μg of purified high-quality plasmid to a 2 mL round-bottom centrifuge tube, followed by 100 μL of the resuspended protoplasts. Then add 110 μL of transformation buffer TP, slowly invert to mix, and incubate at room temperature in the dark for 15 minutes.

[0111] (2) Add 1 ml of culture medium CM, gently invert to mix, and terminate the transformation. Centrifuge at 150 rpm for 3 minutes at room temperature, increasing speed by 3 and decreasing speed by 3, and remove the supernatant as much as possible without losing protoplasts.

[0112] Example 8: VIGS silencing test and detection of related disease resistance indicators

[0113] 1. VIGS Silencing Experiment

[0114] Based on the GhICDH3 gene sequence, specific VIGS primers were designed (Table 5). The target fragment was amplified by PCR and directionally cloned into the pTRV2 vector to construct the recombinant expression plasmid pTRV2-GhICDH3. The recombinant plasmid and the helper vector pTRV1 were transformed into Agrobacterium GV3101 competent cells using a freeze-thaw method. After culturing Agrobacterium containing different vectors to the logarithmic growth phase, the bacterial concentration was adjusted and mixed at a 1:1 ratio. The cotyledons of cotton seedlings were then infected via leaf dorsal puncture injection. A positive control group injected with pTRV2-CLA1 (for observing leaf albinism phenotype) and a negative control group injected with the empty vector pTRV2 were simultaneously set up. The VIGS system was considered successfully induced when the new true leaves of the positive control plants showed a typical reticulated albinism phenotype. Subsequently, true leaf tissues from the silencing group and the control group, with consistent growth stages, were collected. Total RNA was extracted using an RNA extraction kit and reverse transcribed into cDNA. The relative expression level of GhICDH3 in each group was detected using qRT-PCR to calculate the gene silencing efficiency. The experiment was conducted in three biological replicates to ensure the scientific validity of the silencing effect and the consistency of the data.

[0115] 2. Detection of relevant disease resistance indicators

[0116] Staining and content detection of reactive oxygen species in cotton leaves:

[0117] Leaf reactive oxygen species staining (Solepro Plant Tissue Reactive Oxygen Detection Kit):

[0118] (1) Collect plant seedlings or leaves inoculated with Verticillium dahliae Vd991 and treated with clean water, wash them with tap water, and place them on filter paper to absorb excess water.

[0119] (2) Immerse the experimental sample in NBT staining solution, treat with negative pressure of -0.1 MPa for 30 minutes, and then stain at room temperature in the dark for 4-12 hours until the positive part turns dark blue and the rest of the part is light blue or nearly colorless or the color of the plant itself.

[0120] (3) Carefully remove the sample with tweezers, rinse it in distilled water 3-5 times, place it on filter paper to absorb excess water, and then immerse it in tissue decolorization solution in a water bath at 70-80℃ for 20-40 minutes until the tissue background color is completely removed. If the decolorization solution is too dark during the treatment, fresh tissue decolorization solution can be used.

[0121] Leaf reactive oxygen species content detection (Solepro hydrogen peroxide content detection kit):

[0122] (1) Weigh about 0.1 g of plant leaf tissue, add 1 ml of reagent one and homogenize in an ice bath; centrifuge at 8000 rpm and 4℃ for 10 minutes, take the supernatant and place it on ice for testing.

[0123] (2) Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 415 nm, and zero the spectrophotometer with distilled water.

[0124] (3) Place reagents two, three and four in a water bath at 25°C for more than 10 minutes.

[0125] (4) Dilute the 1 mmol / mL standard with acetone to a 2 μmol / mL standard.

[0126] (5) Add the corresponding reagents to the test tube, blank tube and standard tube in sequence, and calculate ∆A_test = A_test tube - A_blank tube, ∆A_standard = A_standard tube - A_blank tube.

[0127] Staining and content detection of callose in cotton leaves:

[0128] Leaf callosity staining (Soluble plant callosity staining solution):

[0129] (1) The true leaves of cotton were directly immersed in AAF fixative for 24 hours.

[0130] (2) Rinse twice with anhydrous ethanol for 1 minute each time, then transfer to at least 10 times the volume of 100% ethanol for storage.

[0131] (3) Before staining, remove the tissue and immerse it in 50% ethanol for 30 minutes to equilibrate. Then remove and drain slightly.

[0132] (4) Then immerse in 1×PBS for 30 minutes to equilibrate, then remove and drain slightly.

[0133] (5) Prepare the staining working solution before use. Add or immerse the tissue sections in the callosity staining working solution and stain at room temperature in the dark for 1 hour.

[0134] (6) Add 25 μL of water-based gelatin mounting medium or anti-fluorescence decay mounting medium to a glass slide, carefully transfer the stained tissue to the glass slide, add a small amount of mounting medium, and then mount the slide for observation.

[0135] Leaf callosity content detection (Yubo Biotechnology callosity content test kit):

[0136] (1) Weigh about 0.1 g of plant leaf tissue, add 1 ml of reagent one and homogenize in an ice bath; centrifuge at 8000 rpm and 4℃ for 10 minutes, take the supernatant and place it on ice for testing.

[0137] (2) Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 570 nm, and zero the spectrophotometer with distilled water.

[0138] (3) Add the corresponding reagents to the test tube, blank tube and standard tube in sequence, place them in a boiling water bath for 15 minutes, cool them and repeatedly invert the EP tube several times, measure the absorbance at 570 nm, and calculate ∆A_test = A_test tube - A_blank tube, ∆A_standard = A_standard tube - A_blank tube.

[0139] Lignin staining and content detection of cotton stems:

[0140] Stem lignin staining (Soleb lignin staining kit):

[0141] (1) Place the cut slices on a glass slide, add 100 μL of lignin acidification solution, and acidify for 1 minute.

[0142] (2) Add an equal amount of phloroglucinol staining solution to the acidified material above, gently shake the slide or gently blow and aspirate the staining solution with a pipette, and let it stand for 1 minute.

[0143] (3) Cover with a coverslip and quickly place under a microscope for observation.

[0144] Detection of lignin content in stems (Solebo lignin content detection kit):

[0145] (1) Dry the sample at 80°C to constant weight, grind it, pass it through a 30-50 mesh sieve, and weigh about 5 mg into a 1.5 mL EP tube.

[0146] (2) Preheat the spectrophotometer for more than 30 minutes, adjust the wavelength to 280 nm, and zero it with glacial acetic acid.

[0147] (3) Add the corresponding reagents to the test tube and blank tube in sequence and mix thoroughly. Incubate in an 80°C water bath for 40 minutes to carry out acetylation. Shake for 30 seconds every 10 minutes and then cool naturally to room temperature.

[0148] (4) Pipette the reaction solution into a 1 mL quartz cuvette and measure the absorbance value A at 280 nm. Record it as A test tube and A blank tube. Calculate ∆A = A test tube - A blank tube.

[0149] Browning detection of cotton stem segments: On day 25 after inoculation with Verticillium dahliae, stem segments of 8 TRV :: 00 and TRV :: ICDH3 plants were randomly selected. Tissue from the same location on the stem was cut, and the stem segment was longitudinally cut into two equal parts with a knife. The degree of browning of the stem segment was observed under a microscope, and the relevant phenotypes were recorded by taking pictures.

[0150] Example 9: Creation and Disease Resistance Identification of GhICDH3 Transgenic Arabidopsis

[0151] 1. Creation of transgenic Arabidopsis thaliana

[0152] Creation and screening of transgenic Arabidopsis thaliana

[0153] (1) Vector transformation and strain activation: The pCAMBIA2300-GhICDH3 recombinant plasmid was introduced into Agrobacterium GV3101 competent cells by freeze-thaw method. Positive clones were obtained by PCR identification and activated culture was performed.

[0154] (2) Agrobacterium amplification and collection: Activated Agrobacterium was inoculated into LB liquid medium and cultured with shaking at 28°C until OD. 600 The bacterial culture was centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded to collect the bacterial precipitate.

[0155] (3) Preparation of infection solution: The bacterial cells were washed and resuspended using a resuspension solution (containing 5% sucrose and 0.02% Silwet L-77). The final concentration of the infection solution was adjusted to OD using a UV spectrophotometer. 600 It is 0.8-0.9.

[0156] (4) Arabidopsis thaliana transformation: Arabidopsis thaliana plants with consistent growth and in the early flowering stage (only flower buds and no pods) were selected and transformed using the inflorescence immersion method. The inflorescences were completely immersed in the inoculum solution for 45 seconds, followed by 24 hours of light-protected and moisturized treatment. After normal light was restored, the plants were continued to be cultivated in a greenhouse until the seeds matured.

[0157] (5) Resistance screening and identification: T0 generation seeds were collected, surface sterilized, and sown on MS solid medium containing kanamycin screening pressure. After germination in a light incubator for 7-10 days, suspected positive seedlings with well-developed root systems and dark green leaves were selected and transplanted to nutrient soil.

[0158] (6) Homozygous selection: DNA and RNA levels were used to identify transplanted plants to confirm transgenic positive plants. The identified positive plants were harvested individually and subjected to resistance screening again. The T2 generation lines were screened according to the Mendelian segregation ratio (3:1) to finally obtain stable homozygous transgenic lines.

[0159] 2. Identification of disease resistance in transgenic and mutant Arabidopsis thaliana

[0160] To evaluate the disease resistance of GhICDH3 transgenic Arabidopsis and its mutant Arabidopsis, three-week-old wild-type Arabidopsis (Col-0), transgenic Arabidopsis, and mutant Arabidopsis (20 plants per group) were inoculated with Verticillium dahliae. Col-0 plants inoculated with water served as a negative control, and Col-0 plants inoculated with Verticillium dahliae served as a positive control. After 14 days, the Arabidopsis phenotype was observed, and the disease index was calculated (the method for calculating the disease index is as described in Example 5).

[0161] Example 10: GhICDH3 Screening Experiment for Interacting Proteins

[0162] (1) Construction and identification of the bait expression vector: Specific primers were designed based on the GhICDH3 gene sequence, and the target fragment was amplified by PCR. The fragment was then cloned into the yeast DNA binding domain vector pGBKT7 BD using homologous recombination technology to construct the bait plasmid pGBKT7-GhICDH3. The recombinant plasmid was transformed into the yeast recipient strain Y2HGold, and the accuracy of the vector construction was verified by colony PCR and sequencing.

[0163] (2) Toxicity and self-activation detection of bait protein: To eliminate interference from the bait protein on the yeast system, pGBKT7-GhICDH3 and empty vector pGADT7 AD were co-transformed into Y2HGold yeast competent cells. The cells were inoculated onto double-deficient medium (SD / -Trp / -Leu) and quadruple-deficient medium containing X-α-Gal and AbA (SD / -Trp / -Leu / -His / -Ade / X / A), respectively. If the strain grew normally only on the double-deficient medium and did not grow or show a blue color on the quadruple-deficient medium, it proved that the bait protein GhICDH3 was non-toxic to yeast cells and had no self-activation activity, and could be used for subsequent library screening.

[0164] (3) Yeast two-hybrid library screening: Cotton cDNA libraries were screened using yeast hybridization. Y2HGold strain containing the bait plasmid was hybridized with Y187 strain containing the AD-cDNA library plasmid in 2×YPDA medium. After centrifugation and resuspending, the hybridization products were evenly spread on highly selective medium (SD / -Trp / -Leu / -His / -Ade / X / A) for initial screening. Clones that grew well and showed a blue color were selected as candidate positive clones after incubation at 30℃ for 3-5 days.

[0165] (4) Verification and sequencing analysis of positive clones: To rule out false positives, the positive clones obtained from the initial screening were streaked twice on the same selective medium for identification. The yeast plasmids identified as positive were extracted, transformed into E. coli DH5α for amplification and extraction, and then PCR amplification and sequencing were performed using universal primers for the library. The obtained sequencing sequences were compared and analyzed by BLAST in the NCBI database to screen for candidate proteins that have potential interactions with GhICDH3.

[0166] Example 11: Yeast two-hybrid assay (Y2H), in vivo bimolecular complementation assay (BiFC), and in vivo luciferase assay (LCI) to verify protein interactions.

[0167] (1) Yeast two-hybrid experiment: to further verify the relationship between GhICDH3 and GhHSP 70The physical interaction was investigated using a point-to-point hybridization method. First, the full-length CDS sequence of GhICDH3 was cloned into the bait vector pGBKT7 (pGBKT7-GhICDH3), and then GhHSP was... 70 The full-length CDS sequence was cloned into the activation vector pGADT7 (pGADT7-GhHSP). 70 Then the above recombinant plasmid pair (pGBKT7-GhICDH3 + pGADT7-GhHSP) was used. 70 The yeast strain GhICDH3 was co-transformed into the recipient yeast strain Y2HGold. A positive control (pGBKT7-p53 + pGADT7-T) and a negative control (pGBKT7-Lam + pGADT7-T) were also established. The transformed yeast was then diluted to different gradients and inoculated into double-deficient medium (SD / -Trp / -Leu) and quadruple-deficient medium (SD / -Trp / -Leu / -His / -Ade / X / A). If the experimental group grew normally and turned blue on the quadruple-deficient medium, while the negative control did not grow, it confirmed the interaction between GhICDH3 and GhHSP. 70 There is a direct interaction within the yeast cell.

[0168] (2) Bimolecular fluorescence complementation experiment: The interaction between GhICDH3 and GhHSP in plant cells was observed using BiFC technology. 70 To investigate the subcellular localization and interactions of the fluorescent protein, fusion expression vectors containing the N-terminus and C-terminus of the fluorescent protein were first constructed. GhICDH3 was ligated into the pXY106-nYFP vector, and GhHSP was ligated into the vector. 70 The pXY106-cYFP vector was ligated into the recombinant plasmid. The constructed recombinant plasmids were then transformed into Agrobacterium GV3101. Agrobacterium containing both plasmids were mixed in a 1:1 ratio and resuspended at OD200. 600 =0.6. The mixed bacterial solution was then injected into young leaves of *Nicotiana benthamiana* using an injection method. After 24 hours of dark treatment, the tobacco plants were cultured under light for 48 hours. Tobacco leaves were then collected, and yellow fluorescence signals were observed using a laser confocal scanning microscope. If fluorescence was observed at specific locations within the cells, it indicates that the two proteins are spatially adjacent within the plant and interact with each other.

[0169] (3) Luciferase Complementation (LCI) Assay: The reliability of protein-protein interactions was further verified at the in vivo level using the LCI assay. First, GhICDH3 and GhHSP were... 70 nLUC-GhICDH3 and cLUC-GhHSP were constructed by ligating the cLUC-GhICDH3 vector into the pCAMBIA1300-nLuc vector and the pCAMBIA1300-cLuc vector, respectively. 70The expression frame was fused. Then, the recombinant plasmid was transformed into Agrobacterium and co-infected with Tobacco Benzoenta leaves. Two vectors were set up: “nLUC-GhICDH3+cLUC empty vector” and “nLUC empty vector+cLUC-GhHSP”. 70 "As a negative control, after 48-72 hours of infection, a fluorescein substrate was evenly sprayed onto the underside of tobacco leaves and placed in the dark for 5 minutes. The chemiluminescence signal was captured using a plant in vivo imaging system. If a strong luminescence signal was observed in the experimental group area, while no obvious luminescence was observed in the control group, then the interaction between GhICDH3 and GhHSP was biochemically confirmed." 70 Interactions within the body.

[0170] Example 12 Detection of the effect of Verticillium dahliae infection on GhICDH3 gene expression level

[0171] (1) The cotton planting and inoculation methods are the same as those in Examples 1 to 3.

[0172] (2) The methods for extracting plant RNA and detecting gene expression levels are the same as in Example 6.

[0173] Table 5 Primers used in this study

[0174]

[0175]

[0176] Example 1: Results of cotton protein ubiquitination modification proteomics assay

[0177] Protein ubiquitination modification analysis revealed that some proteins in cotton underwent ubiquitination modification during pathogen infection, such as the isocitrate dehydrogenase GhICDH3 in the succinate signaling pathway. Furthermore, the ubiquitination level of this protein changed one day after inoculation with *Verticillium dahliae*. Mass spectrometry further identified the ubiquitination site, detecting one site: lysine residue 407 (_DKYLNTEEFIDAVAADLK(gl)AR_). Figure 1 (As shown).

[0178] Example 2: Effect of Verticillium dahliae infection on GhICDH3 gene expression level

[0179] qRT-PCR results showed that the expression level of GhICDH3 was significantly upregulated by *Verticillium dahliae*. In the early stage of infection (0-6 hours), the gene expression level rose slowly; then it increased rapidly within 6-24 hours, reaching a peak at 24 hours post-infection, at which point its expression level was approximately 3.5 times that of the control group, with a highly significant difference. With prolonged infection time, although the expression level decreased somewhat by 48 hours, it was still significantly higher than the initial level (e.g., ...). Figure 2(As shown in the image). The above results indicate that the GhICDH3 gene exhibits a strong early response to Verticillium dahliae infection, suggesting that this gene may play an important regulatory role in the defense response of cotton against Verticillium dahliae-induced infection.

[0180] Table 6

[0181]

[0182] Table 6 corresponds to Figure 2 The data.

[0183] Example 3: Subcellular localization results of GhICDH3 protein

[0184] Constructing the pCAMBIA2300-GhICDH3 recombinant plasmid and point-mutating pCAMBIA2300-GhICDH3 K407R The recombinant plasmid was simultaneously transformed into the protoplasts of cotton. The results are as follows: Figure 3 As shown, the green fluorescence exhibited by the empty vector was distributed in the cell membrane, cytoplasm, and nucleus, and overlapped with the purple autofluorescence of the chloroplasts. However, the protoplasts expressing the pCAMBIA2300-GhICDH3 fusion gene showed green fluorescence in the cytoplasm, which did not overlap with the purple autofluorescence of the chloroplasts. Furthermore, the point mutation pCAMBIA2300-GhICDH3... K407R The fusion gene also only showed green fluorescence in the cytoplasm. The results indicate that the GhICDH3 protein is normally localized in the cytoplasm, and that the location of the GhICDH3 protein did not change after the lysine mutation at the ubiquitination site of position 407 was changed to arginine, suggesting that ubiquitination modification does not affect the localization of the GhICDH3 protein (e.g., Figure 3 (As shown).

[0185] Example 4: Results of VIGS silencing test and related disease resistance indicators

[0186] This study investigated the function of the GhICDH3 gene in cotton disease resistance using gene silencing technology induced by tobacco brittle virus. Ten days after bacterial injection, the true leaves of the positive control TRV::CLA1 plants showed a distinct albinism phenotype (e.g., ...). Figure 4 As shown in Figure A), this indicates that the VIGS system is functioning normally. Silencing efficiency was detected by qRT-PCR, revealing that compared to the control group TRV::00, the expression level of this gene in the TRV::GhICDH3 treatment group was significantly reduced, with a silencing efficiency exceeding 70% (e.g., Figure A). Figure 4As shown in Figure C), this indicates that the GhICDH3 gene was effectively silenced. Twenty-five days after inoculation with *Verticillium dahliae*, the plants exhibited varying degrees of Verticillium wilt symptoms. The control group (TRV::00) plants showed only mild leaf yellowing, while the silenced group (TRV::GhICDH3) plants showed more severe wilting, extensive leaf yellowing, and necrosis (as shown in Figure C). Figure 4 As shown in B). The statistical results of the disease index show (e.g., Figure 4 As shown in Figure D), the disease index of the control group was approximately 18, while the disease index of the silenced group was significantly higher, reaching around 50, with a highly significant difference. These results indicate that silencing the GhICDH3 gene significantly increases the susceptibility of cotton to Verticillium dahliae and significantly reduces its resistance. Based on this, it is speculated that the GhICDH3 gene plays a crucial positive regulatory role in cotton's resistance to Verticillium wilt.

[0187] Table 7

[0188]

[0189] Table 7 corresponds to Figure 4 The data for C.

[0190] Table 8

[0191]

[0192] Table 8 corresponds to Figure 4 The data for D.

[0193] Example 5

[0194] To further explore the physiological and biochemical mechanisms by which the GhICDH3 gene regulates cotton's resistance to Verticillium dahliae, this study systematically detected immune response indicators in silent plants (TRV::GhICDH3) and control plants (TRV::00) after inoculation. Reactive oxygen species (ROS) bursts are a key early signal for plant resistance to pathogen infection. NBT staining results showed that 12 hours after inoculation with Verticillium dahliae, the number and intensity of blue spots on the leaves of the control group plants significantly increased over time, while the staining area and intensity in the silent group plants did not change significantly (e.g., ...). Figure 5 (As shown in A). Quantitative detection results confirmed that the H2O2 content in the leaves of the control group rose rapidly after inoculation and reached a peak at 12 hpi, while the H2O2 level in the silent group was significantly lower than that in the control group during the period of 6-24 hpi (as shown in A). Figure 5 As shown in Figure B), silencing GhICDH3 inhibited the ROS outbreak in cotton after infection. Callose accumulation is an important means for plants to strengthen cell wall defense. Aniline blue staining results showed that after inoculation with Vd991, the control group had a large number of bright callose deposition spots in the leaves, while the silencing group had sparse and low-brightness fluorescent spots (as shown in Figure B). Figure 5(As shown in C). Statistical analysis revealed that during the 9-12 hpi phase, the callosity content in the control group was significantly higher than that in the silenced group, indicating that gene silencing limited the strengthening of the physical barrier (e.g., Figure 5 As shown in D). Simultaneously, by observing the degree of lignification in the stem through phloroglucinol staining, it was found that after induction by the pathogen, the xylem in the control group showed a deeper color (e.g., D). Figure 5 As shown in Figure E), lignin content peaked at 9 hpi, while the silent group showed lighter xylem staining and significantly insufficient lignin accumulation (as shown in Figure E). Figure 5 (As shown in F). Finally, the damage to the cotton vascular bundles and the amount of fungi were evaluated. Longitudinal stalk observation revealed that the browning of the vascular bundles in the silent group was more severe than that in the control group, exhibiting dark brown necrosis (as shown in F). Figure 5 (As shown in H). The results of the stem segment fungal recovery experiment showed that the number and growth rate of *Verticillium dahliae* colonies germinating from the silent group stem segments significantly exceeded those of the control group (e.g., H). Figure 5 (As shown in G). In summary, silencing the GhICDH3 gene significantly inhibited reactive oxygen species bursts, callose accumulation, and lignin synthesis in cotton, leading to a decrease in vascular bundle defense capabilities and thus promoting the colonization and spread of pathogens. The results indicate that GhICDH3 is a positive regulator in cotton's resistance to Verticillium dahliae infection.

[0195] Table 9

[0196]

[0197] Table 9 corresponds to Figure 5 The data for B.

[0198] Table 10

[0199]

[0200] Table 10 corresponds to Figure 5 The data for D.

[0201] Table 11

[0202]

[0203] Table 11 corresponds to Figure 5 The data for F.

[0204] Example 6: Results of disease resistance identification of ICDH3 transgenic and mutant Arabidopsis thaliana

[0205] To further verify the function of the GhICDH3 gene and its key sites in plant resistance to Verticillium dahliae infection, we created ICDH3 overexpression and site-mutant ICDH3 formulations. K407RArabidopsis thaliana materials were used. First, PCR amplification was performed on the selected T3 generation homozygous lines using specific primers for verification. Electrophoresis results showed that all transgenic lines showed specific bands of the same size as the target fragment, while no bands were observed in the negative control, preliminarily proving that the exogenous gene had been successfully integrated into the Arabidopsis thaliana genome (e.g., ...). Figure 6 (As shown in A). Subsequently, the transcriptional levels of each transgenic line were quantitatively analyzed using qRT-PCR. The results showed that in the selected OE1-4 ::ICDH3 and OE1-4 ::ICDH3 lines... K407R In all transgenic lines, the relative expression level of the ICDH3 gene was significantly increased compared to the wild-type (WT), with the OE2 and OE3 lines showing particularly high expression levels, confirming the successful creation of transgenic plants (e.g., Figure 6 (As shown in B). Subsequently, this study inoculated non-bolting Arabidopsis thaliana lines with Verticillium dahliae Vd991 and observed their disease resistance phenotypes. Phenotypic observation 21 days after inoculation showed that wild-type Arabidopsis thaliana exhibited obvious symptoms of disease, with large-scale wilting, yellowing, and severe chlorosis of the leaves; while the icdh3 deletion mutant plants were the most severely affected, exhibiting extreme stunting and death of the entire plant. In contrast, the overexpression lines OE::ICDH3 and OE::ICDH3... K407R The symptoms of damage have significantly lessened, and the plants are growing relatively well, with only a few leaves showing slight yellowing at the edges (e.g. Figure 6 (C). The disease index statistical results further quantified the differences in resistance (e.g., Figure 6 The results showed that the disease index of the icdh3 mutant was significantly higher than that of WT, while that of OE::ICDH3 and OE::ICDH3... K407R The disease index of all strains was significantly lower than that of WT. It is noteworthy that OE :: ICDH3 K407R The disease resistance of the strains was basically the same as or even slightly different from that of OE::ICDH3, indicating that the mutation at position 407 did not result in the loss of the gene's disease resistance function. These results fully demonstrate that overexpression of the GhICDH3 gene significantly enhances Arabidopsis' resistance to Verticillium dahliae, while deletion of icdh3 leads to loss of resistance. In conclusion, GhICDH3 plays an important positive regulatory role in plant resistance to Verticillium dahliae infection, and its lysine residue at position 407 may be related to the fine-tuning of its disease resistance function.

[0206] Table 12

[0207]

[0208] Table 12 corresponds to Figure 6 The data for B.

[0209] Table 13

[0210]

[0211] Table 13 corresponds to Figure 6 The data for D.

[0212] Example 7: GhICDH3 and GhHSP 70 Validation results of protein-protein interactions

[0213] To identify the interacting proteins of GhICDH3 in cotton disease resistance, various molecular biological techniques were used to verify the interaction between GhICDH3 and the heat shock protein GhHSP. 70 The interaction between them. First, point-to-point verification was performed using a yeast two-hybrid system, and the results showed that simultaneous transformation of PGBKT7-GhICDH3 and PGADT7-GhHSP... 70 The yeast strains grew well and turned blue on the four-deficient medium (SD / -Leu / -Trp / -Ade / -His), while none of the negative control groups grew, preliminarily confirming the interaction between GhICDH3 and GhHSP. 70 Interactions exist at the yeast level. To further clarify GhHSP... 70 The key regions interacting with GhICDH3 are identified in this study based on GhHSP. 70 The structural features of GhICDH3 were used to divide it into an ATPase domain (71-445 aa) and a matrix-binding domain (569-668 aa) for truncated interaction experiments. Y2H results showed that GhICDH3 exhibited a strong interaction signal only with fragments containing the matrix-binding domain (569-668 aa), while showing no interaction with the ATPase domain (71-445 aa), indicating that GhHSP… 70 The C-terminal matrix-binding domain is the core region mediating their interaction. Furthermore, GhICDH3... K407R The mutant can also interact with GhHSP 70 The interaction indicates that the mutation at position 407 does not affect the physical binding between proteins (e.g., Figure 7 (As shown in A). Subsequently, the interaction space of the two in plant cells was verified in tobacco leaves by bimolecular fluorescence complementation experiments. Fluorescence microscopy revealed co-expression of pXY106-nYFP-GhICDH3 and pXY104-cYFP-GhHSP. 70 Strong yellow fluorescence signals were observed in the cytoplasm of the groups with the positive vector, while no fluorescence was observed in any of the empty control groups, confirming that the two interact in the cytoplasm (e.g., Figure 7 (As shown in B). Finally, the luciferase complementation assay results showed that co-infection of nLUC-GhICDH3 and cLUC-GhHSP70 Significant luciferase activity signals were detected in the tobacco leaf region, while no signal was detected in the control region (e.g., Figure 7 (As shown in C). In summary, through three independent experimental systems—yeast two-hybrid, bimolecular fluorescence complementation, and luciferase complementation—it was consistently demonstrated that GhICDH3 and GhHSP... 70 There are direct interactions between them both in vivo and in vitro, and GhHSP 70 The matrix-binding domain is the key site for their interaction. Establishing this interaction relationship lays the foundation for further elucidating the molecular mechanism by which GhICDH3 regulates cotton disease resistance.

[0214] Example 8: Effect of Verticillium dahliae infection on GhHSP 70 Effects of gene expression levels

[0215] GhHSP in upland cotton infected with Verticillium dahliae was detected using qRT-PCR. 70 Gene expression characteristics. Results showed that in the early stages of inoculation, GhHSP... 70 There was no significant difference in expression between the treatment and control groups. Three hours after inoculation, the gene expression level increased significantly; during the period from 6 hpi to 24 hpi, GhHSP... 70 It maintained a highly significant expression level, with two distinct expression peaks around 6 hpi and 24 hpi, where the expression level at 6 hpi was approximately 3.4 times that of the control group. By 48 hpi, its expression level returned to the control level (e.g., ...). Figure 8 (As shown). The above results indicate that GhHSP 70 The gene is strongly induced by Verticillium dahliae and mainly plays a response role in the early and middle stages after infection.

[0216] Table 14

[0217]

[0218] 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. The application of the GhICDH3 gene or its encoded GhICDH3 protein in any of the following: (a) Application in improving plant resistance to Verticillium wilt; (b) Application in the cultivation of new germplasm with high resistance to Verticillium wilt; The nucleotide sequence of the GhICDH3 gene is shown in SEQ ID NO:1; The amino acid sequence of the GhICDH3 protein is shown in SEQ ID NO:

2.

2. The application as described in claim 1, characterized in that, The method for improving plant resistance to Verticillium wilt involves overexpressing the GhICDH3 gene in plants.

3. The application as described in claim 1 or 2, characterized in that, The plant mentioned includes: cotton.

4. A method for improving plant resistance to Verticillium wilt, characterized in that, include: The GhICDH3 gene in plants overexpressed as described in any one of claims 1 to 3.

5. A method for cultivating plants with high resistance to Verticillium wilt, characterized in that, include: The GhICDH3 gene in plants overexpressed as described in any one of claims 1 to 3.

6. A GhICDH3 gene mutant, characterized in that, It has the following characteristics: (1) An amino acid sequence as shown in SEQ ID NO:3; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more amino groups as shown in (1), and which has the same or similar function as the amino acid sequence shown in (1).

7. A nucleic acid molecule encoding the GhICDH3 gene mutant as described in claim 6, characterized in that, It has the following characteristics: (3) A nucleotide sequence as shown in SEQ ID NO:4; or (4) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (3); or (5) A sequence having at least 80% homology to the nucleotide sequence described in (3) or (4); or (6) The complementary sequence of the nucleotide sequence described in (3), (4) or (5).

8. The use of the GhICDH3 gene mutant as described in claim 6 and / or the nucleic acid molecule as described in claim 7 in any of the following: (c) Application in improving plant resistance to Verticillium wilt; (d) Application in cultivating new germplasm with high resistance to Verticillium wilt; the step of improving plant resistance to Verticillium wilt is to overexpress the nucleic acid molecule in the plant; the plant includes: cotton.

9. A method for improving plant resistance to Verticillium wilt, characterized in that, include: Overexpression of the nucleic acid molecule as described in claim 7 in plants.

10. A method for cultivating plants with high resistance to Verticillium wilt, characterized in that, include: Overexpression of the nucleic acid molecule as described in claim 7 in plants.

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