Application of GhCaM53 gene in improving plant resistance to verticillium wilt

Abstract

The present application relates to the field of plants, in particular to application of GhCaM53 gene in improving resistance of plants to verticillium wilt. The protein encoded by the mutant of GhCaM53 gene has the sequence of the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4. The present application aims to analyze the ubiquitination modification characteristics of GhCaM53 protein and its function in cotton resistance to verticillium wilt, to clarify the immune response signal pathway mediated by it, to provide a new target gene (GhCaM53) and theoretical support for molecular breeding of cotton resistance to verticillium wilt, and to make up for the deficiency of the prior art in exploring new disease-resistant genes and key regulatory mechanisms.

Description

Technical Field

[0001] This invention relates to the field of plants, and more particularly to the application of the GhCaM53 gene in improving plant resistance to Verticillium wilt. Background Technology

[0002] Cotton, a vital global economic crop and a core source of renewable textile fibers, is particularly important in my country. Upland cotton, with its high yield and high-quality fiber, accounts for 95% of global cotton production. However, it is a crucial agricultural product for the national economy and people's livelihood. Yet, its production has long been threatened by Verticillium wilt. This disease, caused by Verticillium dahliae, invades the cotton root system through germ tube differentiation, breaking through the root barrier and multiplying in the vascular bundles. It produces conidia, which cause disease by either blocking the vascular bundles or producing wilt toxins that damage cell membranes. Symptoms appear in the seedling stage and peak during flowering and boll formation, leading to sparse buds and bolls, premature boll splitting, and severely reduced yield and fiber quality. Furthermore, the microsclerotia formed by the pathogen remain dormant in the soil, making eradication extremely difficult after infection. To resist the pathogen, plants have evolved a two-layered immune system: PTI (PRR recognizes PAMPs and activates MAPKs, CDPKs, etc.) and ETI (NLR recognizes effector factors and activates a strong immune response). Ca2+ is one of the key components of this immune system. 2+ As a key secondary messenger, it can decode signals through sensors such as calmodulin (CaM). CaM binds to Ca... 2+ Post-conformational changes regulate defense-related target proteins, and studies have confirmed that tobacco NtCaM13 and wheat TaCaM3 play a role in disease resistance.

[0003] Following infection of upland cotton by *Verticillium dahliae*, the cotton initiates a series of signal transduction mechanisms to respond to pathogen stress. Among these, protein ubiquitination, as an important post-translational regulatory mechanism, plays a crucial role in disease resistance signal transduction. This study, through proteomics analysis, revealed that calmodulin GhCaM53 in the cAMP signaling pathway undergoes ubiquitination modification after pathogen infection, and the ubiquitination level dynamically changes with infection time, suggesting its potential involvement in regulating the cotton's defense response against Verticillium wilt.

[0004] Current technical solutions for cotton resistance to Verticillium wilt primarily focus on plant hormones (JA, SA), reactive oxygen species (ROS), and lignin pathways. For example, overexpression of JA synthesis genes, ROS-related genes, or lignin synthesis genes enhances resistance. Molecular breeding relies on genes from these known pathways. Traditional hybridization breeding is limited by the lack of deciduous Verticillium wilt resistance genes in upland cotton, its long cycle, and low efficiency. It does not involve the resistance regulation mechanism mediated by GhCaM53 ubiquitination modification. Overall, there is a lack of exploration of novel resistance genes and related regulatory pathways, making it difficult to meet the demand for diverse targets in resistance breeding. Summary of the Invention

[0005] In view of this, this invention provides the application of the GhCaM53 gene in improving plant resistance to Verticillium wilt. This invention addresses the problems in existing cotton Verticillium wilt research, including the lack of superior resistance genes for deciduous Verticillium wilt in upland cotton, the long breeding cycle and low selection efficiency of traditional methods leading to poor resistance in most bred varieties, frequent mutations of the Verticillium wilt pathogen causing resistant varieties to easily lose their resistance, and the fact that existing research mainly focuses on known pathways such as plant hormones, reactive oxygen species, and lignin, with insufficient exploration of the resistance regulation mechanism mediated by the ubiquitination modification of calmodulin GhCaM53. This invention systematically analyzes the ubiquitination modification characteristics of the GhCaM53 protein and its function in cotton Verticillium wilt resistance, clarifying the immune response signaling pathway mediated by it. This provides theoretical support for a novel target gene (GhCaM53) and related regulatory pathways for molecular breeding of cotton for Verticillium wilt resistance, solving the core technical problems of lacking novel resistance gene discovery and key regulatory mechanism analysis in cotton Verticillium wilt resistance research, and the difficulty in obtaining efficient genetic improvement targets.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a protein encoded by a mutant of the GhCaM53 gene, the sequence of which is as shown in SEQ ID NO:3 or SEQ ID NO:4.

[0008] In some embodiments of the present invention, the sequence of SEQ ID NO:3 in the above-mentioned protein is: MADQLTDEQISEFKEAFSLFDRDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIREADVDGDGQINYDEFVKVMMAKRREKATSHVNSTTKSSKKSKGKQSCKCIFL.

[0009] In some embodiments of the present invention, the sequence of SEQ ID NO:4 in the above-mentioned protein is: MADQLTDEQISEFKEAFSLFDKDGDGCITTRELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIREADVDGDGQINYDEFVKVMMAKRREKATSHVNSTTKSSKKSKGKQSCKCIFL.

[0010] The present invention also provides a nucleic acid molecule encoding the above-mentioned protein, the sequence of which is: a nucleotide sequence as shown in SEQ ID NO:5 or SEQ ID NO:6.

[0011] In some embodiments of the present invention, the sequence of SEQ ID NO:5 in the above-mentioned nucleic acid molecule is:

[0012] In some embodiments of the present invention, the sequence of SEQ ID NO:6 in the above-mentioned nucleic acid molecule is:

[0013] This invention also provides the use of the above-described protein and / or nucleic acid molecule in any of the following:

[0014] (a) Application in improving plant resistance to Verticillium wilt;

[0015] (b) Application in the cultivation of 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 is a dicotyledonous plant.

[0016] The present invention also provides a method for improving plant resistance to Verticillium wilt, comprising: overexpressing the above-mentioned nucleic acid molecules in plants.

[0017] The present invention also provides a method for cultivating plants with high resistance to Verticillium wilt, comprising: overexpressing the nucleic acid molecule described in the plant.

[0018] In some embodiments of the present invention, the plant in the above method is cotton.

[0019] In some embodiments of the present invention, the nucleotide sequence of the GhCaM53 gene is shown in SEQ ID No:1; and the amino acid sequence of the GhCaM53 protein is shown in SEQ ID No:2.

[0020] The beneficial effects of this invention include:

[0021] (1) For the first time, the ubiquitination modification characteristics of calmodulin GhCaM53 in the cotton cAMP signaling pathway were identified by protein ubiquitination modification proteomics technology. It was found that the ubiquitination level changed 1 day after infection with Verticillium dahliae, and the two ubiquitination sites (K22 and K31) at lysine residues 22 and 31 were precisely located. This provides a core molecular target for elucidating the disease resistance regulation mechanism mediated by GhCaM53 and fills the gap in the existing research on the association between calmodulin ubiquitination modification and cotton resistance to Verticillium wilt.

[0022] (2) The functional correlation of ubiquitination modification of GhCaM53 protein was revealed. Subcellular localization experiments confirmed that wild-type GhCaM53 is located in the cell membrane and cell nucleus, while the protein after mutation at K22 and K31 sites is only located in the cell membrane. This showed that ubiquitination modification directly affects its subcellular localization and may regulate its disease resistance function. At the same time, transgenic Arabidopsis experiments showed that the K22 site has a more significant effect on the plant's disease resistance, which clarified the importance of key functional sites.

[0023] (3) A complete verification system for the disease resistance function of the GhCaM53 gene was established. The VIGS silencing experiment confirmed that the cotton disease index increased from 20 to 60 after silencing the gene, and the disease resistance decreased significantly. The heterologous overexpression experiment confirmed that Arabidopsis thaliana overexpressing GhCaM53 and site mutants showed significantly enhanced resistance to Verticillium dahliae. It was clarified that GhCaM53 plays a positive regulatory role by regulating key disease resistance indicators such as reactive oxygen species burst, callus deposition and lignin formation, providing sufficient experimental support for the application of gene function.

[0024] (4) The application value of GhCaM53 as a novel disease resistance target was clarified. The expression level of this gene began to increase 3 hours after infection with Verticillium dahliae and reached its peak at 48 hours. Its expression dynamics were highly consistent with the disease resistance response. Moreover, the disease resistance of cotton was greatly reduced after silencing and significantly improved after overexpression. This broke through the limitations of existing research that relied on traditional pathway genes, provided a highly efficient novel target gene for molecular breeding of cotton against Verticillium wilt, and enriched the cotton disease resistance gene resource library.

[0025] (5) A multi-dimensional and reusable cotton disease resistance gene function verification technology system was constructed, which integrates protein ubiquitination modification proteomics detection, qRT-PCR quantitative analysis, VIGS transient silencing, transgenic creation, subcellular localization and detection methods of multiple disease resistance phenotypes (reactive oxygen species, callose, xylem and stem browning), realizing the whole chain verification from protein modification, gene expression to phenotypic characteristics, and providing a standardized technical paradigm for the functional research of similar disease resistance genes. Attached Figure Description

[0026] 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.

[0027] Figure 1 This demonstrates the cAMP signaling pathway;

[0028] Figure 2 The K22 ubiquitination site of cotton calmodulin GhCaM53 is shown.

[0029] Figure 3 The K31 ubiquitination site of cotton calmodulin GhCaM53 is shown.

[0030] Figure 4 Indicates the expression level of the GhCaM53 gene;

[0031] Figure 5 The results show the subcellular localization of GhCaM53 protein;

[0032] Figure 6 The phenotype and disease index of cotton after transient silencing of the GhCaM53 gene are shown; where: A shows that the positive control gene was effectively silenced; B shows the silencing efficiency of the GhCaM53 gene; C shows the susceptibility phenotype of cotton; and D shows the statistical cotton disease index.

[0033] Figure 7 The results of the test for cotton-related disease resistance indicators are shown below; A shows the reactive oxygen species bloom in the leaves; B shows the integrated optical density of cotton leaves; C shows the callose deposition in cotton leaves; D shows the number of callose fluorescent spots; E shows the color of the xylem in cotton; F shows the width of the xylem; and G shows the degree of browning in the cotton stem.

[0034] Figure 8 The results of disease resistance identification of CaM53 transgenic Arabidopsis thaliana are shown; where: A shows the identification results of positive Arabidopsis thaliana plants; B shows the expression level of GhCaM53 gene in positive plants; C shows the disease phenotype of Arabidopsis thaliana; and D shows the statistical Arabidopsis thaliana disease index. Detailed Implementation

[0035] This invention discloses the application of the GhCaM53 gene in improving plant resistance to Verticillium wilt.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] The experimental materials involved in this invention are:

[0042] (1) Plant materials

[0043] The plant materials used in this invention are upland cotton and Arabidopsis thaliana, with the upland cotton material being JM-11 and the Arabidopsis thaliana material being Columbia (Col). The cotton was sown in a 1:1 mixture of potting soil and vermiculite and cultured in a constant temperature and humidity chamber at 28°C with 16 hours of light / 8 hours of darkness. The Arabidopsis thaliana was sown in a 1:1 mixture of potting soil and vermiculite and cultured in a constant temperature and humidity chamber at 22°C with 16 hours of light / 8 hours of darkness.

[0044] (2) Vector and strain

[0045] Escherichia coli DH5α and Agrobacterium GV3101 were purchased from Shanghai Weidi Company. The cotton deciduous wilt strain was Verticillium dahliae Vd991 (Vd991), a common strain preserved in our laboratory. Vectors used included pTRV1, pTRV2, and pCAMBIA2300, all common vectors preserved in our laboratory and available commercially.

[0046] (3) Main reagents

[0047] Plant total RNA mini-extraction kit, reverse transcription kit, and real-time fluorescence kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; plasmid mini-extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; DNA Marker was purchased from Monad Biotechnology Co., Ltd.; high-fidelity PCR Mix was purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases EcoRI, BamHI, KpnI, and SalI were purchased from NEB (Beijing) Co., Ltd.

[0048] In Examples 1 to 9 and the effect examples of this invention, all raw materials and reagents used can be purchased from the market.

[0049] The present invention will be further illustrated below with reference to the embodiments:

[0050] Example 1 Planting of Plant Materials

[0051] Cotton seeds were delinted with concentrated sulfuric acid, thoroughly rinsed in clean water, and air-dried in a cool place. After soaking in clean water for 24 hours, the seeds were sown in soil substrate and transferred to a growth chamber for cultivation. Arabidopsis seeds were washed with 75% ethanol solution for 3 minutes, then rinsed three times with sterile distilled water for 2 minutes each time. After rinsing three times with sterile distilled water, they were washed with 5% sodium hypochlorite solution for 30 seconds, and then rinsed three times with sterile distilled water for 2 minutes each time. They were then sown on 1 / 2 MS solid medium. After vernalization at 4℃ for 48 hours, they were transferred to a growth chamber for cultivation.

[0052] Example 2 Activation and culture of Verticillium wilt

[0053] The laboratory-preserved *Verticillium dahliae* Vd991 was removed and thawed. 10 μL of the culture was placed in the center of PDA solid medium and incubated upside down at 25°C in the dark for 7–14 days. Then, the mycelial cake was evenly transferred to Czapek agar medium and incubated with shaking at 25°C for 7 days. The mycelium in the culture was filtered through gauze, and the spore concentration was calculated using a hemocytometer and adjusted to 1 × 10⁻⁶ spores. 7 1 / mL for subsequent experiments.

[0054] Example 3: Inoculation treatment of cotton seedlings with Verticillium wilt

[0055] Two days before inoculation, watering of cotton seedlings should be suspended to keep the soil relatively dry. Once the seedlings have grown to the stage of two true leaves and one central leaf, select plants of uniform growth and inoculate them using the root-dip method (root injury method), adjusting the spore concentration to 1×10⁻⁶. 7 Spores were inoculated at a concentration of 1 spore per ml, and then 5 ml of the spore suspension was evenly poured into the soil. As a control, the control group seedlings were treated with the same amount of sterile distilled water. After inoculation, all seedlings were transferred to a culture room with a controlled environment for cultivation.

[0056] Example 4: Cotton Protein Ubiquitination Modification Omics Determination

[0057] 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.

[0058] Example 5: Observation of cotton disease index

[0059] During the two-leaf-one-heart stage of cotton seedlings, the control group was treated with clean water, while some seedlings were inoculated with a fungicide. Two days after inoculation, samples were taken from the cotton tissue, recorded as hour 0. Subsequent samples were taken at 3, 6, 12, 24, and 48 hours for later use. On day 15 after inoculation, some cotton leaves showed symptoms of disease. Based on the severity of the disease, diseased plants were classified into four grades: Grade 0, no disease; Grade 1, 10-20% of leaves showing wilting and yellowing; Grade 2, 20%-50% of leaves showing wilting and yellowing; Grade 3, 50%-75% of leaves showing wilting and yellowing; Grade 4, over 75% of leaves showing wilting and yellowing or plant death. The disease index was calculated using the following formula: Disease Index DI = [Σ(Disease Grade × Number of Diseased Plants at Each Grade) / (Total Number of Plants Surveyed × 4)] × 100.

[0060] Example 6: RNA extraction from plant leaves and qRT-PCR experiment

[0061] 1. RNA extraction (Novizan FastPure® Universal Plant Total RNA Isolation Kit)

[0062] (1) Take an appropriate amount of plant tissue that has been ground with liquid nitrogen and immediately add 600 μL of Buffer PSL. Vortex vigorously for 30 seconds to ensure that the sample and lysis buffer are thoroughly mixed. Centrifuge at 12,000 rpm for 5 minutes.

[0063] (2) Take about 500 μL of the supernatant into the FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III) collection tube, centrifuge at 12,000 rpm for 30 seconds, discard the FastPure gDNA-Filter Columns III, and collect the filtrate.

[0064] (3) Add 0.5 times the volume of the filtrate of anhydrous ethanol (about 250 μL, adjust according to the actual situation of the supernatant) to the collection tube and shake to mix for 15 seconds.

[0065] (4) Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0066] (5) Add 700 μL of Buffer RWA to FastPure RNA Columns V, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0067] (6) Add 500 μL of Buffer RWB to FastPure RNA Columns V (please check that 48 mL of anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate.

[0068] (7) Repeat step (6).

[0069] (8) Place the FastPure RNA Columns V back into the collection tube and centrifuge at 12,000 rpm for 2 minutes.

[0070] (9) Transfer FastPure RNA Columns V to a new RNase-free Collection Tubes 1.5 mL centrifuge tube, add 30-100 μL of ultrapure water to the center of the adsorption column membrane, and centrifuge at 12,000 rpm for 1 minute.

[0071] 2. RNA reverse transcription (using Novizan HiScript® III All-in-one RT SuperMix Perfectfor qPCR kit)

[0072] Table 1 Reverse transcription reaction system

[0073]

[0074] Table 2 Reverse transcription reaction procedure

[0075]

[0076] 3. qRT-PCR experiment (using PerfectStart® Green qPCR SuperMix dye premix for real-time PCR)

[0077] 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.

[0078] Table 3 qRT-PCR reaction system

[0079]

[0080] Table 4 qRT-PCR amplification program

[0081]

[0082] Example 7 Subcellular localization of GhCaM53 protein

[0083] To achieve subcellular localization of the GhCaM53 protein, the complete CDS sequence and corresponding protein sequence of GhCaM53 were downloaded from the online website Cotton MD. The CDS sequence of the GhCaM53 gene was amplified using GFP-GhCaM53-F / R primers (Table 6). 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 introduced into Agrobacterium GV3101 via cryogenic transformation and stored for later use. For injection, Agrobacterium was added to LB liquid medium containing Kan / Rif and cultured on a shaker (28°C, 180 rpm) until the OD600 reached approximately 1.0. The bacterial suspension resuspended in MMA was mixed with a membrane / nucleus localization Maker labeled with YFP at a ratio of 1:1, and then injected into the back of 4-week-old tobacco leaves using a sterile syringe. The leaves were cultured in the dark for 24 hours and then transferred to a normal culture environment for 24 hours. The samples were then observed using a laser scanning confocal microscope (Olympus FV3000).

[0084] CDS sequence of GhCaM53 gene: ATGGCGGACCAGCTTACCGATGAGCAGATCTCTGAGTTCAAGGAGGCCTTTAGCCTCTTTGACAAGGATGGCGATGGTTGCATTACTACCAAGGAACTGGGTACTGTAATGCGGTCACTCGGACAGAACCCTACTGAGGCAGAACTCCAAGATATGATTAATGAAGTTGACGCTGACGGAAATGGGACCATCGATTTCCCTGAATTTCTCAACCTTATGGCAAGGAAAATGAAAGATACCGATTCTGAGGAGGAACTTAAAGAAGCTTTCAGGGTGTTCGATAAGGATCAGAACGGTTTCATATCTGCTGCCGAGCTTCGTCACGTTATGACAAATCTTGGCGAGAAGCTTACCGATGAGGAAGTCGATGAGATGATCCGTGAAGCCGATGTTGATGGTGACGGCCAGATCAACTACGACGAATTCGTCAAAGTCATGATGGCCAAGAGGAGAGAGAAGGCAACATCACATGTGAACTCCACCACCAAGAGTAGTAAAAAATCTAAAGGAAAACAGTCTTGTAAATGCATTTTCCTTTAA (as shown in SEQ ID NO:1);

[0085] Amino acid sequence of GhCaM53 protein: MADQLTDEQISEFKEAFSLFDKDGDGCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMARKMKDTDSEEELKEAFRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIREADVDGDGQINYDEFVKVMMAKRREKATSHVNSTTKSSKKSKGKQSCKCIFL (as shown in SEQ ID NO:2).

[0086] Example 8 VIGS silencing experiment and detection of related disease resistance indexes

[0087] 1. VIGS silencing experiment

[0088] First, specific primers for VIGS-GhCaM53-F / R were designed (Table 6). The VIGS target fragment of the GhCaM53 gene was amplified by PCR and cloned into the multiple cloning site region of the pTRV2 vector. The recombinant plasmid pTRV2-GhCaM53 was introduced into Agrobacterium strain GV3101 using cryotransformation. Subsequently, the prepared VIGS bacterial solution was injected into cotton leaves, and the leaf whitening phenotype of the control plants injected with pTRV2-CLA1 bacterial solution was observed. When the second true leaf of the plant exhibited a reticulated whitening characteristic, the silencing efficiency could be tested. The first true leaf of the same size from both the silencing and control plants was collected, and total RNA was extracted. cDNA templates were synthesized via reverse transcription. Primers qRT-PCR-GhCaM53-F / R (Table 6) were used to quantitatively analyze the gene silencing efficiency using qRT-PCR. The TRV::CLA1 vector was used to detect the gene silencing effect.

[0089] 2. Detection of relevant disease resistance indicators

[0090] DAB staining detection of reactive oxygen species (ROS) production and accumulation: Twelve hours after inoculation with *Verticillium dahliae*, leaves from each group of plants were randomly selected and stained with DAB staining solution in the dark for 8 hours. The leaves were then completely immersed in 95% ethanol for a boiling water bath until the green color completely faded. Subsequently, they were immersed in 70% glycerol, and the production and accumulation of ROS were observed under a microscope.

[0091] Determination of callose accumulation: Forty-eight hours after inoculation with strain Vd991, leaves from each group were randomly selected for callose content detection. Leaves were treated with a 3:1 (v / v) ethanol-acetic acid mixture for 3 hours to remove chlorophyll; subsequently, they were soaked in 70% ethanol and 50% ethanol for 3 hours each, rinsed with distilled water, and soaked overnight. The next day, the leaves were treated in a 10% sodium hydroxide solution for 1.2 hours, gently rinsed, stained with 0.01% aniline blue solution, and incubated in the dark for 3 hours. Callose accumulation in each group of leaves was observed under ultraviolet excitation light using a fluorescence microscope.

[0092] Detection of xylem deposition by phloroglucinol staining: A 10% phloroglucinol staining solution was used to detect xylem deposition in TRV::00 and TRV::GhCaM53 plants 72 hours after inoculation. Five plants were randomly selected from each treatment group, and sections were prepared at the same location on the stem. The sections were placed on a glass slide, and phloroglucinol staining solution was added to completely cover the sections. After 2 minutes, concentrated sulfuric acid was added, and the sections were immediately observed under a microscope and photographed quickly.

[0093] Stem segment browning detection: On day 25 after inoculation with Verticillium dahliae, stem segments of 8 TRV :: 00 and TRV :: GhCaM53 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.

[0094] Example 9: Creation and Disease Resistance Identification of GhCaM53 Transgenic Arabidopsis thaliana

[0095] 1. Creation of transgenic Arabidopsis thaliana

[0096] (1) The pCAMBIA2300-GhCaM53 recombinant plasmid was transformed into Agrobacterium competent cells GV3101, and the positive strain was activated.

[0097] (2) Inoculate the activated Agrobacterium into an Erlenmeyer flask and place it on a shaker at 28°C for amplification. When the bacterial solution OD 600 When the value reaches 1.5~2.0, dispense the bacterial solution into 50 ml centrifuge tubes.

[0098] (3) Centrifuge the bacterial solution in the centrifuge tube at 5000 rpm for 10 minutes, then discard the supernatant, add about 20 ml of resuspension solution to the centrifuge tube to fully resuspend, then centrifuge at 5000 rpm for 5 minutes, and discard the supernatant.

[0099] (4) Resuspend the bacterial culture in the centrifuge tube and adjust the OD600 of the bacterial culture resuspension to 0.8~0.9 in a UV spectrophotometer. The resuspension preparation system is shown in Table 5.

[0100] Table 5. Preparation system of resuspended transgenic Arabidopsis thaliana.

[0101]

[0102] (5) Using the flower dipping method, the resuspended strain was transferred into Arabidopsis thaliana (selecting Arabidopsis thaliana that has not grown pods but only flower buds), the flower buds were soaked in the resuspended solution for 45 seconds, then treated in the dark for 24 hours, and then placed in a greenhouse for cultivation.

[0103] (6) After the Arabidopsis thaliana matures, the seeds collected are of generation T0. Then wash the seeds and spread them on solid plates of Kana resistant MS medium. Place them in a light incubator for 7-10 days. If the leaves of the newly grown Arabidopsis thaliana seedlings are dark green, they may be overexpression positive seedlings.

[0104] (7) Transplant the dark green seedlings into nutrient soil for further cultivation, and then perform DNA and RNA level identification to determine whether they are positive seedlings. Then, collect the mature seeds of the identified positive seedlings individually and spread them on MS medium plates. When the segregation ratio is 3:1, T2 generation positive seedlings are obtained.

[0105] 2. Disease resistance identification of transgenic Arabidopsis thaliana

[0106] To assess the disease resistance of GhCaM53 transgenic Arabidopsis thaliana, three-week-old wild-type Arabidopsis thaliana Col-0 and overexpressing genotype OE (20 plants per group) were inoculated with Verticillium dahliae. Col-0 plants inoculated with water served as a negative control, while Col-0 plants inoculated with Verticillium dahliae served as a positive control. After 14 days, the phenotype of Arabidopsis thaliana was observed, and the disease index was calculated (the method for calculating the disease index is as described in Example 5).

[0107] Table 6 Primers used in this study

[0108]

[0109] Results and Analysis of Effects

[0110] 1. Results of cotton protein ubiquitination modification proteomics assay

[0111] Protein ubiquitination modification analysis revealed that some proteins in cotton underwent ubiquitination modification during pathogen infection, such as calmodulin GhCaM53 in the cAMP signaling pathway (e.g., Figure 1 As shown), the ubiquitination level of this calmodulin changed one day after inoculation with *Verticillium dahliae*. Mass spectrometry further identified its ubiquitination sites, detecting two sites: lysine at position 22 (EAFSLFDKDGDGCITTK(gl)ELGTVMR) and lysine at position 31 (EAFSLFDK(gl)DGDGCITTK) (as shown). Figure 2 and Figure 3 (As shown).

[0112] 2. Effects of Verticillium dahliae infection on GhCaM53 gene expression levels

[0113] qRT-PCR results showed that inoculation with *Verticillium dahliae* significantly increased the expression level of the GhCaM53 gene. The expression level began to gradually increase 3 hours after infection, reaching its maximum at 48 hours. This indicates that *Verticillium dahliae* induction promotes the production of more GhCaM53 protein in cotton, suggesting its potential involvement in the cotton's defense response against Verticillium wilt. Figure 4 (As shown in Table 7).

[0114] Table 7

[0115]

[0116] 3. Subcellular localization results of GhCaM53 protein

[0117] Tobacco leaves were transformed using Agrobacterium to induce point mutations in the 35S::GhCaM53-GFP fusion gene and the 35S::GhCaM53 gene. K22R -GFP fusion gene, point mutation 35S :: GhCaM53 K31R -GFP fusion gene and 35S::GFP empty vector were mixed 1:1 with a cell membrane / nuclear localizer tagged with YFP and transiently co-expressed in tobacco leaf cells to determine the localization of GhCaM53. Results are as follows: Figure 5 As shown, the fluorescence of empty GFP is distributed in the cell membrane, cytoplasm, and nucleus. In tobacco leaf epidermal cells expressing the 35S::GhCaM53-GFP fusion gene and the Maker gene, green fluorescence is visible in the cell membrane and nucleus, which overlaps with the red fluorescence of the membrane / nuclear Maker gene, resulting in yellow light. However, the point mutations in the 35S::GhCaM53K22R-GFP fusion gene and the point mutations in the 35S::GhCaM53... K31R The -GFP fusion gene only exhibits green fluorescence on the cell membrane. The results indicate that the GhCaM53 protein is normally localized on the cell membrane and nucleus. However, after the lysine mutation at the ubiquitination sites of positions 22 and 31 to arginine, the localization of the GhCaM53 protein changed from nuclear and membrane localization to membrane-only localization. This suggests that ubiquitination modification affects the localization of the GhCaM53 protein, and its function in the cell may also be affected (e.g., Figure 5 (As shown).

[0118] 4. Results of VIGS silencing test and related disease resistance indicators

[0119] Ten days after the bacterial injection, the true leaves of the TRV :: CLA1 control plants began to show leukoplakia. Figure 6 (Figure A in the diagram) Five silent and five control plants were selected, and RNA was extracted from their leaves for qRT-PCR to detect the silencing efficiency of the GhCaM53 gene. The silencing efficiency of both plants reached approximately 50%. Figure 6 (See Figure B and Table 8 in the table). The results indicate that the GhCaM53 gene was effectively silenced, allowing for the study of its function during the seedling stage. Twenty-five days after injection of the bacterial solution, plants in both the control and treatment groups began to show varying degrees of wilting. The control group plants exhibited yellowing and wilting of individual leaves with downward curling of leaf margins. The treatment group plants showed more severe symptoms of Verticillium wilt, including wilting, yellowing, and leaf drop. Figure 6 (See Figure C in the diagram). After calculating the disease index, it was found that the disease index of the control group was around 20, while the disease index of the treatment group was around 60. Figure 6Figure D in the diagram shows that silencing the GhCaM53 gene significantly increases the disease index and reduces the disease resistance of cotton when infected with Verticillium dahliae. It is speculated that the GhCaM53 gene plays an important regulatory role in cotton's resistance to Verticillium wilt.

[0120] Reactive oxygen species (ROS) burst is one of the indicators for evaluating plant disease resistance. DAB staining results showed that at 6 and 9 hours after inoculation, control plants stained more deeply and over a larger area than silent plants, indicating that the ROS level in the leaves of TRV::GhCaM53 plants was lower than that of TRV::00 plants starting at 6 hours after inoculation. Furthermore, according to integrated optical density (IOD) statistics, the IOD value of TRV::00 plants was consistently higher than that of TRV::GhCaM53 plants during the infection period of Verticillium dahliae, and the difference reached its maximum at 9 hpi. Figure 7 (See Figures A and B in the diagram). Callose is a type of glucan, often used in plants as an indicator of disease resistance. After inoculation with the pathogen Vd991, the true leaf callose deposition in TRV :: 00 plants was more concentrated than that in TRV :: GhCaM53 plants, and the number of true leaf callose spots in TRV :: 00 plants was also greater than that in TRV :: GhCaM53 plants. Figure 7 (See Figures C and D in the original text). Phloroglucinol staining results showed that the stained area of ​​the xylem in the stems of plants infected with Vd991 was larger than that in uninfected plants. After infection with the pathogen, the stained area of ​​the xylem in TRV::GhCaM53 plants was smaller than that in TRV::00 plants (…). Figure 7 Figures E and F in the diagram). When the cotton stalks were longitudinally sectioned and their vascular bundles were observed, it was found that the browning of the vascular bundles in the TRV :: GhCaM53 plants was more severe. Figure 7 (See Figure G in the diagram). The above results indicate that silencing the GhCaM53 gene weakens the resistance of cotton to Verticillium dahliae, suggesting that GhCaM53 may be a positive regulator in the process of cotton resisting Verticillium dahliae infection, and the regulatory process may involve changes in reactive oxygen species, lignin, and callose content.

[0121] Table 8

[0122]

[0123] The data in Table 8 corresponds to... Figure 6 B.

[0124] Table 9

[0125]

[0126] The data in Table 9 corresponds to... Figure 6 D.

[0127] Table 10

[0128]

[0129] The data in Table 10 corresponds to Figure 7 B.

[0130] Table 11

[0131]

[0132] The data in Table 11 corresponds to Figure 7 D.

[0133] Table 12

[0134]

[0135] The data in Table 12 corresponds to Figure 7 F.

[0136] 5. Results of disease resistance identification in CaM53 transgenic Arabidopsis thaliana

[0137] PCR amplification was performed on the selected T3 generation Arabidopsis thaliana strains overexpressing the GhCaM53 gene and those with ubiquitination site mutations. All bands were detected and matched the target band size. Figure 8 (See Figure A in the original text). The obtained T3 generation transgenic Arabidopsis plants were named OE1::CaM53, OE2::CaM53, OE3::CaM53, OE4::CaM53, and OE::CaM53. K22R and OE :: CaM53 K31R RNA was extracted from leaves and qRT-PCR was performed. The results showed that the relative expression level of the CaM53 gene was increased to varying degrees in different lines, proving that the transformation was successful. Figure 8 (See Figure B in the original text). Then, after inoculating unbolted wild-type Arabidopsis and transgenic Arabidopsis with Vd991, phenotypic observations and disease index measurements were performed. It was found that the wild-type lines inoculated with *Verticillium dahliae* exhibited severe leaf wilting, yellowing, lodging, and chlorosis, while the OE::CaM53 lines... K22R strains and OE :: CaM53 K31R Compared with wild-type Arabidopsis, the wilting and yellowing of the strains were reduced to varying degrees. Figure 8 (Figure C in the text), and the disease index was also significantly lower than that of wild-type Arabidopsis thaliana (Figure C in the text). Figure 8(See Figure D in the original text). The results show that the resistance of transgenic Arabidopsis thaliana overexpressing GhCaM53 to Verticillium dahliae was significantly increased, while the plants still exhibited resistance after the ubiquitination site mutation. Furthermore, the ubiquitination site at position 22 had a greater impact on the plant's resistance level, indicating that the overexpression of the CaM53 gene plays a positive regulatory role in Arabidopsis thaliana's resistance to Verticillium dahliae infection, with the ubiquitination site at position 22 being more closely related to the plant's disease resistance function.

[0138] Table 13

[0139]

[0140] The data in Table 13 corresponds to Figure 8 B.

[0141] Table 14

[0142]

[0143] The data in Table 14 corresponds to Figure 8 D.

[0144] 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. A protein encoded by a mutant of the GhCaM53 gene, characterized in that, Its sequence is as shown in SEQ ID NO:3, which contains amino acids.

2. A nucleic acid molecule encoding the protein as described in claim 1, characterized in that, Its sequence is as shown in SEQ ID NO:5, which is a nucleotide sequence.

3. The use of the protein as described in claim 1 and / or the nucleic acid molecule as described in claim 2 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 method of improving plant resistance to Verticillium wilt involves overexpressing the nucleic acid molecule in the plant. The method for cultivating new germplasm with high resistance to Verticillium wilt involves overexpressing the nucleic acid molecule in the germplasm. The plant and / or the germplasm are: Arabidopsis thaliana and / or cotton.

4. A method for improving plant resistance to Verticillium wilt, characterized in that, include: Overexpression of the nucleic acid molecule as described in claim 2 in a plant; the plant is Arabidopsis thaliana and / or cotton.

5. 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 2 in a plant; the plant is Arabidopsis thaliana and / or cotton.

Images