Grape gene VlPUB4 and its application in genetic improvement of plant resistance to gray mold

Abstract

This invention discloses the grape gene VlPUB4 and its application in the genetic improvement of plant resistance to gray mold. The VlPUB4 gene was cloned from the disease-resistant grape variety "Beda," and its sequence is shown in SEQ ID NO.1; the amino acid sequence encoded by the grape VlPUB4 gene is shown in SEQ ID NO.2. This invention first analyzed the expression pattern of this gene after gray mold induction in grape varieties with different disease resistance. Then, by achieving overexpression and silencing of this gene in grapes, plants with enhanced and weakened resistance to gray mold were obtained. The VlPUB4 gene provided by this invention can provide technical support for the improvement of grape varieties with poor resistance to gray mold, thereby reducing the damage caused by gray mold to grapes; at the same time, this gene can provide important genetic resources and theoretical guidance for the creation of high-quality, disease-resistant new grape varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and genetic engineering technology, specifically relating to the grape gene VlPUB4 and its application in the genetic improvement of plant resistance to gray mold. Background Technology

[0002] Grape cultivation has a long history, and its fruits are rich in nutrients, can be processed in various ways, and are beneficial to human health while also possessing significant economic value. Gray mold is one of the most damaging diseases in grape cultivation, infecting leaves, fruits, and other tissues and organs, severely impacting plant growth, fruit quality, and yield. Improving grapes' resistance to gray mold has become a critical issue urgently needing to be addressed in the grape industry. Currently, gray mold control in production mainly relies on chemical control methods, primarily pesticides, which suffer from high costs, environmental pollution, and negative impacts on fruit quality. Therefore, discovering and utilizing the plant's own disease-resistant genes and using molecular breeding techniques to cultivate high-quality, disease-resistant grape varieties has become an important measure for sustainable disease control.

[0003] VlPUB4 belongs to the U-box ubiquitin ligase (PUB) family. It plays a key role in immune regulation by mediating a series of complex ubiquitination modification reactions. PUB can ubiquitinate and modify core proteins in the plant immune defense system, thereby effectively maintaining homeostasis in the plant. Summary of the Invention

[0004] The purpose of this invention is to provide the grape gene VlPUB4 and its application in the genetic improvement of plant resistance to gray mold, so as to solve the problems mentioned in the background art.

[0005] The objective of this invention is achieved through the following technical solution: a grape gray mold resistance gene VlPUB4, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of the VlPUB4 protein encoded by the grape gray mold resistance gene VlPUB4, as shown in SEQ ID NO.2;

[0006] The coding region of the grape gray mold resistance gene VlPUB4 is 1020 bp in length, encoding 339 amino acids, and the amino acid sequence contains a total of 5 ARM domains.

[0007] The sequence SEQ ID NO.1 is shown below:

[0008]

[0009] The amino acid sequence of the protein, SEQ ID NO.2, is shown below:

[0010] SEQ ID NO.2

[0011] MGQSSGDHNDYSSTFSDCNSDRSGEFPTTSSQGRRLLLACAADNCDDLIRQLVSDLDSCSIDEQKQAAMEIRLLAKNKPENRLKIAGAGAIKPLISLISSSDAQLQENGVTAILNLSLCDENKELIASSGAIKPLVRALKTGTSTAKENAACALLRLSQIEENKIVIGM AGAIPLLVNLLGYGSFRGKKDASTTLYSLCSVKENKIRAIQAGIMKPLVELMADFGSNMVDKAAYVLSQLVSLPEGRTSLVEEDGIPVLVEILEDGSQRQKEIAVAILLQICEDSLAYRNMVAREGAIPPLVALSQSSANRSKQKAEALIDLLRQPRSGNVAARTSDVSV 339.

[0012] Furthermore, it includes a primer pair for amplifying the VlPUB4 gene, the primer sequences of which are:

[0013] VlPUB4-F:

[0014] ATGGGGCAGAGCTCTGGTG;

[0015] VlPUB4-R:

[0016] TTAGACTGACACATCTGACGTTCTGG.

[0017] The application of the grape gene VlPUB4 in the genetic improvement of plant resistance to gray mold includes a recombinant vector or recombinant plasmid containing the gene VlPUB4.

[0018] Furthermore, the CDS sequence of the grape VlPUB4 gene was cloned, using cDNA from the "Betta" grape as a template, and amplified using a high-fidelity enzyme. The amplification primer sequences are as follows:

[0019] VlPUB4-F: 5'-ATGGGGCAGAGCTCTGGTG-3'

[0020] VlPUB4-R: 5'-TTAGACTGACACATCTGACGTTCTGG-3'

[0021] After obtaining the full-length sequence of the VlPUB4 gene by PCR, the PCR product was purified and recovered and ligated into the pCAMBIA2300 vector. The ligation product was then transformed into competent E. coli DH5α cells by heat shock. Recombinant plasmids were extracted from bacterial cultures with the correct sequencing results.

[0022] Furthermore, the application of the grape VlPUB4 gene or the grape gray mold resistance gene VlPUB4 encoded protein or recombinant vector in regulating plant resistance to gray mold, wherein the plants are grape, Arabidopsis thaliana and tobacco.

[0023] Furthermore, the plant expression vector is a pCAMBIA-2300 series vector.

[0024] Furthermore, the method for constructing the overexpression vector of the gene VlPUB4 is as follows:

[0025] Based on the CDS sequence of the VlPUB4 gene, specific amplification primers with restriction enzyme sites were designed. The VlPUB4 gene was integrated into the overexpression vector pCAMBIA2300 using homologous recombination to construct the pCAMBIA2300-VlPUB4 recombinant plasmid. This plasmid was then transformed into Agrobacterium GV3101 using the heat shock method. The primer sequences are as follows:

[0026] pC2300-VlPUB4-F:

[0027] GAGCTCGGTACCCGGGGATCCATGGGGCAGAGCTCTGGTG;

[0028] pC2300-VlPUB4-R:

[0029] CTTGCTCACCATGGTGTCGACTTAGACTGACACATCTGACGTTCTGG;

[0030] The method for constructing the gene VlPUB4 silencing vector is as follows:

[0031] Based on the CDS sequence of the VlPUB4 gene, specific amplification primers with restriction enzyme sites were designed. The VlPUB4 gene was integrated into the silencing vector pTRV2 using homologous recombination to construct the pTRV2-VlPUB4 recombinant plasmid. This plasmid was then transformed into Agrobacterium GV3101 using the heat shock method. The primer sequences are as follows:

[0032] pTRV2-VlPUB4-F:

[0033] GTGAGTAAGGTTACCGAATCCGGTGGATAAAGCAGCTTACGTGT;

[0034] pTRV2-VlPUB4-R:

[0035] GAGACGCGTGAGCTCGGTACCCCGGATCTTGGTTGCCGT.

[0036] Furthermore, the host cell of the recombinant vector is Agrobacterium cells.

[0037] Furthermore, this is achieved by introducing the grape gene VlPUB4 into grape cells, tissues, or plants to enable its expression.

[0038] Furthermore, the method for introducing the grape gene VlPUB4 into grape cells, tissues, or plants is Agrobacterium-mediated transformation.

[0039] This invention analyzes the expression pattern of the gene PUB4 after being induced by gray mold in grape varieties with different resistance. It finds that the gene expression level of PUB4 in the susceptible grape variety "Seedless White" is significantly lower than that in the resistant variety "Beda", indicating that the gene PUB4 is related to the resistance of grapes to gray mold.

[0040] This invention establishes genetically modified materials that stably overexpress VlPUB4 by transiently overexpressing or silencing VlPUB4 in grape leaves or fruits, heterologously overexpressing VlPUB4 in Arabidopsis thaliana, and establishing materials that stably overexpress VlPUB4 in grape callus. The results showed that after inoculation of plant materials with Botrytis cinerea, the disease incidence was significantly reduced in grape leaves or fruits, Arabidopsis thaliana, and grape callus overexpressing VlPUB4 compared to the control group. Measurements of electrolyte permeability and chlorophyll content in grape leaves overexpressing VlPUB4 revealed that, compared to the control group, VlPUB4 overexpression reduced the upregulation of electrolyte permeability and inhibited the decrease in chlorophyll content, indicating that VlPUB4 can alleviate the stress damage caused by Botrytis cinerea to plants. Analysis of reactive oxygen species, malondialdehyde (MDA), a peroxidation product, and the activities of antioxidant enzymes such as SOD, POD, and CAT in grape leaves overexpressing VlPUB4 revealed that, compared with the control group, overexpression of the VlPUB4 gene in grape leaves could slow down the accumulation of membrane lipid peroxidation product MDA and significantly enhance the activities of endogenous antioxidant enzymes such as SOD, POD, and CAT. Silencing VlPUB4 showed the opposite trend, indicating that VlPUB4 can enhance the tolerance of grapes to oxidative stress induced by Botrytis cinerea.

[0041] This invention introduces a recombinant vector containing the VlPUB4 promoter into tobacco leaves and inoculates them with Botrytis cinerea. The promoter activity of the VlPUB4 gene before and after treatment is detected. It is found that compared with the control group, the promoter activity of proVlPUB4 is significantly enhanced after induction by Botrytis cinerea, while the promoter activity of proVvPUB4 does not change significantly.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] This invention is the first to conduct research on the relationship between the ubiquitin ligase gene VlPUB4 and gray mold resistance in grapes. The gene VlPUB4 was cloned, isolated and its disease resistance function was verified in grapes. It was found that it can enhance the ability of grapes to resist gray mold, providing richer gene resources for grape breeding. It can provide important gene resources and theoretical references for the future use of molecular breeding methods to cultivate new disease-resistant grape varieties. Attached Figure Description

[0044] Figure 1 This is a diagram analyzing the expression pattern of the grape PUB4 gene in response to Botrytis cinerea under induction conditions.

[0045] Figure 2 This is an analysis of the promoter activity of the grape VlPUB4 gene;

[0046] Among them: A is the qualitative analysis of promoter activity after tobacco conversion using the PUB4 promoter, conducted using histochemical staining; B is the quantitative analysis of the activity of the grape PUB4 promoter.

[0047] Figure 3 This is an agarose gel electrophoresis image of the grape VlPUB4 gene. The size of the bands indicates the successful cloning of grape VlPUB4.

[0048] Figure 4 This study obtained grape callus overexpressing the VlPUB4 gene through genetic transformation and compared its phenotype before and after Botrytis cinerea inoculation. A represents the expression level analysis of VlPUB4 in transgenic grape callus; B represents the phenotype observation of transgenic grape callus before and after Botrytis cinerea inoculation.

[0049] Figure 5 Transgenic Arabidopsis thaliana lines overexpressing the VlPUB4 gene were obtained through genetic transformation, and their phenotypic characteristics were observed after inoculation with Botrytis cinerea.

[0050] Figure 6This study investigated the overexpression of the VlPUB4 gene in the leaves of the "Seedless White" grape variety and analyzed its effect on disease resistance. Specifically: A) Analysis of the VlPUB4 gene expression level in "Seedless White" grape leaves overexpressing the VlPUB4 gene; B) Phenotypic changes in "Seedless White" grape leaves overexpressing the VlPUB4 gene before and after inoculation with *Botrytis cinerea*; C) Electrolyte osmotic pressure analysis in "Seedless White" grape leaves overexpressing the VlPUB4 gene before and after inoculation with *Botrytis cinerea*; and D) Phenotypic changes in "Seedless White" grape leaves overexpressing the VlPUB4 gene. E shows the chlorophyll content of leaves before and after inoculation with Botrytis cinerea; F shows the MDA content of leaves of "seedless white" grapes overexpressing the VlPUB4 gene before and after inoculation with Botrytis cinerea; G shows the peroxidase (POD) activity of leaves of "seedless white" grapes overexpressing the VlPUB4 gene before and after inoculation with Botrytis cinerea; H shows the catalase (CAT) activity of leaves of "seedless white" grapes overexpressing the VlPUB4 gene before and after inoculation with Botrytis cinerea.

[0051] Figure 7 This study analyzed the reactive oxygen species (ROS) content in the leaves of "seedless white" grapes overexpressing the VlPUB4 gene before and after inoculation with Botrytis cinerea. A was NBT staining to determine the superoxide anion content; B was DAB staining to determine the hydrogen peroxide content. Both A and B were based on histochemical staining methods for ROS content determination.

[0052] Figure 8 This study analyzed the resistance of 'Betta' grapes to gray mold after silencing the VlPUB4 gene in the leaves. Specifically: A) Analysis of VlPUB4 gene expression levels in 'Betta' grape leaves with silenced VlPUB4; B) Phenotypic observation of 'Betta' grape leaves with silenced VlPUB4 before and after inoculation with gray mold; C) Electrolyte osmotic pressure measurement of 'Betta' grape leaves with silenced VlPUB4 before and after gray mold inoculation; D) [Further details about the effects of VlPUB4 gene silencing on the resistance of 'Betta' grapes.] E represents the determination of chlorophyll content in grape leaves before and after inoculation with Botrytis cinerea; F represents the determination of MDA content in leaves of "Betta" grape with silenced VlPUB4 gene before and after inoculation with Botrytis cinerea; G represents the determination of POD activity in leaves of "Betta" grape with silenced VlPUB4 gene before and after inoculation with Botrytis cinerea; H represents the determination of CAT activity in leaves of "Betta" grape with silenced VlPUB4 gene before and after inoculation with Botrytis cinerea.

[0053] Figure 9These are chemical histological stainings of Betta grape leaves with the silenced VlPUB4 gene before and after inoculation with Botrytis cinerea. Among them: A is NBT staining; B is DAB staining. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0055] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, so that those skilled in the art can understand and implement the present invention. Unless otherwise stated, the specific experimental methods involved in the following embodiments are conventional methods, or implemented according to the conditions recommended in the instructions of the manufacturers of the reagents and instruments used.

[0056] Example 1: Gene expression analysis of the grape PUB4 gene was performed after treating the disease-resistant grape variety "Betta" and the disease-susceptible grape variety "Seedless White" with Botrytis cinerea.

[0057] To analyze the expression pattern of the VlPUB4 gene, this study employed quantitative real-time PCR (qRT-PCR). Total RNA was extracted from grape leaves treated with the fungus using a plant total RNA extraction kit. Reverse transcription was performed using the HiScriptII Q Select RT SuperMix kit (Vazyme, China) to synthesize cDNA. Gene-specific primers were designed and synthesized using cDNA from the disease-resistant grape variety 'Beda' as a template. The qRT-PCR reaction was performed according to the instructions of the SYBR-qPCR-Mix-kit (Vazyme, China), with three technical replicates for each sample. Relative gene expression levels were analyzed by 2... −ΔΔCt The calculations were performed using the method described in Table 1. The reaction system is detailed in Table 1, and the reaction procedure is detailed in Table 2.

[0058] Using the grape Actin gene as an internal reference gene, the primers used are as follows:

[0059] Actin-F: 5'-ACTCCTACGGGAGGCAGCAG-3'

[0060] Actin-R: 5'-ATTACCGCGGCTGCTGG-3'

[0061] VlPUB4-qPCR-F: 5'-CTCTCTGTGCGACGAGAATAAG-3'

[0062] VlPUB4-qPCR-R: 5'-ATCTCCACGAGTACCGGAATA-3'

[0063] Table 1 Quantitative PCR reaction system

[0064]

[0065] Table 2 Quantitative PCR reaction procedure

[0066]

[0067] The results showed that the expression level of the VlPUB4 gene was strongly induced by Botrytis cinerea. At 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h after inoculation with Botrytis cinerea, the gene expression level of VlPUB4 in the resistant grape variety "Beda" was significantly higher than that in the susceptible grape variety "Seedless White". The gene expression level reached its peak at 120 h, which was nearly 8 times higher than the initial level. This indicates that the VlPUB4 gene is significantly upregulated by Botrytis cinerea and may play an important role in the process of plant resistance to Botrytis cinerea.

[0068] Example 2: Analysis of promoter activity of the grape VlPUB4 gene

[0069] Using the DNA of grape "Beda" as a template, a high-fidelity enzyme was used for amplification. The amplification system is shown in Table 3, and the amplification procedure is shown in Table 4.

[0070] The amplification primer sequences are:

[0071] pC0390-VlPUB4-F: 5'-CAAGCTTGGCTGCAGGTCGACTCACTTTTTTCACCTAAAAAATAC-3'

[0072] pC0390-VlPUB4-R: 5'-GGTCTTAGAATTCCCGGATCCGTAGATATAGTTGGCCGGAATCTC-3'

[0073] After obtaining the promoter sequences of the VlPUB4 and VvPUB4 genes by PCR, the PCR products were purified and recovered and ligated into the pC0390-GUS vector. The specific vector construction method was performed according to the instructions of the One-Step Cloning Kit (Vazyme, China). The ligation product was transformed into competent E. coli DH5α cells by heat shock. Positive clones with correct sequencing were selected and recombinant plasmids were extracted for subsequent promoter activity analysis.

[0074] Table 3 Gene amplification system

[0075]

[0076] Table 4 Gene Amplification Procedure

[0077]

[0078] The constructed pC0390-VlPUB4 and pC0390-VvPUB4 recombinant vector plasmids were transformed into Agrobacterium GV3101, and the concentration of Agrobacterium culture containing the recombinant plasmids was adjusted to OD. 600 After adding 0.7-0.8 μL of the solution, it was injected into the underside of the leaves of 5-week-old Nicotiana benthamiana. The plants were then cultured at 22°C, 60% humidity, and under 16 h light / 8 h dark conditions for 48 h. Histochemical staining of Nicotiana benthamiana was performed using a GUS staining kit (Coolaber, China), and quantitative analysis of GUS protein activity was performed using a GUS gene quantification kit (Coolaber, China).

[0079] The results showed that in tobacco leaves not inoculated with Botrytis cinerea, the GUS activity of the VlPUB4 promoter was significantly stronger than that of the VvPUB4 promoter. After inoculation with Botrytis cinerea, the GUS activity of the VlPUB4 promoter was significantly enhanced compared with that before inoculation, while the GUS activity of the VvPUB4 promoter did not differ significantly before and after inoculation.

[0080] Example 3: Cloning of the CDS sequence of the grape VlPUB4 gene

[0081] Using cDNA from "Betta" grapes as a template, a high-fidelity enzyme was used for amplification. The amplification system and procedure were the same as above.

[0082] The amplification primer sequences are:

[0083] VlPUB4-F: 5'-ATGGGGCAGAGCTCTGGTG-3'

[0084] VlPUB4-R: 5'-TTAGACTGACACATCTGACGTTCTGG-3'

[0085] After obtaining the full-length sequence of the VlPUB4 gene by PCR, the PCR product was purified and recovered, and ligated into the pCAMBIA2300 vector. The ligation product was then transformed into competent E. coli DH5α cells using a heat shock method. Recombinant plasmids were extracted from bacterial cultures with correctly sequenced sequences after amplification and culture. The coding region of this gene is 1020 bp long, encoding 339 amino acids. The amino acid sequence contains five ARM domains, which are characteristic domains of members of the U-box ubiquitin ligase PUB family.

[0086] Example 4: Genetic transformation and functional verification of grape callus

[0087] Agrobacterium containing the pCAMBIA2300-VlPUB4 fusion plasmid was activated in a shaker at 28°C and 180 rpm for 12-16 h. Then, 1 mL of the activated bacterial culture was transferred to 30 mL of YEP (containing Rif and Kan antibiotics) and shaken in a shaker at 28°C and 180 rpm until OD was reached. 600 When the concentration of callus is 0.4-0.6, centrifuge at 5000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Wash the bacterial cells twice with 30 mL of sterile water, centrifuge at 5000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in 30 mL of sterile water (containing 200 μM acetylsylgenone) and incubate at 28℃ in the dark for 3 h. Add 'Cabernet Sauvignon' grape callus that has grown for about 15 days to the resuspension, incubate at 24℃ with shaking at 120 rpm for 20 min, filter the callus with sterile gauze, discard the infection solution, place the filtered callus on sterile filter paper, dry it for about 30 min, and then spread it evenly on B5 agar medium (containing 200 μM acetylsylgenone). After 2 days of dark culture, the callus tissue was washed three times in sterile water, dried, and then laid flat on B5 agar selection medium (containing Kan antibiotic). After about 30 days of culture, total RNA was extracted from the newly grown callus tissue for qRT-PCR verification.

[0088] The gene expression level of VlPUB4 in transgenic grape callus was detected by qRT-PCR, using the same primers as in Example 1. The results showed that, compared with the control group, the transgenic gene expression level was nearly 5 times that of the empty negative control group, indicating a high expression level. This demonstrates that the VlPUB4 gene was successfully overexpressed in grape callus and can be used for subsequent disease resistance verification experiments.

[0089] Transgenic grape callus that had been identified was inoculated with Botrytis cinerea and observed at 0 h, 72 h, and 120 h. It was found that, compared with the control group, the lesion area of ​​transgenic callus overexpressing VlPUB4 was significantly smaller than that of the empty vector control group, indicating that VlPUB4 can enhance the resistance of grapes to Botrytis cinerea.

[0090] Example 5: Genetic transformation and functional verification of Arabidopsis thaliana, a model plant

[0091] Agrobacterium containing the pCAMBIA2300-VlPUB4 fusion plasmid was used to genetically transform Arabidopsis thaliana. The genetic transformation was carried out by dipping the inflorescence in Agrobacterium. After successive generations of screening in a kanamycin-resistant medium, Arabidopsis thaliana T3 generation seeds were finally obtained. After the seedlings were propagated, the entire T3 generation and control group Arabidopsis thaliana plants were sprayed with a suspension of Botrytis cinerea spores.

[0092] Preparation of *Botrytis cinerea* spore suspension: Mycelia were scraped from the surface of the culture medium using an autoclaved applicator and filtered through double-layer sterile gauze to obtain a homogeneous spore suspension. Spore counting was performed using a hemocytometer: 30 μL of the suspension was added to the counting chamber, and the number of spores was counted in five randomly selected fields of view, with the average value calculated. Based on the counting results, the spore concentration was adjusted to 1 × 10⁻⁶. 7 / mL, prepare fresh for immediate use. When inoculating, select leaves at the same node for inoculation; all experiments were repeated three times.

[0093] The results showed that 72 h after inoculation with gray mold, the transgenic lines exhibited significantly less yellowing compared to the control group, confirming that VlPUB4 could enhance the plants' resistance to gray mold.

[0094] Example 6: Obtaining transgenic grape leaves overexpressing the VlPUB4 gene and verifying their disease resistance function.

[0095] Agrobacterium containing the pCAMBIA2300-VlPUB4 fusion plasmid was activated and shaken, as described above, in a shaker at 28°C and 180 rpm until OD was reached. 600 When the OD value is 0.8-1.0, centrifuge at 5000 rpm for 10 min, discard the supernatant and collect the bacterial cells. Wash twice with sterile water, then resuspend the bacterial cells in buffer (1 M MgCl2, 500 mM MES, 50 mM acetylsylcholine) and adjust to OD value. 600 After reaching a pressure of 0.8, leaves of seedless white grapes of uniform growth and size were immersed in the suspension. A circulating water vacuum pump was used to vacuum the leaves at 0.08 MPa for 30 minutes. When the leaf surface showed obvious water stains, the leaves were removed and the residual bacterial solution was gently wiped away with sterile filter paper. The petioles were wrapped with moistened sterile absorbent cotton and placed in a petri dish for dark incubation for 1 day. Afterward, they were inoculated with *Botrytis cinerea*, and disease resistance was comprehensively assessed by measuring phenotype, electrolyte osmotic pressure, chlorophyll content, reactive oxygen species content, and antioxidant enzyme activity.

[0096] The expression level of VlPUB4 in transgenic grape leaves overexpressing the VlPUB4 gene was analyzed using qRT-PCR, with the same primers as in Example 1. The results showed that, compared to the control group leaves, the transgenic gene expression level was nearly 5 times that of the empty negative control group, indicating a high expression level. This demonstrates that the VlPUB4 gene was successfully overexpressed in the leaves of "seedless white" grapes and can be used for subsequent functional verification experiments.

[0097] The disease resistance phenotypes of grape leaves were recorded at 0 h, 72 h, and 120 h. It was found that the diseased area of ​​grape leaves overexpressing VlPUB4 was smaller than that of the negative control, the upregulation of electrolyte permeability was smaller, the decrease in chlorophyll content was less, the increase in MDA was smaller, and the upregulation of antioxidant enzymes SOD, POD, and CAT was higher. The accumulation of reactive oxygen species in grape leaves overexpressing VlPUB4 was observed by NBT staining and DAB staining. It was found that overexpression of the VlPUB4 gene in grapes can significantly reduce the accumulation of reactive oxygen species. The above results indicate that the VlPUB4 gene can enhance the plant's resistance to gray mold.

[0098] Example 7: Validation of disease resistance function of "Betta" grape after silencing the VlPUB4 gene

[0099] Agrobacterium containing the pTRV2-VlPUB4 fusion plasmid was infecting and transforming "Betta" grape leaves, using the same method as in Example 6. The comprehensive disease resistance of grapes was assessed by identifying the disease resistance phenotype after inoculation with Botrytis cinerea, and measuring electrolyte permeability, chlorophyll content, peroxidation products, and antioxidant enzyme activity.

[0100] The gene expression level of VlPUB4 in grape leaves with the VlPUB4 gene silenced was detected using qRT-PCR. The primers for quantitative real-time PCR are as follows:

[0101] VlPUB4-qPCR-F: 5'-GGAAGATGGTATTCCGGTACTC-3'

[0102] VlPUB4-qPCR-R: 5'-GGATCTTGGTTGCCGTAGAA-3'

[0103] The results showed that, compared with the control group leaves, the expression level of the VlPUB4 gene in the silent group grape leaves was nearly 0.1 times that of the empty negative control group, and the expression level was extremely low. This indicates that the VlPUB4 gene was successfully silenced in the leaves of "Beda" grapes and can be used for subsequent verification of disease resistance function.

[0104] Leaf disease resistance phenotypes were monitored at 0 h, 72 h, and 120 h. It was found that the diseased area of ​​leaves of the 'Beda' grape with the PUB4 gene silenced was larger than that of the negative control, the electrolyte permeability was increased more significantly, the chlorophyll content was decreased more significantly, the MDA was increased more significantly than that of the empty control group, and the activities of antioxidant enzymes SOD, POD, and CAT were increased less significantly. The accumulation level of reactive oxygen species in the leaves of 'Beda' grape with the VlPUB4 gene silenced was analyzed by NBT staining and DAB staining. It was found that silencing the VlPUB4 gene could significantly increase the content of reactive oxygen species, indicating that silencing the VlPUB4 gene can weaken the plant's resistance to gray mold.

[0105] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0106] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A grapevine downy mildew resistance gene V1PUB4, characterized in that: The nucleotide sequence of the grape gray mold resistance gene VlPUB4 is shown in SEQ ID NO.1, and the amino acid sequence of the VlPUB4 protein encoded by the grape gray mold resistance gene VlPUB4 is shown in SEQ ID NO.2; The coding region of the grape gray mold resistance gene VlPUB4 is 1020 bp in length, encoding 339 amino acids, and the amino acid sequence contains a total of 5 ARM domains.

2. The grape gene VlPUB4 according to claim 1, characterized in that: Includes a primer pair for amplifying the VlPUB4 gene, the primer sequences of which are: VlPUB4-F: ATGGGGCAGAGCTCTGGTG; VlPUB4-R: TTAGACTGACACATCTGACGTTCTGG.

3. The application of the grape gene VlPUB4 according to claim 1 in the genetic improvement of plant resistance to gray mold, characterized in that: A recombinant vector or recombinant plasmid containing the gene VlPUB4 as described in claim 1.

4. The application of the grape gene VlPUB4 according to claim 3 in the genetic improvement of plant resistance to gray mold, characterized in that: The CDS sequence of the grape VlPUB4 gene was cloned using cDNA from the "Betta" grape as a template and amplified with a high-fidelity enzyme. The primer sequences were as follows: VlPUB4-F: 5'-ATGGGGCAGAGCTCTGGTG-3' VlPUB4-R: 5'-TTAGACTGACACATCTGACGTTCTGG-3' After obtaining the full-length sequence of the VlPUB4 gene by PCR, the PCR product was purified and recovered and ligated into the pCAMBIA2300 vector. The ligation product was then transformed into competent E. coli DH5α cells by heat shock. Recombinant plasmids were extracted from bacterial cultures with the correct sequencing results.

5. The application of the grape gene VlPUB4 according to claim 1 or 3 in the genetic improvement of plant resistance to gray mold, characterized in that: The application of the grape VlPUB4 gene or the grape gray mold resistance gene VlPUB4 encoded protein or recombinant vector in regulating plant resistance to gray mold, wherein the plants are grape, Arabidopsis thaliana and tobacco.

6. The application of the grape gene VlPUB4 according to claim 5 in the genetic improvement of plant resistance to gray mold, characterized in that: The plant expression vector is the pCAMBIA-2300 series vector.

7. The application of the grape gene VlPUB4 according to claim 6 in the genetic improvement of plant resistance to gray mold, characterized in that: The method for constructing the overexpression vector of the gene VlPUB4 is as follows: Based on the CDS sequence of the VlPUB4 gene, specific amplification primers with restriction enzyme sites were designed. The VlPUB4 gene was integrated into the overexpression vector pCAMBIA2300 using homologous recombination to construct the pCAMBIA2300-VlPUB4 recombinant plasmid. This plasmid was then transformed into Agrobacterium GV3101 using the heat shock method. The primer sequences are as follows: pC2300-VlPUB4-F: GAGCTCGGTACCCGGGGATCCATGGGGCAGAGCTCTGGTG; pC2300-VlPUB4-R: CTTGCTCACCATGGTGTCGACTTAGACTGACACATCTGACGTTCTGG; The method for constructing the gene VlPUB4 silencing vector is as follows: Based on the CDS sequence of the VlPUB4 gene, specific amplification primers with restriction enzyme sites were designed. The VlPUB4 gene was integrated into the silencing vector pTRV2 using homologous recombination to construct the pTRV2-VlPUB4 recombinant plasmid. This plasmid was then transformed into Agrobacterium GV3101 using the heat shock method. The primer sequences are as follows: pTRV2-VlPUB4-F: GTGAGTAAGGTTACCGAATCCGGTGGATAAAGCAGCTTACGTGT; pTRV2-VlPUB4-R: GAGACGCGTGAGCTCGGTACCCCGGATCTTGGTTGCCGT.

8. The application of the grape gene VlPUB4 according to claim 7 in the genetic improvement of plant resistance to gray mold, characterized in that: The host cell for the recombinant vector is Agrobacterium cells.

9. A method for improving the resistance of grapes to gray mold, characterized in that: This includes introducing the grape gene VlPUB4 as described in claim 1 into grape cells, tissues, or plants to induce its expression.

10. The method for improving grape resistance to gray mold according to claim 9, characterized in that: The method for introducing the grape gene VlPUB4 into grape cells, tissues, or plants is the Agrobacterium-mediated method.

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