Disease-resistant gene VaWRKY14 of Vitis amurensis and its application

Abstract

The present invention belongs to the field of biotechnology and specifically relates to a Vitis vinifera disease-resistance gene, VaWRKY14, and its applications. The present invention analyzes the expression patterns of this gene in grape varieties with different resistance to gray mold induction. Heterologous and transient overexpression were used to verify the role of VaWRKY14 in regulating gray mold resistance in grapes. The results showed that, compared with control leaves, grape leaves overexpressing VaWRKY14 exhibited reduced reactive oxygen species bursts, enhanced antioxidant capacity, and increased expression of disease-resistance-related genes. This indicates that this gene can regulate gray mold resistance in grapes and has a positive guiding role in the breeding of new gray mold-resistant grape varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a Vitis vinifera disease-resistant gene VaWRKY14 and an application thereof in regulating gray mold resistance. Background Art

[0002] Grape (Vitis) is one of the four major fruits in the world. According to OIV statistics, my country's grape planting area reached 78.3 hectares in 2021, ranking third in the world. Grapes occupy a very important position in my country's agricultural industry and regional economic development. Gray mold (Botrytis cinerea) is a fungal disease that seriously harms the grape industry. Globally, gray mold causes a 20% to 40% grape production reduction each year, and severe cases can reach more than 60%. It is becoming more and more serious with the widespread use of facility cultivation. At present, gray mold is mainly controlled by chemical pesticides and biological control, but the effect is not good. It not only increases the cost of grape cultivation management and pollutes the environment, but also seriously affects the quality of grape products. Domestic and foreign research and breeding practices have proved that using the plant's own disease-resistant genes to cultivate high-quality disease-resistant grape varieties is one of the important measures for long-term, effective and safe control of grape fungal diseases.

[0003] WRKY transcription factors (WRKYs) are zinc-finger transcriptional regulators unique to higher plants. Members of the WRKY family are components of signaling networks that regulate many plant responses to biotic and abiotic stresses, as well as growth and developmental processes. In recent years, the role of WRKYs in plant responses to Botrytis cinerea has garnered increasing attention. However, whether WRKYs play a key role in plant resistance to Botrytis cinerea, particularly in grapevine, remains to be determined. Summary of the Invention

[0004] The present invention aims to provide a Vitis vinifera disease-resistant gene VaWRKY14 and its application in regulating gray mold resistance.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a Vitis amurensis disease resistance gene VaWRKY14, wherein the DNA sequence of the gene VaWRKY14 is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a protein encoded by the gene VaWRKY14, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned gene VaWRKY14.

[0009] Preferably, the recombinant vector is an Agrobacterium overexpression vector.

[0010] In a fourth aspect, the present invention provides a primer for amplifying the gene VaWRKY14, wherein the primer sequence is:

[0011] VaWRKY14-SAK-F:

[0012] ATCCAAAGAATTCCCCGGTACCATGGCATCGGGGGATTACTCGCAGG;

[0013] VaWRKY14-SAK-R:

[0014] CATGATCTTTGTAATCCTCGAGTTAATAAAAAGGAGGGTGAGAAGAA.

[0015] In a fifth aspect, the present invention provides a method for subcellular localization of the gene VaWRKY14, specifically comprising:

[0016] Specific primers with restriction enzyme sites were designed to amplify the CDS sequence of the VaWRKY14 gene. The VaWRKY14 gene was integrated into the subcellular localization vector pCAMBIA2300-35S-GFP using homologous recombination. The pCAMBIA2300-35S-VaWRKY14-GFP recombinant plasmid was constructed and transformed into GV3101 Agrobacterium using the heat shock method. The primer sequences are as follows:

[0017] VaWRKY14-2300-GFP-F:

[0018] ACGGGGGACGAGCTCGGTACCATGGCATCGGGGGATTACTCGCAGG;

[0019] VaWRKY14-2300-GFP-R:

[0020] GGTGTCGACTCTAGAGGATCCATAAAAAGGAGGGTGAGAAGAAGTT.

[0021] In a sixth aspect, the present invention provides a method for constructing a VaWRKY14 gene overexpression vector, specifically comprising:

[0022] Specific primers with restriction enzyme sites were designed to amplify the CDS sequence of the VaWRKY14 gene. The VaWRKY14 gene was integrated into the overexpression vector pSAK277 using homologous recombination to construct the pSAK277-VaWRKY14 recombinant plasmid, which was then transformed into GV3101 Agrobacterium using the heat shock method. The primer sequences are as follows:

[0023] VaWRKY14-SAK-F:

[0024] ATCCAAAGAATTCCCCGGTACCATGGCATCGGGGGATTACTCGCAGG;

[0025] VaWRKY14-SAK-R:

[0026] CATGATCTTTGTAATCCTCGAGTTAATAAAAAGGAGGGTGAGAAGAA.

[0027] In a seventh aspect, the present invention provides a use of the gene VaWRKY14 or the protein in improving plant resistance to gray mold.

[0028] Preferably, the gene VaWRKY14 or the protein is used to alleviate the extent of reactive oxygen species burst, improve antioxidant capacity, or activate the expression of disease-resistant related genes.

[0029] In an eighth aspect, the present invention provides a method for cultivating plants with improved resistance to gray mold, which is achieved by promoting the expression of the gene VaWRKY14.

[0030] Preferably, the method for cultivating plants with improved resistance to gray mold comprises the following steps:

[0031] constructing an overexpression vector containing the gene VaWRKY14;

[0032] The overexpression vector containing the VaWRKY14 gene is transformed into competent cells, cultured, and inoculated onto plants to obtain a plant strain resistant to gray mold.

[0033] Preferably, the plant is tobacco or Vitis amurensis.

[0034] The present invention verified the function of VaWRKY14 by heterologous overexpression in tobacco and transient overexpression in grape leaves. The study found that the gene can improve the resistance of grapes to gray mold, which is specifically manifested as: the diseased area of ​​grape leaves is reduced, H2O2 and O2 .- The production and accumulation of tau2+ increased, the activities of CAT, POD and AAO enzymes increased, and defense-related genes such as NtPOD, NtPAL, NtPDF1.2 and NtPR1a / b were activated earlier and more strongly.

[0035] Advantages of the present invention:

[0036] The present invention discloses a gene, VaWRKY14, and its application in regulating resistance to gray mold. The gene's expression pattern after induction by gray mold in grape varieties with different resistances was analyzed. Heterologous overexpression and transient overexpression were used to verify the role of VaWRKY14 in regulating gray mold resistance. The results showed that, compared with control leaves, grape leaves overexpressing VaWRKY14 showed significantly reduced reactive oxygen species bursts, significantly enhanced antioxidant capacity, and significantly increased expression of disease-resistance-related genes, thereby reducing the degree of leaf necrosis and enhancing resistance to gray mold. This indicates that the gene can regulate grape leaf resistance to gray mold, providing a positive guiding role in the breeding of new disease-resistant grape varieties. This study helps clarify the molecular mechanism of the role of the Vitis amurensis transcription factor VaWRKY14 in regulating gray mold resistance, providing a theoretical basis and gene resources for molecular breeding of disease-resistant grapes.

[0037] For some grape varieties that are extremely susceptible to gray mold during cultivation and production, new disease-resistant varieties can be bred by overexpressing the transcription factor VaWRKY14 gene or its homologous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The expression of WRKY family genes in grapevine in different resistant materials after treatment with Botrytis cinerea;

[0039] Figure 2 The effect of exogenous H2O2 treatment on leaves of Vitis amurensis 'Shuangyou';

[0040] Figure 3 This is the electrophoresis diagram of the full-length PCR amplification of VaWRKY14 gene;

[0041] Figure 4 is the subcellular localization analysis of VaWRKY14 gene;

[0042] Figure 5 The figure shows the generation of tobacco lines overexpressing the VaWRKY14 gene. Figure A. Generation of tobacco lines overexpressing VaWRKY14; (a) Pre-culture of leaves on culture medium; (b) Infected leaves placed on resistance selection medium for callus induction; (c) Callus formation from infected leaves; (d) Callus differentiation and bud formation; (e) Screening of resistant seedlings; (f) Transplantation of resistant seedlings. B. Genome-wide identification of three transgenic plants. C. Real-time quantitative PCR analysis of the expression of the grape VaWRKY14 gene in tobacco overexpressing lines (#2, #4, #36) and wild type (WT).

[0043] Figure 6Figure 3. Effect of overexpression of the grape VaWRKY14 gene on resistance to gray mold in tobacco. Figure 3. A. Phenotype of tobacco lines overexpressing VaWRKY14-OE after infection with gray mold. B. Lesion diameter. C. Trypan blue tissue staining.

[0044] Figure 7 This is the histochemical staining of tobacco leaves overexpressing the grape VaWRKY14 gene at different stages after infection; in the figure, A. DAB staining; B. NBT staining.

[0045] Figure 8 This is a quantitative analysis of antioxidant indicators in tobacco leaves after inoculation with gray mold; in the figure, A. H2O2 content; B. O2 .- content; C.CAT enzyme activity; D.POD enzyme activity; E.AAO enzyme activity; F:MDA content.

[0046] Figure 9 It is a quantitative analysis of resistance genes in tobacco leaves after inoculation with Botrytis cinerea;

[0047] Figure 10 Figure 3. Effects of transient overexpression of the VaWRKY14 gene on resistance to gray mold in 'Red Globe' grape leaves. Figure 3. A. Phenotypic observation after inoculation with gray mold. Figure 3. Analysis of the ratio of lesion area to leaf area at different stages after infection. DETAILED DESCRIPTION

[0048] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. However, the specific experimental methods involved in the following examples, unless otherwise specified, are all conventional methods or are performed under the conditions recommended by the manufacturer's instructions.

[0049] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The experimental methods in the following examples are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be purchased from the market.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0051] Unless otherwise indicated, the practice of the present invention will utilize conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination, and bioinformatics that are apparent to those skilled in the art and are fully explained in the published literature.

[0052] As used herein, the terms "nucleic acid," "nucleic acid sequence," "nucleotide," "nucleic acid molecule," or "polynucleotide" are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-stranded or double-stranded structures. These nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences in non-coding regions. These terms include a gene. "Gene" or "gene sequence" is widely used to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in a genomic sequence, and / or include coding sequences in a cDNA, and / or include cDNA and its regulatory sequences. In specific embodiments, such as with respect to isolated nucleic acid sequences, it is preferably assumed to be cDNA.

[0053] "Expression vectors" refers to a vector that adds expression elements (such as promoter, RBS, terminator, etc.) to the basic skeleton of a cloning vector to enable the expression of the target gene.

[0054] Agrobacterium-mediated transformation refers to the technique of inserting the target gene into the modified T-DNA region, using Agrobacterium infection to achieve the transfer and integration of the exogenous gene into plant cells, and then regenerating transgenic plants through cell and tissue culture technology.

[0055] This study used transcriptome sequencing and real-time quantitative analysis of the disease-resistant Chinese wild Vitis 'Shuangyou' and susceptible European Vitis 'Red Globe' treated with Botrytis cinerea to analyze and screen for highly significantly differentially expressed genes involved in the response to Botrytis cinerea. The candidate genes were cloned and overexpression vectors constructed. Gene function was then verified in tobacco and grape leaves. The results revealed that overexpression of VaWRKY14 can alleviate reactive oxygen species (ROS) bursts, enhance antioxidant capacity, and activate the expression of disease-resistance-related genes. The specific experiments are as follows:

[0056] 1. Analysis of WRKY family gene expression in grapes after treatment with Botrytis cinerea

[0057] The present invention analyzed the transcriptomes of disease-resistant Chinese wild Vitis amurensis 'Shuangyou' and susceptible European Vitis 'Red Globe' after infection with Botrytis cinerea. It was found that 14 WRKY family genes were extremely significantly involved in the response to Botrytis cinerea. RT-qPCR was used to detect their expression induced by Botrytis cinerea in three susceptible materials: European Vitis 'Flame Seedless', 'Seedless White', and 'Red Globe'; and four disease-resistant materials: wild Vitis amurensis 'Shuangyou', Qinling Vitis 'Pingli-5', Vitis 'Taishan-2', and European and American hybrid cultivar 'Giant Rose'. Figure 1 shown.

[0058] RT-qPCR steps: using the plant total RNA extraction kit EZNA Total RNA was extracted from grape leaves using Plant RNA Kit #R6827-01 (Omega Bio-Tek, USA), and cDNA was synthesized using the reverse transcription kit HiScriptⅢqRT SuperMixfor qPCR (+gDNAwiper) (Vazyme, China). This cDNA was used as a template and detected using the real-time fluorescence quantitative PCR kit ChamQ Universal SYBR qPCR MasterMix (Vazyme, China) using the real-time fluorescence quantitative PCR instrument Applied Biosystems 7500FAST fluorescence quantitative instrument (Applied Biosystems, USA). VvActin1 was selected as the internal reference gene, and the cDNA was synthesized using the reverse transcription kit HiScriptⅢqRT SuperMixfor qPCR (+gDNAwiper) (Vazyme, China). -ΔΔCt The relative gene expression was calculated using the formula.

[0059] PCR reaction system 20 μL: SYBR qPCR MasterMix 10 μL, template cDNA 1 μL, upstream and downstream primers 1 μL each, primer concentration 10 μmol·L -1 , ddH2O 7μL.

[0060] PCR reaction program: 95°C for 5 min, 40 cycles of 95°C for 10 s, 60°C for 30 s.

[0061] The primer sequences are as follows:

[0062] VaWRKY14-F: GATGCCAAGTGAAGAAGAGG, as shown in SEQ ID NO. 3;

[0063] VaWRKY14-R: TGTGATGTGGTTGCGAGGG, as shown in SEQ ID NO. 4;

[0064] VvActin1-F: GATTCTGGTGATGGTGTGAG, as shown in SEQ ID NO. 5;

[0065] VvActin1-R: GACAATTTCCCGTTCAGCAGT, as shown in SEQ ID NO.6.

[0066] The expression of WRKY family genes in grapes after treatment with Botrytis cinerea Figure 1As shown, VvWRKY3, 14, and 44 were all induced to express at high levels in the three susceptible materials, but only WRKY14 had the highest expression level among the four resistant materials, reaching its peak at the earliest stage of the interaction. Among them, VaWRKY14 in Vitis amurensis performed the most prominently and was considered to be the key WRKY gene in grape response to gray mold infection.

[0067] 2. Expression analysis of the Vitis amurensis transcription factor gene VaWRKY14 in response to exogenous H2O2 induction

[0068] The present invention analyzed the expression of VaWRKY14 in 'Shuangyou' grape leaves 0 to 48 hours after H2O2 treatment. The real-time fluorescence quantitative PCR steps, reaction system, procedure and primer sequences were the same as those described in 1.

[0069] The expression of VaWRKY14 in response to exogenous H2O2 is shown in Figure 2 As shown, VaWRKY14 transcription is activated by H₂O₂. Its expression level increases significantly 3 hours after treatment and reaches a peak 6 hours after treatment, which is 10 times higher than the control at the same time. Although it decreases thereafter, it still maintains an upward expression trend. This indicates that VaWRKY14 can respond to H₂O₂ and also suggests that VaWRKY14 may play a role in disease resistance through the ROS signaling pathway.

[0070] 3. Sequence analysis of the Vitis amurensis transcription factor gene VaWRKY14

[0071] According to the annotation information of Vitis vinifera in the NCBI database, the CDS sequence of Vitis vinifera VvWRKY14 was used as a reference, and the cDNA of 'Shuangyou' was used as a template. Specific primers were designed using Primer Premier 5.0 to amplify the full-length gene.

[0072] PCR reaction system 50 μL: 2× PhantaFlash MasterMix 25 μL, template cDNA 2 μL, upstream and downstream primers 2 μL each, primer concentration 10 μmol·L -1 , ddH2O 19μL.

[0073] RCR reaction program: 98°C for 30 s; 35 cycles: 98°C for 10 s, 58°C for 5 s, 72°C for 5 s; 72°C for 2 min.

[0074] The full-length sequence of VaWRKY14 was obtained by PCR. The electrophoresis diagram of PCR amplification is shown in Figure 2. Figure 3 The PCR products were purified and recovered. The ligation product was then ligated with a -Blunt vector (TransGen Biotech, China). The ligation product was transformed into competent E. coli DH5α cells using the heat shock method. The correctly sequenced bacterial cultures were selected for amplification and culture to extract the recombinant plasmid. The full length of the gene is 411 bp, and the sequence is shown in SEQ ID NO. 1, encoding 136 amino acids. The amino acid sequence of the transcription factor VaWRKY14 encoded by this gene is shown in SEQ ID NO. 2.

[0075] SEQ ID NO.1:

[0076] ATGGCATCGGGGGATTACTCGCAGGAGGAGATTATGGGCGGTGATATTCAG

[0077] TTCTCAAGTAGTGCTACTCCAACAAATGGGGGAGTGAAGAGACGAGGGGA

[0078] AGATATGGGGATTAGGGTTTTTGCATTGAGGACAAGATCCGAGGAGGATAC

[0079] CATAGATGATGGGTTTAAGTGGAGAAAATATGGAAAAAAGAAGATCAAGA

[0080] GCAACCCGATTTATCCAAGGAACTACTATAGATGTTCAAGCAGAGGATGCC

[0081] AAGTGAAGAAGAGGGTAGAAAGGGATAGGGATGACTCAAGTTGTGTAATA

[0082] ACTACATATGAGGGAGTCCACAACCACCCCACCCCTCGCAACCATATCACA

[0083] CTTCCTATTAATTATTGGGCTTTGCAACAAACTTCTTCTCACCCTCCTTTTTA

[0084] TTAA

[0085] SEQ ID NO.2:

[0086] MASGDYSQEEIMGGDIQFSSSATPTNGGVKRRGEDMGIRVFALRTRSEEDTID

[0087] DGFKWRKYGKKKIKSNPIYPRNYYRCSSRGCQVKKRVERDRDDSSCVITTYE

[0088] GVHNHPTPRNHITLPINYWALQQTSSHPPFY

[0089] 4. Subcellular localization analysis of the Vitis amurensis transcription factor VaWRKY14

[0090] The present invention uses the pCAMBIA2300-35S-GFP vector for subcellular localization. Specific primers with restriction enzyme sites were designed to amplify the CDS sequence of the VaWRKY14 gene, and the VaWRKY14 gene was integrated into pCAMBIA2300-35S-GFP using homologous recombination. The specific vector construction method refers to the homologous recombination kit. The pCAMBIA2300-35S-VaWRKY14-GFP subcellular localization vector plasmid was transformed into Agrobacterium GV 3101 according to the instructions of the II OneStep Cloning Kit (Vazyme, China). The primer sequences are as follows:

[0091] VaWRKY14-2300-GFP-F:

[0092] ACGGGGGACGAGCTCGGTACCATGGCATCGGGGGATTACTCGCAGG, as shown in SEQ ID NO:7;

[0093] VaWRKY14-2300-GFP-R:

[0094] GGTGTCGACTCTAGAGGATCCATAAAAAGGAGGGTGAGAAGAAGTT, as shown in SEQ ID NO:8.

[0095] The OD value of the Agrobacterium culture containing the recombinant plasmid was adjusted to 0.7-0.8. The culture was injected into the dorsal surface of leaves of well-grown, 5-week-old Nicotiana benthamiana plants. After 48 hours of incubation at 22°C, 16 hours of light / 8 hours of darkness, and 60% relative humidity, the cells were observed and photographed using a laser confocal microscope. The subcellular localization of VaWRKY14 was investigated using an empty vector (GFP) as a control and pBi221-mCherry red fluorescence as a nuclear localization marker.

[0096] The results of VaWRKY14 subcellular localization analysis were as follows: Figure 4As shown, detection of red fluorescence signals revealed that all cell nuclei were labeled red. Green fluorescence was detected in both the nuclei and cell membranes of control tobacco leaves, while green fluorescence was detected only in the nuclei of tobacco mesophyll cells transiently expressing VaWRKY14. In the mixed field, the nuclei of cells transiently expressing VaWRKY14 appeared yellow, coinciding with the location of mCherry staining. This indicates that the VaWRKY14 gene is localized to the nucleus and functions there.

[0097] 5. Functional Verification

[0098] To investigate whether VaWRKY14 regulates grape resistance to gray mold, its function was analyzed and identified by heterologous overexpression in tobacco and transient overexpression in grape leaves.

[0099] 5.1. Construction of overexpression vector

[0100] Specific primers with restriction enzyme sites were designed to amplify the CDS sequence of the VaWRKY14 gene. The VaWRKY14 gene was integrated into the overexpression vector pSAK277 using homologous recombination. The specific vector construction method was referred to the homologous recombination kit. The pSAK277-VaWRKY14 recombinant plasmid was transformed into Agrobacterium GV3101 according to the instructions of II One Step Cloning Kit (Vazyme, China).

[0101] The primer sequences are as follows:

[0102] VaWRKY14-SAK-F:

[0103] ATCCAAAGAATTCCCCGGTACCATGGCATCGGGGGATTACTCGCAGG, as shown in SEQ ID NO:9;

[0104] VaWRKY14-SAK-R:

[0105] GGTGTCGACTCTAGAGGATCCATAAAAAGGAGGGTGAGAAGAAGTT, as shown in SEQ ID NO:10.

[0106] 5.2. Obtaining tobacco lines heterologously overexpressing VaWRKY14

[0107] The present invention integrates VaWRKY14 into tobacco by Agrobacterium-mediated transformation, and obtains VaWRKY14 overexpression candidate plants after resistance screening ( Figure 5 Then, the genome level and transcriptional expression level of positive plants were detected by PCR and real-time fluorescence quantitative PCR ( Figure 5A batch of tobacco plants stably transformed with VaWRKY14 were obtained. Three overexpression lines (OE) with higher expression levels were selected, #2, #4 and #36 ( Figure 5 (B, C) The wild-type strain (WT) was used as a control to study the resistance of VaWRKY14 to gray mold.

[0108] 5.3 Effect of heterologous overexpression of VaWRKY14 on resistance to gray mold in tobacco lines

[0109] In this study, leaves from strains overexpressing VaWRKY14-OE and WT were inoculated with gray mold. Detached leaves were sprayed with a suspension of gray mold spores, while controls were sprayed with sterile water. Samples were collected at 0, 4, 8, 18, and 36 hpi after inoculation, and disease resistance was assessed through phenotype, lesion diameter analysis, histochemical staining, and measurements of ROS-related substances, enzyme activity, and defense-related gene expression.

[0110] The leaf phenotype after overexpression of VaWRKY14 gene is as follows Figure 6 As shown in A and B, 24 hpi after inoculation with gray mold, WT showed more severe water-soaked symptoms than VaWRKY14-OE, and the lesion diameter was significantly larger than that of VaWRKY14-OE, at 1.61 mm and 0.63 mm, respectively. By 48 hpi, large areas of soft rot appeared on the leaves of WT, and the lesion diameter was 7.22 mm, significantly larger than that of VaWRKY14-OE (1.78 mm).

[0111] Trypan blue tissue staining results Figure 6 As shown in Figure C, cell death in tobacco leaves was observed 48 hpi after inoculation with Botrytis cinerea. The results showed that WT leaves exhibited a darker blue color than VaWRKY14-OE leaves, indicating that WT cells experienced more severe cell death after infection with Botrytis cinerea. This suggests that overexpression of VaWRKY14 significantly blocked Botrytis cinerea infection and enhanced tobacco leaf resistance to Botrytis cinerea.

[0112] Histochemical staining results Figure 7 As shown in Figure 1, at 12 hpi after inoculation with Botrytis cinerea, the staining position of the WT strain leaves was relatively concentrated in the area in direct contact with the pathogen, indicating that more H2O2 and O2 accumulated in the inoculated area. .- The stained areas of the leaves of the VaWRKY14-OE strain were mostly spotted, with small and uneven distribution on the entire leaf surface, indicating that H2O2 and O2 .- It mainly accumulated in the non-inoculated areas of VaWRKY14-OE leaves and in the inoculated areas of WT leaves.

[0113] Quantitative test results of antioxidant indicators are as follows Figure 8As shown, at 0 hpi, when not inoculated, the VaWRKY14-OE strain accumulated more H2O2, which was significantly higher than that of the WT strain at the same period, and was 1.2 times its content. Afterwards, H2O2 in the leaves of the WT strain increased rapidly and peaked significantly at 12 hpi after inoculation. At this time, its H2O2 accumulation was significantly higher than that of the VaWRKY14-OE strain leaves by 1.3 times. Then, the accumulation of H2O2 in the leaves of the WT strain began to decline continuously, and eventually became significantly lower than that of the VaWRKY14-OE strain; however, after inoculation, H2O2 in the leaves of the VaWRKY14-OE strain increased significantly only at 12 hpi, and there was no significant difference between the other time points ( Figure 8 Middle A). As the infection time of gray mold increases, O2 .- The content of O2 in the leaves of VaWRKY14-OE strain showed an overall upward trend. .- The accumulation amount was always significantly higher than that of the WT strain at the same period ( Figure 8 (B). The CAT, POD, and AAO enzyme activities of the VaWRKY14-OE strain were significantly higher than those of the WT strain. After inoculation with Botrytis cinerea, the CAT enzyme activities of the leaves of the VaWRKY14-OE and WT strains increased significantly, and the former was significantly higher than the latter, especially at 12-24 hpi ( Figure 8 For POD enzyme activity, the VaWRKY14-OE strain was always significantly higher than the WT ( Figure 8 By observing the changes in AAO enzyme activity, it was found that the AAO enzyme activity of the VaWRKY14-OE strain was significantly higher than that of the WT strain. At 0 hpi, the former was 1.36 times that of the latter. After infection with gray mold, the leaves of both VaWRKY14-OE and WT strains showed a significant increase in AAO activity. From 12 to 24 hpi, the former was significantly higher than the latter ( Figure 8 In addition, MDA is one of the important indicators reflecting the degree of oxidative damage in plants. There was no significant difference in MDA content between VaWRKY14-OE and WT strains at 12-24 hpi in the control treatment. The MDA content of VaWRKY14-OE was significantly lower than that of WT at 0 and 48 hpi. However, after inoculation with Botrytis cinerea, the MDA content of VaWRKY14-OE was always significantly lower than that of WT ( Figure 8 Middle F).

[0114] Quantitative analysis of resistance genes Figure 9As shown, the expression levels of defense-related genes such as NtPOD, NtPAL, NtPDF1.2, and NtPR1a / b in VaWRKY14-OE strains were elevated compared to 0 hpi at the early stage of infection by Botrytis cinerea and significantly higher than those in the WT. In particular, at 12 hpi, the expression levels of NtPDF1.2 and NtPAL in VaWRKY14-OE strains were approximately 587-fold and 256-fold higher than those in the WT, respectively, while the expression levels of NtPR1a, NtPR1b, and NtPOD in VaWRKY14-OE strains were approximately 2.12-fold, 13-fold, and 4-fold higher than those in the WT, respectively. In particular, the expression of NtPDF1.2 was almost completely suppressed in the WT strain. In contrast, the expression of these genes in the WT strain was significantly activated at least 12 h later than in the VaWRKY14-OE strain.

[0115] 5.4 Effect of transient overexpression of VaWRKY14 on resistance to gray mold

[0116] VaWRKY14 was transiently overexpressed in the leaves of Red Globe. Agrobacterium GV3101 containing the target gene plasmid and the empty plasmid was activated and cultured, and the cells were collected by centrifugation. The bacterial suspension was resuspended in grape juice to a concentration of OD 600 The value was 0.7-0.8. The grape leaves in good growth condition were cleaned, naturally air-dried, and then immersed in the resuspended bacterial solution with the back facing up (the control used a resuspended empty bacterial solution). Then, the leaves were placed in a vacuum pump for vacuum treatment and allowed to stand for 30 minutes under a vacuum condition of 0.085 MPa. The air was then slowly released. The bacterial solution on the surface of the leaves was wiped dry with sterile filter paper. The petioles were wrapped with wet cotton and placed in a tray. The petioles were covered with plastic wrap and inoculated with gray mold. Wild-type leaves (WT) and leaves transiently overexpressing the empty vector pCAMBIA2300-GFP (GFP) were used as controls. Disease resistance identification was carried out through phenotypic observation and statistical analysis of the severity of the diseased area.

[0117] Phenotypic observation of leaves of grape 'Red Globe' transiently overexpressing VaWRKY14 after inoculation with Botrytis cinerea Figure 10 As shown, at 72 hpi, distinct lesions were observed on leaves of the WT and GFP control, with a diseased area of ​​approximately 9.4%. However, lesions on OE leaves were less pronounced, with a diseased area of ​​only 0.5%, significantly lower than that of the control. By 120 hpi, although the diseased area on OE leaves had increased to 11.0%, it was still significantly lower than the 66.2% and 41.5% lesions on the WT and GFP control leaves. After infection with Botrytis cinerea, not only did OE leaves develop disease later than the control, but their lesion area was also consistently significantly lower than that of the control. This indicates that transient overexpression of VaWRKY14 significantly enhances grapevine resistance to Botrytis cinerea.

[0118] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, it is of course possible to easily make other implementation methods by replacing or changing the technical content disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Disease resistance genes of wild grapes VaWRKY14 The application of the invention in improving plant resistance to gray mold is characterized in that: The plant is tobacco or Vitis amurensis, and the gene VaWRKY14 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The gene VaWRKY14 Used to alleviate the degree of reactive oxygen species outbreak, improve antioxidant capacity or activate the expression of disease-related genes.

3. A method for cultivating plants with improved resistance to gray mold, characterized in that: It is to promote the gene described in claim 1 VaWRKY14 The plant is tobacco or Vitis amurensis. 。 4. The method according to claim 3, characterized in that The method is specifically carried out according to the following steps: Construct containing the gene VaWRKY14 Overexpression vector; will contain the gene VaWRKY14 The overexpression vector was transformed into competent cells, cultured, and inoculated onto plants to obtain plant strains resistant to gray mold.

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