Application of tomato transcription factor SlMYB12

Abstract

The invention discloses application of a tomato transcription factor SlMYB12, and belongs to the technical field of plant antiviral gene engineering. The invention researches and verifies that the protein coded by the SlMYB12 is a tomato protein for regulating and controlling pepper yellow ringspot virus infection for the first time. According to the present invention, the expression of the SlMYB12 gene in the tomato is inhibited by using the virus-induced gene silence (VIGS) technology, and the results show that the active oxygen component in the tomato leaf with the silent SlMYB12 gene is reduced, the antioxidant enzyme and resistance gene expression is improved, the accumulation level of the pepper yellow ringspot virus in the tomato plant with the silent SlMYB12 is significantly reduced, and the resistance of the pepper yellow ringspot virus in the tomato plant with the silent SlMYB12 is significantly improved; therefore, the silent tomato SlMYB12 gene can improve the resistance of the plant to the pepper yellow ringspot virus. Protein interaction analysis shows that interaction exists between the SlMYB12 protein and functional protein NSs of CYRSV, and further shows that the SlMYB12 protein has a regulation effect on CYSRV infection.

Description

Technical Field

[0001] This invention belongs to the field of plant antiviral gene engineering technology, specifically relating to the application of a tomato transcription factor SlMYB12. Background Technology

[0002] Chilli yellow ring spot virus (CYRSV) is a negative single-stranded RNA virus belonging to the genus Orthotospovirus of the family Tospoviridae in the order Bunyavirales. It causes symptoms such as whitening, yellowing, yellow spots, stunted fruit, deformity, and necrosis in tomato fruits.

[0003] Tomatoes are annual or perennial herbaceous plants belonging to the Solanaceae family. They are highly favored by consumers due to their rich content of carotene and other nutrients beneficial to human health. Yuanmou County in Yunnan Province is a region where many vegetables are grown out of season, with tomatoes being one of the main vegetable varieties cultivated. During tomato production, various diseases can affect the industry, with viral diseases being a significant factor impacting the development of the tomato industry and severely restricting local economic growth. Currently, there are few research reports on CYRSV, and its pathogenic mechanism remains unclear. There are currently no effective control methods for CYRSV; prevention is the only option to control its large-scale spread. Planting resistant tomato varieties is the most economical and effective method for controlling CYRSV. However, existing tomato resistance genes show little or no resistance to CYRSV. Therefore, it is urgent to explore new tomato germplasm resources with viral resistance for the control of CYRSV.

[0004] MYB transcription factors are among the most numerous transcription factor families in plants, participating in all aspects of plant growth, development, and metabolism. They play crucial regulatory roles in processes such as cell differentiation, cell cycle, and leaf morphogenesis. Furthermore, MYB transcription factors are involved in various defense and stress responses, including hormone signal transduction and abiotic and biotic stress responses. Numerous studies have demonstrated the important role of MYB in regulating plant stress responses, particularly in responding to abiotic stresses, which has attracted widespread attention. MYB12 belongs to the MYB transcription factor family and regulates flavonoid biosynthesis pathways. Currently, MYB12 has been reported and identified in species such as leeks, passionflowers, lilies, and tobacco. MYB12 plays an important role in regulating plant responses to biotic stresses such as diseases and pests, and abiotic stresses such as drought, low temperature, and salinity. However, its functional role in plants infected with the plant virus CYRSV has not been reported. Therefore, MYB12 is crucial for plant growth, development, and stress adaptation; however, research on its function in plant resistance to CYRSV is limited. Summary of the Invention

[0005] The purpose of this invention is to provide an application of the tomato transcription factor SlMYB12.

[0006] The objective of this invention is achieved as follows: the application of the tomato transcription factor SlMYB12 in the prevention and treatment of CYRSV; the amino acid sequence of the SlMYB12 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the SlMYB12 coding region is shown in SEQ ID NO.2; The target gene can be used in the following (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to cultivate plant varieties resistant to CYRSV; As a target protein, its applications are as follows (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to develop drugs against CYRSV virus diseases as a target.

[0007] Specifically: In a first aspect, the present invention provides the use of the SlMYB12 gene as a target gene in the following (1) or (2): (1) preventing and controlling plant diseases caused by CYRSV; (2) cultivating plant varieties resistant to CYRSV; wherein the nucleotide sequence of the SlMYB12 gene is shown in SEQ ID NO.2, and in the above applications, the plant's resistance to CYRSV is enhanced by silencing or knocking out the SlMYB12 gene.

[0008] In a second aspect, the present invention provides the use of the SlMYB12 protein in (1) or (2) as follows: (1) preventing and controlling plant diseases caused by CYRSV; (2) developing drugs against CYRSV as targets; the amino acid sequence of the SlMYB12 protein is shown in SEQ ID NO.1.

[0009] In a third aspect, the present invention provides the application of a recombinant expression vector or genetically engineered bacteria targeting the SlMYB12 gene in the following (1) or (2): (1) prevention and control of plant diseases caused by CYRSV; (2) cultivation of CYRSV-resistant plant varieties. In the above applications, the recombinant expression vector targeting the SlMYB12 gene is a knockout vector or a silencing vector targeting the SlMYB12 gene. Preferably, the silencing vector is a TRV vector containing a silencing-specific segment of the SlMYB12 gene; the silencing-specific segment of the SlMYB12 gene is the 133-432 bp segment of the sequence shown in SEQ ID NO.2.

[0010] A fourth aspect of the present invention provides a method for improving CYRSV resistance in tomatoes, comprising: knocking out or silencing the SlMYB12 gene in tomato plants; or, inactivating the function of the SlMYB12 protein in tomato plants. In the above method, techniques such as homologous recombination, VIGS technology, T-DNA insertion, CRISPR-Cas9 gene editing, and RNA interference can be used to knock out or silence the SlMYB12 gene.

[0011] In a fifth aspect, the invention provides the use of a substance that inhibits the expression of the SlMYB12 gene in the preparation of a medicament for treating CYRSV. In the above application, the substance that inhibits the expression of the SlMYB12 gene is selected from knockout fragments, silencing fragments, antisense RNA, or siRNA targeting the SlMYB12 gene.

[0012] The beneficial effects of this invention are as follows: This invention is the first to study and confirm that the tomato transcription factor SlMYB12 is a tomato protein that regulates CYRSV infection. By using virus-induced gene silencing (VIGS) technology, this invention inhibits the expression of the SlMYB12 gene in tomatoes. The accumulation level of CYRSV in SlMYB12-silenced tomato plants is reduced, and the reactive oxygen species content in tomato leaves with silenced SlMYB12 is decreased, while the expression of antioxidant enzymes and resistance genes is increased. This indicates that SlMYB12 has the function of promoting virus infection, and that silencing the tomato gene SlMYB12 plays an important role in resisting virus infection, reducing the harm of CYRSV to plants. By combining bimolecular fluorescent labeling and luciferase complementation technology to screen and verify CYRSV functional proteins that interact with tomato SlMYB12, the regulatory mechanism of SlMYB12 in CYRSV-infected tomatoes is further elucidated, providing a theoretical basis and technical support for creating CYRSV-resistant germplasm and breeding new varieties. The tomato transcription factor SlMYB12 provided by this invention can also be used to study the related molecular mechanisms of CYRSV infection in tomatoes. Attached Figure Description

[0013] Figure 1 Phenotypic images of tomato plants after inoculation with CYRSV for control and silencing SlMYB12, respectively. TRV2 is the control group, and TRV2-SlMYB12 is the experimental group with the SlMYB12 gene silenced. Figure 2 The results show the relative expression levels of the SlMYB12 gene in the leaves of tomato plants after inoculation with CYRSV, control group, and SlMYB12-silenced group. In the figure, TRV2 is the control group, and TRV2-SlMYB12 is the experimental group with the SlMYB12 gene silenced. Figure 3The results show the relative expression levels of CYRSV in the leaves of tomato plants after inoculation with the control and silencing of SlMYB12. In the figure, TRV2 is the control group, and TRV2-SlMYB12 is the experimental group with the SlMYB12 gene silenced. Figure 4 The results of detecting reactive oxygen species in the leaves of tomato plants after inoculation with CYRSV and silencing SlMYB12 are shown in the figure. TRV2 is the control group, and TRV2-SlMYB12 is the experimental group that silences the SlMYB12 gene. Figure 5 The results show the detection of antioxidant enzyme (POD and SOD) activities in the leaves of tomato plants after inoculation with CYRSV, control and silenced SlMYB12. In the figure, TRV2 is the control group and TRV2-SlMYB12 is the experimental group with silenced SlMYB12 gene. Figure 6 The results show the relative expression levels of the resistance gene in the leaves of tomato plants after inoculation with CYRSV, control group, and tomato plants with silenced SlMYB12. In the figure, TRV2 is the control group, and TRV2-SlMYB12 is the experimental group with silenced SlMYB12 gene. Figure 7 BiFC was used to detect the absence of interaction between SlMYB12 and N protein; Figure 8 BiFC was used to detect the interaction between SlMYB12 and NSs proteins; Figure 9 BiFC was used to detect the absence of interaction between SlMYB12 and NSm proteins; Figure 10 The results of the luciferase complementation experiment between SlMYB12 and NSs are shown. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments, but this is not intended to limit the present invention in any way. Any modifications or substitutions made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0015] The application of the tomato transcription factor SlMYB12 in the prevention and control of CYRSV according to the present invention, wherein the amino acid sequence of the SlMYB12 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the SlMYB12 coding region is shown in SEQ ID NO.2; The target gene can be used in the following (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to cultivate plant varieties resistant to CYRSV; As a target protein, its applications are as follows (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to develop drugs against CYRSV virus diseases as a target.

[0016] The CYRSV control described herein is achieved by silencing or knocking out the SlMYB12 gene to enhance plant resistance to CYRSV.

[0017] The recombinant expression vector or genetically engineered bacteria targeting the SlMYB12 gene described in this invention may be used in the following (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to cultivate plant varieties resistant to CYRSV.

[0018] The recombinant expression vector targeting the SlMYB12 gene is a knockout vector or a silencing vector targeting the SlMYB12 gene.

[0019] The silencing vector is a TRV vector containing a silencing-specific region of the SlMYB12 gene; the silencing-specific region of the SlMYB12 gene is the 133-432bp segment of the sequence shown in SEQ ID NO.2.

[0020] The method for improving tomato resistance to CYRSV according to the present invention includes the steps of knocking out or silencing the SlMYB12 gene in tomato plants; or, the step of inactivating the function of the SlMYB12 protein in tomato plants.

[0021] The steps for knocking out or silencing the SlMYB12 gene in tomato plants are to use homologous recombination, VIGS technology, T-DNA insertion, CRISPR-Cas9 gene editing, or RNA interference to knock out or silence the SlMYB12 gene.

[0022] The application of the substance that inhibits the expression of the SlMYB12 gene described in this invention in the preparation of drugs against CYRSV virus disease.

[0023] The substance that inhibits the expression of the SlMYB12 gene is selected from knockout fragments, silencing fragments, antisense RNA or siRNA targeting the SlMYB12 gene.

[0024] CYRSV is highly virulent and has an extremely strong ability to spread, posing a serious threat to tomato growers and the tomato industry chain worldwide. Existing tomato resistance genes have weak or no resistance to CYRSV, making its control extremely difficult.

[0025] In view of this, this invention, based on previous research results, identifies CYRSV susceptibility factors, among which the tomato transcription factor SlMYB12 was found to be significantly highly expressed in CYRSV-infected tomato leaves and fruits. The amino acid sequence of the transcription factor SlMYB12 protein is shown in SEQ ID NO.1, and is as follows: MGRTPCCEKVGIKRGRWTAEEDQILTNYIISNGEGSWRSLPKNAGLLRCGKSCRLRWINYLRSDLKRGNITSQEEDIIIKLHATLGNRWSLIAEHLSGRTDNEIKNYWNSHLSRKVDSLRIPSDEKLPKAVVDLAKKGIPKPIKKSSISRPKNKKSNLLEKEALLCCTNM PACDSAMELMQEDLAKIEVPNSWAGPIEAKGSLSSDSDIEWPRLEEIMPDVVIDDEDKNTNFILNCFREEVTSNNVGNSYSCIEEGNKKISSDDEKIKLLMDWQDNDELVWPTLPWELETDIVPSWPQWDDTDTNLLQNCTNDNNNYEEATTMEINNQNHSTIVSWLLS* The nucleotide sequence encoding the SlMYB12 transcription factor is shown in SEQ ID NO.2, as follows: To verify the function of the SlMYB12 transcription factor, this invention used virus-induced gene silencing (VIGS) technology to inhibit the expression of the SlMYB12 gene in tomato. The accumulation level of CYRSV in SlMYB12-silenced tomato plants was reduced, and the reactive oxygen species content in the leaves of tomato plants with silenced SlMYB12 was decreased, while the expression of antioxidant-related indicators and resistance genes was increased. This indicates that SlMYB12 has the function of promoting virus infection, and that silencing the tomato gene SlMYB12 plays an important role in resisting virus infection and can reduce the harm of viruses to plants. Therefore, this demonstrates that knocking out or reducing the expression of the SlMYB12 gene can inhibit CYRSV infection, potentially providing a new approach for the control of CYRSV.

[0026] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0027] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions.

[0028] The amino acid sequence of the SlMYB12 protein involved in the following examples is shown in SEQ ID NO.1, and its encoding gene sequence is shown in SEQ ID NO.2.

[0029] The pTRV1 and pTRV2 vectors used in the following examples were purchased from Yunnan Sinong Vegetable Seed Industry Development Co., Ltd.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] Example 1

[0032] Detection of the preventive effect of silencing the SlMYB12 gene on CYRSV I. Construction of the VIGS vector for the tomato SlMYB12 gene The pTRV2 vector map is shown in Figure 1. Based on the pTRV2 vector sequence and the target fragment of the SlMYB12 gene (positions 133-432 of SEQ ID NO. 2), upstream and downstream specific primers with a 21 bp homologous arm at the BamHI restriction site of the pTRV2 vector were designed to insert the sequence at the BamHI restriction site, as shown below (5'-3'): Upstream primer pTRV2:SlMYB12-F: agaaggcctccatggggatcc GGATTATTGAGATGCGGAAAGAG Downstream primer pTRV2:SlMYB12-1-R: cgtgagctcggtaccggatcc TTTAATTGGCTTCGGTATACCTTTT In the primers mentioned above, the underlined part is the homologous arm sequence, and the sequence following it is the target fragment-specific sequence of the SlMYB12 gene.

[0033] Using the tomato variety "Liangsi" as the research object, total RNA was extracted from the tomato fruit according to the instructions of the RNA extraction kit (Promega).

[0034] Using extracted RNA as a template, RT-PCR was performed using a one-step RT-PCR kit (AG11728, Aike Rui). The RT-PCR system consisted of: 2 μL gDNA Clean Reaction Mix Ver.2, 4 μL 5x Evo M-MLV RT Reaction Mix Ver.2, 1 μg RNA, and up to 20 μL RNase-free water. The reaction procedure was: 37 ℃ for 15 min, 85 ℃ for 5 s, and stored at 4 ℃.

[0035] The obtained cDNA was used as a template to amplify the tomato SlMYB12 gene fragment. The PCR reaction system was as follows: 12.5 μL 2×Taq mix; 0.4 μL upstream primer (10 μM); 0.4 μL downstream primer (10 μM); 2 μL cDNA; ddH2O to a final volume of 25 μL. The PCR reaction steps were: 94 ℃ pre-denaturation for 30 s; 98 ℃ denaturation for 10 s, 58 ℃ annealing for 30 s, 72 ℃ extension for 1.5 min, 35 cycles; 72 ℃ final extension for 2 min; storage at 4 ℃. The PCR product was detected by agarose gel electrophoresis, and the target fragment was approximately 340 bp. The target fragment was recovered using a gel extraction kit (Axygen) for later use.

[0036] The pTRV2 vector was digested with the restriction endonuclease BamHI. The digestion reaction mixture consisted of 2 μL template, 5 μL 10× Buffer, 1 μL BamHI, and 42 μL ddH2O. The reaction program was incubation at 37 °C for 20 min. The digested DNA was purified and its concentration was measured for later use to ensure adequate concentration.

[0037] Following the instructions of the OK Clon DNA Ligation Kit III (Hunan Aike Rui Biotechnology Co., Ltd.), the target gene was ligated to the pTRV2 vector using the 2.5 × OK Clon Ligation Kit II. The reaction mixture consisted of 4 μL of 2.5 × OK Clon Master Mix, 4 μL of pTRV2 vector, and 2 μL of gene fragment, for a total volume of 10 μL. The reaction program was 50 ℃ for 10 min. The ligation product was transformed into *E. coli* DH5α competent cells. Single clones were screened on LB agar plates containing kanamycin resistance. Colony PCR was performed to identify the single clones. Finally, PCR-positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After sequence alignment analysis, plasmid extraction was performed on the correctly sequenced positive clones using the Trelief® Plasmid Mini Kit Plus high-purity plasmid mini-prep kit to obtain the pTRV2-SlMYB12 recombinant plasmid for later use.

[0038] II. Transformation of tomato with SlMYB12 gene VIGS vector and CYRSV inoculation 1. SlMYB12 gene VIGS vector transformation of Agrobacterium tumefaciens The recombinant plasmid pTRV2-SlMYB12 was transformed into Agrobacterium GV3101 competent cells using a liquid nitrogen rapid dissolution method. The procedure was as follows: 3 μL of pTRV2-SlMYB12 plasmid was added to 50 μL of Agrobacterium GV3101 competent cells, gently mixed, and placed on ice for 30 min. Finally, the cells were placed in liquid nitrogen for 1 min, followed by a 37°C water bath for 5 min. Afterward, 800 μL of liquid LB medium was added, and the cells were incubated at 28°C and 200 rpm for 2 h. 300 μL of the bacterial culture was then evenly spread onto solid LB medium (100 μg / mL Kana, 50 μg / mL Rif), and incubated upright at 28°C for 30 min, followed by inverted incubation for 48–72 h. Single colonies were picked and cultured in 1 ml of liquid LB medium (100 μg / mL Kana, 50 μg / mL Rif) at 28°C and 200 rpm for 5-7 h until the culture became turbid. An equal volume of 50% glycerol was added to the positive colony culture, and the mixture was flash-frozen in liquid nitrogen and stored at -80°C for later use. The final result was recombinant Agrobacterium transformed with the pTRV2-SlMYB12 plasmid.

[0039] Meanwhile, the empty pTRV2 plasmid and the pTRV1 plasmid were introduced into Agrobacterium strain GV3101 using the method described above, to obtain Agrobacterium strains containing the empty pTRV2 plasmid and the pTRV1 plasmid, for later use.

[0040] 2. VIGS carrier infection of tomatoes pTRV2-SlMYB12 was injected into tomato leaves using an Agrobacterium-mediated infection method. The procedure was as follows: 50 μL of preserved Agrobacterium pTRV2-SlMYB12, pTRV2, and pTRV1 bacterial cultures were added to 800 μL of liquid LB medium (100 μg / mL kanamycin, 50 μg / mL rifampin) and cultured at 28°C and 200 rpm for 14-18 h. Subsequently, 100 μL of the above-mentioned Agrobacterium pTRV2-SlMYB12, pTRV2, and pTRV1 bacterial cultures were added to 30 mL of liquid LB medium (100 μg / mL kanamycin, 50 μg / mL rifampin) and cultured at 28°C and 200 rpm for 14-18 h. Finally, the pTRV2-SlMYB12, pTRV2, and pTRV1 cells were collected by centrifugation at 3000 × g.

[0041] Tomato seedlings can be used for infection injection after they have grown to 7-8 true leaves. Infection solution preparation: 50 mL sterile water, 50 μL LAS, 25 μL MES, 500 μL MgCl2. Suspend appropriate amounts of bacterial precipitate in the infection solution, measure the OD value using a spectrophotometer, ensuring the OD value of the bacterial solution is 0.6–0.8, and incubate at 28 ℃ in the dark for 6–8 h until ready for use. Injection: Mix the infection solutions of pTRV2-SlMYB12 and pTRV2 with the pTRV1 infection solution at a 1:1 volume ratio. Fill a 1 mL syringe with the infection solution, gently puncture the lower epidermis of the leaf to be injected (select healthy and suitable leaves from tobacco and pepper plants), gently hold the upper epidermis with your left index finger, and slowly inject the solution into the puncture site until the solution has penetrated about one-quarter of the leaf.

[0042] 3. CYRSV vaccination Forty-eight hours after injecting pTRV2-SlMYB12 and pTRV2 infection solutions into tomato leaves, CYRSV was inoculated using carborundum (emery). First, the mortar was placed on ice. Then, the CYRSV virus source stored in our laboratory was taken out from a -80 °C freezer, and an appropriate amount of PBS buffer was added. The mixture was then ground in the mortar and set aside. The carborundum was evenly sprinkled onto the leaves to be inoculated. Then, a cotton swab was used to apply the CYRSV inoculation solution evenly to the leaves, inoculating three leaves per tomato plant. The plants were then cultured in a greenhouse. The groups were set as follows: pTRV2 group: injected with pTRV1 / pTRV2 and inoculated with CYRSV; pTRV2-SlMYB12 group: injected with pTRV1 / pTRV2-SlMYB12 and inoculated with CYRSV.

[0043] III. Resistance of Silent SlMYB12 Tomato Plants to CYRSV 1. Leaf phenotypic observation Observe continuously for 1-4 weeks, noting the phenotype and growth status of tomato leaves at different times after virus infection. Figure 1 As shown in the figure, 12 days after CYRSV inoculation, the leaves and central leaves of tomato plants with silenced SlMYB12 showed chlorosis, yellow spots, and yellowing symptoms; while the leaves and central leaves of the control group tomato plants showed mild yellowing spots. 28 days after CYRSV inoculation, the leaves of tomato plants with silenced SlMYB12 showed yellowing, necrotic spots, and central leaf necrosis; while the control group tomato plants only showed mild yellowing of the leaves. This indicates that silencing SlMYB12 significantly inhibited CYRSV infection.

[0044] 2. Expression analysis of the SlMYB12 gene Three days after CYRSV inoculation, total RNA was extracted from systemic leaves, and cDNA was obtained by reverse transcription as described above. qPCR was then performed using quantitative real-time primers. The primer sequences are as follows: upstream primer for SlMYB12 is GCCAGCTTTGTGATAGTGCCAT, and downstream primer for SlMYB12 is AAGGCTTCCCTTGGCCTCTA. Tomato β-actin was used as an internal reference gene, with upstream primer for β-actin being CCTCAGCACATTCCAGCAG, and downstream primer for β-actin being CCACCAAACTTCTCCATCCC.

[0045] The reaction system was as follows: 5 μL 2×Taq (SYBR) Mix, 0.5 μL upstream primer (10 μM), 0.5 μL downstream primer (10 μM), 1 μL cDNA, and 3 μL ddH2O, for a total volume of 20 μL. Three biological and three technical replicates were performed for each gene in the test samples. The qPCR reaction program was: 95℃ for 30 s; 95℃ for 30 s, 60℃ for 30 s, 40 cycles; melting curves were added. The relative expression level of the SlMYB12 gene was calculated using Formula 2. -△△C t Calculations were performed. All reactions were biologically replicated three times.

[0046] The results are as follows Figure 2 As shown, the expression level of the SlMYB12 gene in the systematic leaves of tomato plants in which the pTRV2-SlMYB12 vector was introduced was significantly reduced, indicating that the SlMYB12 gene was silenced.

[0047] 3. Analysis of CYRSV accumulation Three days after CYRSV inoculation, total RNA was extracted from systemic leaves and subjected to reverse transcription and qPCR detection according to the methods described above. The primer sequences are as follows: CYRSV N upstream primer: GCGGTACTGCAGATGTTGAA; CYRSV N downstream primer: GGTCCAATCTTCTGGTCCAA. Tomato β-actin was used as an internal control gene; β-actin upstream primer: CCTCAGCACATTCCAGCAG; β-actin downstream primer: CCACCAAACTTCTCCATCCC. The reaction system was the same as above. The relative expression level of the CYRSV N gene is expressed by Formula 2. -△△C t Calculations were performed. All reactions were biologically replicated three times.

[0048] The results are as follows Figure 3 As shown, the expression level of CYRSV in the systematic leaves of tomato plants infused with the pTRV2-SlMYB12 vector was significantly reduced, indicating that silencing the SlMYB12 gene inhibited CYRSV virus proliferation.

[0049] 4. Detection of reactive oxygen species accumulation Three days after virus inoculation, tomato leaves were collected for reactive oxygen species (ROS) detection. DAB and NBT staining solutions (Solepro) were used to detect the ROS components H₂O₂ and O₂, respectively. 2- The accumulation of [something] is used for staining.

[0050] DAB staining: Tomato leaves were collected from each group, with three biological replicates per group. The leaves were rinsed briefly with tap water and blotted dry on filter paper. A working DAB staining solution was prepared by mixing DAB staining solution and DAB diluent at a 1:9 ratio. The samples were immersed in the working solution and then stained at room temperature in the dark for 5 hours, until the positive areas turned dark brown, and the remaining areas were pale yellow, nearly colorless, or the plant's natural color. The samples were removed with tweezers and rinsed 3-5 times in distilled water. After blotting dry on filter paper, the samples were immersed in tissue destaining solution in a water bath at 70-80℃ for 30 minutes until the tissue background color was completely removed. After cooling, the samples were removed and rinsed 3-5 times in distilled water. After blotting dry on filter paper, the samples were transferred to an appropriate amount of tissue preservation solution and soaked for 10-30 minutes. They were then removed and photographed.

[0051] NBT staining: Tomato leaves were collected from each group, with three biological replicates per group. The leaves were rinsed slightly with water and blotted dry on filter paper. The samples were then immersed in the NBT staining working solution at room temperature in the dark for 5 hours, until the positive areas turned deep blue, and the remaining areas were light blue, nearly colorless, or the plant's natural color. The samples were removed with tweezers, rinsed 3-5 times in distilled water, blotted dry on filter paper, and then immersed in tissue destaining solution in a water bath at 70-80℃ for 30 minutes until the tissue background color was completely removed. After cooling, the samples were removed, rinsed 3-5 times in distilled water, blotted dry on filter paper, and then transferred to an appropriate amount of tissue preservation solution for 10-30 minutes. Finally, the samples were photographed.

[0052] The results are as follows Figure 4 As shown, the control group of tomato plants showed a significant accumulation of reactive oxygen species (ROS) in their leaves, indicating an oxygen burst phenomenon. However, the tomato plants introduced with the pTRV2-SlMYB12 vector showed a significant reduction in ROS accumulation in their leaves, suggesting that silencing the SlMYB12 gene suppressed the oxygen burst in tomato leaves.

[0053] 5. Determination of antioxidant enzyme activity Superoxide dismutase (SOD) and peroxidase (POD) are the main antioxidant enzymes in plant tissues, which can decompose excess reactive oxygen species in plant tissues. Three days after virus inoculation, SOD and POD activities were measured in tomato leaves.

[0054] POD detection: Weigh 0.1g of tissue and add 1mL of extraction solution for homogenization in an ice bath. Centrifuge at 8000g at 4℃ for 10min, and place the supernatant on ice for analysis. Preheat the spectrophotometer for at least 30min, adjust the wavelength to 470nm, and zero the instrument with distilled water. Before analysis, place Reagent 1, Reagent 2 working solution, and Reagent 3 at 25℃ for 10min. During analysis, add 15μL of sample, 270μL of distilled water, 520μL of Reagent 1, 130μL of Reagent 2 working solution, and 135μL of Reagent 3 to a 1mL glass cuvette in that order, mix immediately, and start timing. Record the initial absorbance value A1 at 470nm for 30s and the absorbance value A2 after 1min30s. Calculate ΔA = A2 - A1. The calculation formula is as follows: POD (U / g mass) = 7133 × ΔA ÷ W, where W is the sample mass.

[0055] SOD detection: Weigh 0.1g of tissue and add 1mL of extraction buffer for homogenization on ice. Centrifuge at 8000g at 4℃ for 10min, and place the supernatant on ice for testing. Measure the absorbance of each tube at 450nm using a microplate reader, following the instructions. First, calculate the inhibition percentage. Inhibition percentage = (ΔA blank - ΔA assay) ÷ ΔA blank × 100%, aiming for a result within the range of 30-70%. Then, calculate the SOD activity. SOD activity (U / g mass) = 10 × inhibition percentage ÷ (1 - inhibition percentage) ÷ W × F, where W is the sample mass and F is the sample dilution factor.

[0056] The results are as follows Figure 5 As shown, the activities of SOD and POD in the leaves of tomato plants in which the pTRV2-SlMYB12 vector was introduced increased, indicating that the activity of antioxidant enzymes in tomato leaves was enhanced after the SlMYB12 gene was silenced, i.e., the resistance was increased.

[0057] 6. Determination of disease resistance-related genes The resistance genes detected in this invention are mainly PR1, PAL, and PAL5. After CYRSV inoculation, total RNA was extracted from system leaves and subjected to reverse transcription and qPCR detection according to the methods described above. The primer sequences are as follows: For tomato PR1, the upstream primer is TCCGAGAGGCCAAGCTATAA, and the downstream primer is GTAAGGACGTTGTCCGATCC; for tomato PAL, the upstream primer is ACGGGTTGCCATCTAATCTG, and the downstream primer is AGCTGTTTTCCTGGCTGAAA; for tomato PAL5, the upstream primer is TTTCGCTGAAGTGATGAACG, and the downstream primer is AGCGCATAACGATCTTGCTT. Tomato β-actin was used as an internal reference gene, with the upstream primer being CCTCAGCACATTCCAGCAG and the downstream primer being CCACCAAACTTCTCCATCCC. The reaction system was the same as above. The relative expression level of the CYRSV N gene was calculated using Formula 2- △△C t Calculations were performed. All reactions were biologically replicated three times.

[0058] The results are as follows Figure 6 As shown, the expression levels of three resistance genes, PR1, PAL, and PAL5, were significantly increased in the leaves of tomato plants in which the pTRV2-SlMYB12 vector was introduced, indicating that silencing the SlMYB12 gene promoted the expression of resistance genes, thereby increasing the resistance of tomatoes to CYRSV.

[0059] Example 2

[0060] Screening and validation of CYRSV major functional proteins interacting with SlMYB12 This example demonstrates the interaction between the SlMYB12 protein and three major functional proteins (N, NSs, NSm) of CYRSV using bimolecular fluorescence complementation (BiFC) and luciferase complementation techniques. This confirms the existence of CYRSV functional proteins that interact with the SlMYB12 protein, providing support for further experiments.

[0061] I. Prediction of the interaction relationships between the three major functional proteins of CYRSV (N, NSs, NSm) and SlMYB12 protein The Alphafold3 online website was used to predict potential interactions between SlMYB12 and the major functional proteins (N, NSs, NSm) of CYRSV. A sum of ipTM and pTM greater than 0.8 was considered a high probability of interaction; a sum of ipTM and pTM greater than 0.5 was considered a moderate probability of interaction; and a sum of ipTM and pTM less than 0.5 was considered to indicate virtually no probability of interaction.

[0062] Predictions revealed that SlMYB12 may interact with N, NSs, and NSm. The highest interaction probability was found between SlMYB12 and NSs, with ipTM and pTM equal to 0.91. Next was SlMYB12 and N, with ipTM and pTM equal to 0.63. Finally, SlMYB12 and NSm interacted, with ipTM and pTM equal to 0.57.

[0063] II. Validation of Bimolecular Fluorescence Complementary (BiFC) Technology Further verification was performed using BiFC experiments. The results showed that SlMYB12 and N protein did not interact in tobacco leaves. Figure 7 As shown, no yellow fluorescence was observed after the interaction; SlMYB12 interacts with NSs protein, as shown in the figure. Figure 8 As shown, obvious yellow fluorescence was observed, and the interaction site may be located on the endoplasmic reticulum; SlMYB12 and NSm proteins do not interact, as... Figure 9 As shown, no yellow fluorescence was observed after the interaction.

[0064] III. Validation by luciferase complementation experiment To further verify the above findings regarding the interaction between SlMYB12 and NSs proteins, we used a luciferase complementation assay, with the following results: Figure 10 As shown, fluorescence signals were observed in the leaf regions where SlMYB12 and NSs luciferin carriers were co-injected in a plant in vivo imaging system, indicating that SlMYB12 and NSs proteins interact.

[0065] The above examples demonstrate that silencing the tomato transcription factor SlMYB12 can inhibit CYRSV infection in tomato plants and improve tomato leaf resistance. Subsequent bimolecular fluorescence complementation (BiFC) and luciferase complementation experiments verified the protein-protein interaction between SlMYB12 and CYRSV's NSs. CYRSV is a spherical virus with a diameter of approximately 80-120 nm, possessing a tripartite genome containing three gene segments: large, medium, and small. Genes encoding the main functional proteins of CYRSV include N, NSs, and NSm genes, as well as Gn / Gc encoding glycoproteins and RdRp encoding RNA polymerase. These main functional genes are closely related to viral classification, viral motility, and systemic infectivity. Among them, NSs encodes a non-structural protein of CYRSV—a viral gene silencing repressor—and plays a crucial role in viral infection of the host. Currently, research on the mechanism of CYRSV infection remains in its infancy. The embodiments of the present invention show that SlMYB12 plays a role in promoting infection during CYRSV infection of tomatoes and is a positive regulator of CYRSV infection. It may interact with the NSs protein of CYRSV to regulate the virus's infection of the host.

[0066] 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. An application of the tomato transcription factor SlMYB12, characterized in that, The application of the tomato transcription factor SlMYB12 in the prevention and control of CYRSV, wherein the amino acid sequence of the SlMYB12 protein is shown in SEQ ID NO.1, and the nucleotide sequence of the SlMYB12 coding region is shown in SEQ ID NO.2; The target gene can be used in the following (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to cultivate plant varieties resistant to CYRSV; As a target protein, its applications are as follows (1) or (2): (1) to prevent and control plant diseases caused by CYRSV; (2) to develop drugs against CYRSV virus diseases as a target.

2. The application according to claim 1, characterized in that, The CYRSV control described herein is achieved by silencing or knocking out the SlMYB12 gene to enhance plant resistance to CYRSV.

3. An application of a recombinant expression vector or genetically engineered bacteria targeting the SlMYB12 gene in the following (1) or (2): (1) prevention and control of plant diseases caused by CYRSV; (2) cultivation of plant varieties resistant to CYRSV.

4. The application according to claim 3, characterized in that, The recombinant expression vector targeting the SlMYB12 gene is a knockout vector or a silencing vector targeting the SlMYB12 gene.

5. The application according to claim 4, characterized in that, The silencing vector is a TRV vector containing a specific silencing region of the SlMYB12 gene; the specific silencing region of the SlMYB12 gene is the 133-432bp segment of the sequence shown in SEQ ID NO.

2.

6. A method for improving CYRSV resistance in tomatoes, characterized in that, include: The steps to knock out or silence the SlMYB12 gene in tomato plants; Alternatively, steps to inactivate the SlMYB12 protein in tomato plants.

7. The method according to claim 6, characterized in that, The steps for knocking out or silencing the SlMYB12 gene in tomato plants are to use homologous recombination, VIGS technology, T-DNA insertion, CRISPR-Cas9 gene editing, or RNA interference to knock out or silence the SlMYB12 gene.

8. The application of a substance that inhibits the expression of the SlMYB12 gene in the preparation of drugs against CYRSV virus disease.

9. The application according to claim 8, characterized in that, The substance that inhibits the expression of the SlMYB12 gene is selected from knockout fragments, silencing fragments, antisense RNA or siRNA targeting the SlMYB12 gene.

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