Tomato disease resistance gene mutants and their use in the control of tobrfv

Abstract

This invention discloses a tomato disease-resistant gene mutant and its application in controlling ToBRFV, belonging to the field of biological control technology for viral diseases. This invention discovers that the tomato Tm-2... 2 The coding sequence of the gene is mutated from G to A at nucleotide position 1927; or, the tomato Tm-2... 2 A mutation of glycine to arginine at amino acid position 643 of the LRR domain of the gene-encoded protein significantly reduced the accumulation level of ToBRFV capsid protein (CP). This gene (named Tm-2) 2 -Mut5) can not only serve as a new ToBRFV resistance gene, but also retains resistance to TMV, ToMV, and ToMMV. Tm-2 2 -Mut5 and the previously screened Tm-2 2 Combinatorial mutations were performed on the -Mut3-1 mutant (tyrosine at position 767 of the LRR domain was replaced with phenylalanine) to obtain the mutant Tm-2. 2 -Mut6. Analysis revealed that Tm-2 2 -Mut6 significantly reduced the accumulation level of ToBRFV capsid protein; compared with the mutant pFGCTm-2 obtained in previous studies. 2 -Mut3-1 and the newly obtained mutant Tm-2 2 Compared to Mut5, Tm-2 2 -Mut6 further enhances resistance to ToBRFV.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for viral diseases, specifically to tomato disease-resistant gene mutants and their application in the control of ToBRFV. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) is a newly emerging virus. The main symptoms of Tomato leaf infection include mosaic patterns, dark green raised bumps, narrow leaves, yellowing and necrosis of leaf veins, reduced flower and fruit numbers, yellow or brown patches on the fruit, smaller fruit size, and wrinkles. Severe cases can lead to necrosis of the fruit stalk, resulting in a significant decrease in fruit yield and marketability. ToBRFV is highly infectious, and currently there are no effective measures to control its infection of plants.

[0003] According to the gene-gene hypothesis, a plant can only exhibit disease resistance when its resistance gene R encounters the pathogen's non-toxic gene Avr (Vander Biezen & Jones, 1998). The Tm-2 of tomatoes... 2 The gene is a disease resistance gene belonging to the NBS-LRR family, Tm-2. 2 The gene exhibits some resistance to various viruses in the Tomato Mosaic Virus (TMV), Tomato Mosaic Virus (ToMV), and Tomato Mottle Mosaic Virus (ToMMV) genera. However, ToBRFV, a virus in the Tomato Mosaic Virus genus, completely disrupts the Tm-2 mutation on tomatoes. 2 Resistance, currently contains Tm-2 2 Tomato varieties with certain genes are susceptible to ToBRFV.

[0004] By analyzing Tm-2 2 Mutating genes can potentially yield mutants resistant to ToBRFV; however, due to Tm-2... 2 The full-length ORF of the gene is 2586 bp, encoding 861 amino acids. There are countless possibilities for the direction of mutation in its nucleotides or amino acids, and the resulting performance is unpredictable, potentially even altering Tm-2. 2 The original resistance of the gene. Therefore, screening for Tm-2 that possesses ToBRFV resistance and can maintain its original viral resistance is important. 2 Gene mutants remain a key technical challenge. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a tomato disease resistance gene mutant and its application in the prevention and control of ToBRFV.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a tomato disease resistance gene mutant, said tomato disease resistance gene mutant being a variant of Tm-2. 2 Nucleotide mutations occur at the positions shown in (1) or (2) in the nucleotide sequence of the gene coding region:

[0008] (1)Tm-2 2 A mutation occurred at nucleotide position 1927 of the coding region sequence of the gene;

[0009] (2)Tm-2 2 The nucleotides at positions 1927, 2300, and 2301 of the coding region of the gene were mutated simultaneously.

[0010] Preferably, the nucleotide sequence of the tomato disease resistance gene mutant is shown in SEQ ID NO.1 or SEQ ID NO.3. Specifically, if the mutation shown in (1) occurs, Tm-2 2 The nucleotide at position 1927 of the coding region of the gene was mutated from G to A, and the tomato disease resistance gene mutant was named Tm-2. 2 -Mut5, whose nucleotide sequence is shown in SEQ ID NO.1; if the mutation shown in (2) occurs, Tm-2 2 The coding sequence of the gene was mutated, with nucleotide 1927 changing from G to A, nucleotide 2300 changing from A to G, and nucleotide 2301 changing from C to G. This tomato disease resistance gene mutant was named Tm-2. 2 -Mut6, whose nucleotide sequence is shown in SEQ ID NO.3.

[0011] In a second aspect, the present invention provides a protein encoded by the above-mentioned tomato disease resistance gene mutant.

[0012] With wild-type Tm-2 2 Compared to the protein encoded by the gene, the protein encoded by the above-mentioned tomato disease resistance gene mutant has a mutation at amino acid position 643; or, amino acids at positions 643 and 767 are mutated simultaneously.

[0013] Specifically, the 643rd amino acid is mutated to arginine or to other amino acids, such as alanine, valine, leucine, isoleucine, proline, serine, threonine, methionine, tyrosine, aspartic acid, glutamic acid, lysine, tryptophan, histidine, phenylalanine, cysteine, asparagine, or glutamine.

[0014] Alternatively, the glycine at position 643 can be mutated to arginine or another amino acid, and the tyrosine at position 767 can be mutated to phenylalanine or another amino acid.

[0015] Preferably, the amino acid sequence of the protein is as shown in SEQ ID NO.2 or SEQ ID NO.4. Specifically, the Tm-2 2 The amino acid sequence of -Mut5 is shown in SEQ ID NO.2; the Tm-2 2 The amino acid sequence of -Mut6 is shown in SEQ ID NO.4.

[0016] The above mutant Tm-2 2 -Mut5 is the tomato Tm-2 2 The glycine (G) at position 643 of the gene-encoded protein is mutated to arginine (R). The above mutant Tm-2 2 -Mut6 is the tomato Tm-2 2 The 643rd amino acid of the gene-encoded protein was mutated from glycine (G) to arginine (R), and the 767th tyrosine (Y) was mutated to tryptophan (W).

[0017] A third aspect of the present invention provides the application of the above-mentioned tomato-resistant disease-resistant gene mutant in the following (1) or (2):

[0018] (1) Prevention and control of tomato brown wrinkled fruit virus;

[0019] (2) Cultivate plant varieties resistant to tomato brown wrinkled fruit virus.

[0020] Preferably, the plant species are crops such as tomatoes, peppers, and tobacco.

[0021] In a fourth aspect, the present invention provides the use of the protein encoded by the above-mentioned tomato disease resistance gene mutant in the following (1) or (2):

[0022] (1) Prevention and control of tomato brown wrinkled fruit virus;

[0023] (2) Prepare products for the prevention and control of tomato brown wrinkled fruit virus.

[0024] A fifth aspect of the present invention provides a method for improving plant resistance to ToBRFV, comprising the following steps:

[0025] Plant Tm-2 2 The 643rd amino acid of the gene-encoded protein is mutated to an amino acid other than glycine.

[0026] Alternatively, plant Tm-2 2 The 643rd amino acid of the gene-encoded protein is mutated to an amino acid other than glycine, and the 767th amino acid is mutated to an amino acid other than tyrosine.

[0027] Preferably, plant Tm-22 The 643rd amino acid in the gene-encoded protein is mutated to arginine; or, the plant Tm-2... 2 The 643rd amino acid of the gene-encoded protein was mutated to arginine, and the 767th amino acid was mutated to phenylalanine.

[0028] The beneficial effects of this invention are:

[0029] (1) This invention is the first to discover that tomato Tm-2 2 In the coding region sequence of the gene, nucleotide 1927 is mutated from G to A; or, the tomato Tm-2... 2 The mutation of glycine to arginine at amino acid position 643 of the gene-encoded protein can significantly reduce the accumulation of ToBRFV capsid protein (CP), and can serve as a new ToBRFV resistance gene, while retaining resistance to TMV, ToMV and ToMMV.

[0030] (2) This invention will use Tm-2 2 Combinatorial mutations were performed at different sites in the coding region of the gene to obtain the mutant Tm-2. 2 -Mut6, the result showed Tm-2 2 -Mut6 significantly reduced the accumulation of ToBRFV capsid protein (CP), compared to the mutant pFGCTm-2 screened in previous studies. 2 -Mut3-1 and the newly acquired mutant pFGCTm-2 2 Compared to -Mut5, it has further improved resistance to ToBRFV. Attached Figure Description

[0031] Figure 1 Mutants that can react with ToBRFV MP to induce cell necrosis were screened, and their sites of action were determined.

[0032] Figure 2 Tm-2 2 -Mut5 reacts with ToBRFV MP, as well as TMV, ToMV, and ToMMV MP, to induce cell necrosis; (Left image: compared to wild-type Tm-2) 2 In comparison, Tm-2 2 -Mut5 can induce an allergic necrosis reaction with ToBRFV MP; Right figure: Tm-2 2 -Mut5 can also recognize MPs of TMV, ToMV, and ToMMV. Western blot analysis was performed 4 days after vaccination.

[0033] Figure 3 Tm-2 2-Mut5 significantly reduces the accumulation of ToBRFV, as well as TMV, ToMV, and ToMMV.

[0034] Figure 4 Tm-2 2 and different Tm-2 2 Comparison of the intensity of cell necrosis induced by the mutant and the ToBRFV MP response.

[0035] Figure 5 Tm-2 2 and different Tm-2 2 The effect of mutants on ToBRFV accumulation. Western blot analysis was performed 4 days post-inoculation. Detailed Implementation

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0037] As mentioned earlier, ToBRFV completely breaks the Tm-2 on tomatoes. 2 Resistance, currently contains Tm-2 2 Tomato varieties with certain genes are susceptible to ToBRFV. This was determined by analyzing the Tm-2 gene. 2 Mutating genes can potentially yield mutants resistant to ToBRFV; however, due to Tm-2... 2 The full-length ORF of the gene is 2586 bp, with four different mutation directions at each site. Combined with the coding relationship between the nucleotide and amino acid sequences, therefore, Tm-2... 2 Genes have countless mutation possibilities; the key is to extract meaningful Tm-2 mutations from these numerous possibilities. 2 Gene mutants are extremely difficult to synthesize.

[0038] The inventors regarding Tm-2 2 Gene mutants have been studied for many years, and four Tm-2 mutants with ToBRFV resistance have been identified. 2 The gene mutant is related to Tm-2 with NCBI accession number AF536201.1. 2 Gene sequence comparison, Tm-2 2 -Mut1's Tm-2 2 Three nucleotide mutations occurred in the coding region of the gene: A138G, T418C, and A1425G, corresponding to amino acid mutations of R46R, C140R, and S475S. Tm-2 2 -Mut2's Tm-2 2A single nucleotide mutation occurred in the coding region of the gene, changing to A1062G, with the corresponding amino acid mutation being I354M. Tm-2 2 -Mut3-1's Tm-2 2 Two nucleotide mutations occurred in the coding region of the gene, A2300G and C2301G, corresponding to the amino acid mutation Y767W. Tm-2 2 -Mut4's Tm-2 2 Three nucleotide mutations occurred in the gene coding region: A1062G, A2300G, and C2301G, with corresponding amino acid mutations of I354M and Y767W.

[0039] However, the number of the aforementioned mutants is insufficient to meet actual production needs, and Tm-2 2 The anti-ToBRFV effect of gene mutants also needs further improvement.

[0040] In view of this, the present invention further addresses Tm-2 2 Gene mutants were screened and investigated, and a new ToBRFV-resistant mutant plasmid was discovered. Sequencing of this plasmid revealed a similarity to the Tm-2 mutant plasmid with NCBI accession number AF536201.1. 2 Gene sequence comparison revealed three nucleotide mutations in the mutant: A521G, G924A, and G1927A, corresponding to amino acid mutations of Q174R, L308L, and G643R. Validation showed that the nucleotide mutation G1927A, with the corresponding amino acid mutation G643R, was responsible for the hypersensitive response to ToBRFV MP.

[0041] Subsequently, a new mutant plasmid was constructed using G1927A as the mutation site and named pFGCTm-2. 2 -Mut5. pFGCTm-2 2 Tm-2 in Mut5 2

[0042] The encoded amino acid sequence is shown in SEQ ID NO.2, as follows: MAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMRHIRSYVDNAKAKEAGGDSRVKNLLKDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRSSFADEFAMEIEKIKRRVVDIDRIRKTYNIIDTDNNNDDCVLLDRRRLFLHADETEIIGLDDDFNMLQAKLLNQDLHYGVVSIVGMPGLGKTTLAKKLYRLIRDQFECSGLVYVSQQPRASEILLDIAKQIGLTEQKMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKVGSRMIITSRNSNVGRYIGGESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASPDLVNIGRNIVGRCGGIPLAIVVTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASRPCFLYFGLYPEDHEIRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLNDLVSRNLIQLAKRTYNGRISSCRIHDLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMIEFFRSNPKLEKLRVLFCFAKDPSIFSHMAYFDFKLLHTLVVVMSQSFQAYVTIPSKFGNMTCLRYLRLEGNICGKLPNSIVKLTRLETIDIDRRSLIQPPSGVWESKHLRHLCYRDYRQACNSCFSISSFYPNIYSLHPNNLQTLMWIPDKFFEPRLLHRLINLRKLGILGVSNSTVKMLSIFSPVLKALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNSQSFPPNLIKLTLAYFSVDRYILAVLKTFPKLRKLKMFICKYNEEKMDLSGEANGYSFPQLEVLHIHSPNGLSEVTCTDDVSMPKLKKLLLTGFHCRISLSERLKKLSK

[0043] To further enhance the resistance to ToBRFV, we combined Tm-2 2 -Mut3-1 with Tm-22 Combining these two mutation sites in -Mut5 yields Tm-2. 2 mutant plasmid pFGCTm-2 2 -Mut6.

[0044] With NCBI accession number AF536201.1, Tm-2 2 Gene sequence comparison, Tm-2 2 Three nucleotide mutations occurred in the coding region of -Mut6: mutation G1927A, corresponding to the amino acid mutation G643R; mutations A2300G and C2301G, corresponding to the amino acid mutation Y767W.

[0045] pFGCTm-2 2 Tm-2 in Mut6 2 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.3, as follows:

[0046] atggctgaaattcttcttacatcagtaatcaataaatctgtagaaatagctggaaatttactgattcaagaaggaaagcgtttatattggttgaaa

[0047] gaggatatcgattggctccagagagaaatgagacacattcgatctttatgttgacaacgcaaaggccaaggaagctggaggtgattcaagg

[0048] gtcaaaaacttattgaaagatattcaagaattggcaggtgatgtggaggatctcttagatgacttccttccaaaaattcaacaatccaataagtt

[0049] caattattgccttaagaggagttcttttgcagatgagtttgctatggagatgagaagataaagagaagggttgttgacattgaccgaataagg

[0050] aaaacttacaacatcatagatacagataacaataatgatgattgtgttctgctggatcggagaagattattcctacatgctgatgaaacagaga

[0051] tcatcggtttggatgatgacttcaatatgctacaagccaaattacttaatcaagatttgcattatggagttgtttccatagttggcatgcccggtct

[0052] ggggaaaacaactcttgccaagaaactttataggctcattcgtgatcaatttgagtgttctggactggtctacgtttcacaacagccaagagc

[0053] gagtgaaatcttacttgacattgccaaacaaattggactgacggaacagaaaatgaaggaaaatttggaggacaacctgcgatcactcttga

[0054] aaataaaaaggtatgttatcctcctagatgacatttgggatgtggaatttgggatgatctgaaacttgtccttcctgaatgtgattcaaaagtcg

[0055] gcagtagaatgataatcacgtctcgaaatagtaatgtaggcagatacataggaggggaatcctccctccatgcattgcaacccctagaatcc

[0056] gagaaaagctttgaactctttaccaagaaaatctttaattttgatgataataatagttgggccaatgcttcacctgacttggtgaatattggtaga

[0057] aatatagttgggagatgtggaggtataccgctagccatagtggtgactgcaggcatgttaagggcaagagaaagaacagaacatgcgtgg

[0058] aacagagtacttgagagtatgggccataaagttcaagatggatgtgctaaggtattggctctcagttacaatgatttacctattgcctcaaggc

[0059] catgtttcttgtactttggcctttaccccgaggaccatgaaattcgtgcttttgatttgataaatatgtggattgctgagaagtttatagtagtaaat

[0060] agtggtaataggcgagaggctgaggatttggcggaggacgtcctaaatgatttggtttctagaaacttgattcaacttgccaaaaggacatat

[0061] aatggaagaatttcaagttgtcgcatacatgacttgttacatagtttgtgtgtggacttggctaaggaaagtaacttctttcacaccgcgcatgat

[0062] gcatttggtgatcccggcaatgttgctaggctccgaaggattacattctactctgacaatgtcatgattgagttcttccgttcaaatcctaagctt

[0063] gagaagcttcgtgtacttttctgtttcgcaaaagacccttccatattttctcatatggcttattttgacttcaaattgttgcacacattggttgtagtca

[0064] tgtctcaaagttttcaagcatatgtcactatcccaagcaaatttgggaacatgacttgcttacgctatctgagattggaggggaatatttgtgga

[0065] aaactgccaaatagtattgtcaagctcacacgtctagagaccatagacattgatcgacgtagcctcattcaacctccttctggtgtttgggagt

[0066] ctaaacatttgagacatctttgttatagagattatagacaagcatgtaacagttgcttttctataagctcattttacccaaatatttactcattgcatc

[0067] ctaacaatctacaaaccttgatgtggatacctgataaattttttgaaccgaggttgttgcaccgattgatcaatttaagaaaactgggtatactgg

[0068] gagtgtccaattctaccgttaagatgttatcaatatttagccctgtgcttaaggcgctggaggttctgaagctcagtttttccagtgacccgagt

[0069] gaacaaataaagttgtcatcgtatccacatattgctaagttgcatttgaatgttaacagaacaatggccttgaactctcaatcatttcctccaaat

[0070] ctcatcaagcttactctagcctggtttagtgtagaccgttatatactggcagtacttaagacatttcccaaattaagaaaacttaaaatgttcatct

[0071] gcaagtataatgaagaaaagatggatctctcgggcgaggcaaatggttatagctttccgcaacttgaagttttgcatattcatagcccgaatg

[0072] ggttgtctgaagtaacgtgcacggatgatgtcagtatgcccaaattgaaaaagctgttacttacaggattccattgccgaatcagtttatcgga

[0073] acggcttaaaaagctgagtaaatga

[0074] The encoded amino acid sequence is shown in SEQ ID NO.4, specifically as follows:

[0075] MAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMRHIRSYVDNAKAKEAG

[0076] GDSRVKNLLKDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRSSFADEFAMEIEKIKRR

[0077] VVDIDRIRKTYNIIDTDNNNDDCVLLDRRRLFLHADETEIIGLDDDFNMLQAKLLNQDL

[0078] HYGVVSIVGMPGLGKTTLAKLYRLIRDQFECSGLVYVSQQPRASEILLDIAKQIGLTEQ

[0079] KMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKVGSRMIITSRNSNV

[0080] GRYIGGESSLHALQPLESEKSFELFTKKIFNDDNNSWANASPDLVNIGRNIVGRCGGIPL

[0081] AIVVTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASRPCFLYFG

[0082] LYPEDHEIRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLNDLVSRNLIQLAKRTYNG

[0083] RISSCRIHDLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMIEFFRSNP

[0084] KLEKLRVLFCFAKDPSIFSHMAYFDFKLLHTLVVVMSQSFQAYVTIPSKFGNMTCLRYL

[0085] RLEGNICGKLPNSIVKLTRLETIDIDRRSLIQPPSGVWESKHLRHLCYRDYRQACNSCFSI

[0086] SSFYPNIYSLHPNNLQTLMWIPDKFFEPRLLHRLINLRKLGILGVSNSTVKMLSIFSPVLK

[0087] ALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNSQSFPPNLIKLTLAWFSVDRYI

[0088] LAVLKTFPKLRKLKMFICKYNEEKMDLSGEANGYSFPQLEVLHIHSPNGLSEVTCTDDV

[0089] SMPKLKKLLLTGFHCRISLSERLKKLSK

[0090] The mutant plasmid pFGCTm-2 2 -Mut6 and ToBRFV co-expression revealed Tm-2 2 -Mut6 significantly reduced the accumulation of ToBRFV capsid protein (CP), compared to the mutant pFGCTm-2 screened in previous studies. 2 -Mut3-1 and the newly acquired mutant pFGCTm-2 2 Compared to -Mut5, it has further improved resistance to ToBRFV and has a synergistic effect.

[0091] 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.

[0092] 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 were performed according to conventional test methods or the supplier's recommended operating instructions. The Tm-2 used in this invention... 2 The gene's NCBI accession number is AF536201.1. The ToBRFV-SD isolate used in this invention has the NCBI accession number MT018320; TMV has the NCBI accession number MH595920; ToMMV has the NCBI accession number MW37351; and ToMV has the GenBank accession number KY967221.1.

[0093] Example 1: Tm-2 2 Construction and screening of gene mutant libraries

[0094] 1. Tm-2 2 Construction of a gene mutant library:

[0095] Constructing Tm-2 using error-prone PCR 2 The gene mutant library is as follows:

[0096] First, prepare a 50 μL reaction mixture, which includes: 15 μL buffer, 5 μL error-prone 2.5 mM dNTPs, 1 μL 10 μM forward primer (5'-ttacaattaccatggatggctgaaattcttcttacatc-3'), 1 μL 10 μM reverse primer (5'-actcacctaggatcctcatttactcagctttttaagccg-3'), 2.5 μL 20 mM MnCl2, 0.3 μL 5 U / μL DNA polymerase, and 30 ng / μL template (wild-type plasmid pFGCTm-2). 2 0.5 μL, double-distilled water 24.7 μL.

[0097] The reaction was then carried out according to the following procedure: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 15 s, 55℃ annealing for 8 s, 72℃ extension for 2 min 35 s, 35 cycles, followed by a final extension at 72℃ for 2 min. The amplified fragment was cloned into the binary expression vector pFGC5941 via homologous recombination, and then transformed into *E. coli* DH5α competent cells. Plasmids were extracted from the cells, and plasmids of the correct size were selected to obtain 1800 Tm-2 cells. 2 Mutant plasmid.

[0098] 2. Screening for Tm-2 cells that induce hypersensitive cell necrosis in response to ToBRFV MP. 2 mutant

[0099] Construction of the infectious clone pCBToBRFV using conventional PCR (Reference: Yan ZY, Ma HY, Wang L, Tettey C, Zhao MS, Geng C, Tian YP, and Li XD. Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-2) 2 MP was amplified in the drug *resistance gene*. *Molecular Plant Pathology*. 2021, 11, (22): 1347–1357. The amplified product was cloned into the pCam-35S binary expression vector between the 35S promoter and the NOS terminator to obtain pCamToBRFV-MP. The sequence of MP is encoded by the ToBRFV-SD isolate (NCBI accession number MT018320).

[0100] The 1800 Tm-2 obtained 2 The mutant plasmid and the pCamToBRFV-MP plasmid expressing ToBRFV MP were transformed into Agrobacterium GV3101 competent cells, respectively. The cells carrying Tm-2... 2 OD of Agrobacterium with mutant plasmid and Agrobacterium with pCamToBRFV-MP 600 Adjust the concentration to 0.5, then mix the two at a 1:1 volume ratio. Inject the mixed Agrobacterium into the leaves of Tobacco Benedict. Two days after inoculation, observe the leaf necrosis in the inoculated area.

[0101] The results showed that 1800 Tm-2 molecules were obtained. 2 One mutant plasmid was found to induce cell necrosis upon reaction with ToBRFV MP. ​​The necrosis-inducing mutant plasmid was sequenced. Sequencing results showed that the coding region of this mutant contained three nucleotide mutations: A521G, G924A, and G1927A, corresponding to amino acid mutations of Q174R, L308L, and G643R. Analysis identified the G643R site as the site of action that enables the mutant to recognize ToBRFV MP and elicit a hypersensitive response. Figure 1 ).

[0102] Example 2: Tm-2 2 Construction and Disease Resistance Study of the Mut5 Mutant

[0103] 1. Tm-2 2 Construction of the Mut5 mutant plasmid:

[0104] Tm-2 was amplified using the following primers via directed mutagenesis. 2 Amplified fragment of the mutation at nucleotide position 1927 of the gene.

[0105] Primer pair 1-Tm-2 2 -G1927A-F:gattatagacaagcatgtaacagttgcttttct;

[0106] Primer pair 1-Tm-2 2 -G1927A-R:tgcttgtctataatctctataacaaagatgtctcaaa.

[0107] First, prepare a 50 μL mutant PCR reaction mixture, which includes: 25 μL buffer, 1 μL 10 mM dNTPs, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 1 μL 1 U / μL DNA high-fidelity polymerase, and 30 ng / μL pFGCTm-2. 2 0.5 μL, double-distilled water 20.5 μL.

[0108] Subsequently, a mutant PCR reaction was performed, with the following reaction program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 2 min 35 s, 15 cycles, 48℃ annealing for 30 s, and 72℃ final extension for 5 min. The amplified product was transformed into *E. coli* DH5α competent cells, and plasmids were extracted. Plasmids of the correct size were screened to construct the unit point mutant plasmid (Tm-2). 2 -Mut5 mutant plasmid).

[0109] mutant Tm-2 2 The nucleotide mutations that occurred in -Mut5 were G1927A and the corresponding amino acid mutations were G643R.

[0110] 2. Tm-2 2 -Mut5 mutant disease resistance study:

[0111] pFGCTm-2 2 and pFGCTm-2 2 -Mut5 and ToBRFV MP are co-expressed. Specifically, it will carry Tm-2. 2 mutant plasmid (or pFGCTm-2) 2 OD of Agrobacterium and pCamToBRFV-MP 600 Adjust the concentration to 0.5, then mix the two at a 1:1 volume ratio. Inject the mixed Agrobacterium into the leaves of Tobacco Benedict. Two days after inoculation, observe the leaf necrosis in the inoculated area.

[0112] Using the same method described above, the obtained pFGCTm-2 2 -Mut5 was co-expressed with plasmids expressing TMVMP, ToMVMP, and ToMMVMP, respectively.

[0113] Phenotypic observation results showed that Tm-2 2 It does not react with ToBRFV MP, while Tm-2 2 -Mut5 and ToBRFV MP response induces cell necrosis ( Figure 2 A). Furthermore, co-expression experimental results indicate that Tm-2 2 -Mut5 can also react with ToMV and ToMMV MP to induce cell necrosis. Figure 2 B). The above results indicate that Tm-2 2 -Mut5 can mediate resistance to ToBRFV while retaining resistance to TMV, ToMV, and ToMMV.

[0114] The obtained pFGCTm-2 2-Mut5, empty vector pFGC5941, wild-type pFGCTm-2 2 The infectious clone pCBToBRFV was transformed into Agrobacterium GV3101 competent cells. The cells carrying pFGCTm-2 were then... 2 Agrobacterium-1, pFGC5941, and pFGCTm-2 2 OD of Agrobacterium and pCBToBRFV 600 Adjust to 0.5, then carry pFGCTm-2 2 -Mut5 Agrobacterium, pFGCTm-2 2 Agrobacterium and pFGC5941 Agrobacterium were mixed with Agrobacterium carrying pCBToBRFV at a 1:1 volume ratio. The mixed Agrobacterium was injected into leaves of *Tobacco Benedict*, and total protein was extracted from the inoculated area 4 days later. Western blot was used to detect coat protein (CP). Figure 3 ).

[0115] Using the same method described above, pFGCTm-2 2 -Mut5, empty vector pFGC5941, pFGCTm-2 2 Co-expression with infectious clones of TMV, ToMV, and ToMMV was performed, respectively. The construction of the TMV infectious clone is described in the reference: Yan ZY, Ma HY, Wang L, Tettey C, Zhao MS, Geng C, Tian YP, and Li XD. Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harboring the Tm-2 virus. 2resistance gene. Molecular Plant Pathology. 2021, 11, (22): 1347–1357; The construction of the ToMV infectious clone was achieved by using primers PCB301-ToMV-F: TTTCATTTGGAGA GGGTATTTATTATTACAACAATTACC, PCB301-ToMV-R: GATATAGTACAGACTGGGCCCCTACCGGGGGTTCCGG to amplify the full length of ToMV and clone it into the binary expression vector pCB301 between the 35S promoter and the NOS terminator; The construction of the ToMMV infectious clone was based on the reference Tettey Carlos, Yan, ZY, Ma, HY, Zhao MS, Geng C, Tian YP, Li XD. Tomato mottle mosaic virus: characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system. Journal of Integrative Agriculture. 2022, 21, (9): 2641-2651. The accumulation of coat protein (CP) was detected using Western blot.

[0116] The results are as follows Figure 3 As shown, the results indicate that compared with the control (empty vector pFGC5941, wild-type pFGCTm-2)... 2 Compared to Tm-2 2 -Mut5 significantly reduced the accumulation of ToBRFV capsid protein (CP). Furthermore, co-expression experiments showed that Tm-2... 2 -Mut5 can also significantly reduce the accumulation of TMV, ToMV, and ToMMV.

[0117] Example 3: Tm-2 2 Construction and Disease Resistance Study of the Mut6 Mutant

[0118] 1. Tm-2 2 Construction of the Mut6 mutant:

[0119] The mutant Tm-2 was obtained in Example 2 by amplification using the following primers via directed mutagenesis. 2 -Amplified fragment of the mutation at amino acid position 767 of Mut5.

[0120] Primer pair 2-Tm-2 2-Y767W-F:tagcctggtttagtgtagaccgttatatactggc;

[0121] Primer pair 2-Tm-2 2 -Y767W-R:actaaaccaggctagagtaagcttgatgagattt.

[0122] First, prepare a 50 μL mutant PCR reaction mixture, which includes: 25 μL buffer, 1 μL 10 mM dNTPs, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 1 μL 1 U / μL DNA high-fidelity polymerase, and 30 ng / μL pFGCTm-2. 2 0.5 μL, double-distilled water 20.5 μL.

[0123] Subsequently, a mutant PCR reaction was performed, with the following program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 2 min 35 s, 15 cycles, 48℃ annealing for 30 s, and 72℃ final extension for 5 min. The amplified product was transformed into *E. coli* DH5α competent cells, and plasmids were extracted. Plasmids of the correct size were screened to construct Tm-2. 2 -Mut6 mutant.

[0124] Tm-2 was amplified using the above primers and the method of directed mutagenesis. 2 The amplified fragments of the mutations at nucleotide positions 2300 and 2301 of the gene were used to construct the single-point mutant plasmid (Tm-2) using the same method. 2 -Mut3-1 mutant plasmid).

[0125] 2. Tm-2 2 - Disease resistance study of the Mut6 mutant:

[0126] pFGCTm-2 2 and pFGCTm-2 2 -Mut6、pFGCTm-2 2 -Mut5、pFGCTm-2 2 -Mut3-1 is co-expressed with ToBRFV MP. ​​Specifically, it carries Tm-2. 2 mutant plasmid (or pFGCTm-2) 2 OD of Agrobacterium and pCamToBRFV-MP 600 Adjust the concentration to 0.5, then mix the two at a 1:1 volume ratio. Inject the mixed Agrobacterium into the leaves of Tobacco Benedict. Two days after inoculation, observe the leaf necrosis in the inoculated area.

[0127] Phenotypic observation results are as follows Figure 4 Tm-2 shown 2 It does not react with ToBRFV MP; pFGCTm-2 2 -Mut6、pFGCTm-2 2 -Mut5、pFGCTm-2 2 Mut3-1 reacts with ToBRFV MP to induce cell necrosis; mutant Tm-2 2 The necrosis reaction induced by Mut6 and ToBRFV MP is more significant than that induced by Tm-2. 2 -Mut5、Tm-2 2 -Mut3-1 is obvious.

[0128] The obtained pFGCTm-2 2 -Mut6、pFGCTm-2 2 -Mut5、pFGCTm-2 2 -Mut3-1 Wild-type pFGCTm-2 2 The infectious clone pCBToBRFV was transformed into Agrobacterium GV3101 competent cells. The cells carrying pFGCTm-2 were then... 2 -Agrobacterium tumefaciens of Mut6, pFGCTm-2 2 -Mut5 Agrobacterium, pFGCTm-2 2 Agrobacterium-1-Mut3-1, pFGCTm-2 2 The OD600 of Agrobacterium and pCBToBRFV was adjusted to 0.5, and then pFGCTm-2 was carried. 2 Mutant Agrobacterium and pFGCTm-2 2 Agrobacterium and Agrobacterium carrying pCBToBRFV were mixed at a 1:1 volume ratio. The mixed Agrobacterium was injected into tobacco leaves, and total protein was extracted from the inoculated area 4 days after inoculation. The accumulation of coat protein (CP) was detected by Western blot.

[0129] The results are as follows Figure 5 As shown, the results indicate that on day 4 post-inoculation, compared to the control (wild-type pFGCTm-2)... 2 Compared to Tm-2 2 -Mut3-1、Tm-2 2 -Mut5、Tm-2 2 -Mut6 significantly reduces the accumulation of ToBRFV capsid protein (CP). Tm-2 2 -Mut6 compared to Tm-2 2 -Mut5、Tm-2 2 Mut3-1 has a lower viral load. We believe Tm-2...2 -Mut6 compared to Tm-2 2 -Mut5、Tm-2 2 -Mut3-1 enhances the recognition of ToBRFV MP. ​​In actual production, Tm-2 2 -Mut6 is more conducive to plants quickly recognizing ToBRFV in the early stages of viral infection and inhibiting viral infection.

[0130] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A tomato disease resistance gene mutant, characterized in that, The nucleotide sequence of the tomato disease resistance gene mutant is shown in SEQ ID NO.1 or SEQ ID NO.

3.

2. The protein encoded by the tomato disease resistance gene mutant according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

3. The use of the tomato disease resistance gene mutant according to claim 1 in the following (1) or (2): (1) Prevention and control of tomato brown wrinkled fruit virus; (2) Cultivate plant varieties resistant to tomato brown wrinkled fruit virus; The plant species are tomatoes or tobacco.

4. The use of the protein encoded by the tomato disease resistance gene mutant according to claim 2 in the following (1) or (2): (1) Prevention and control of tomato brown wrinkled fruit virus; (2) Prepare products for the prevention and control of tomato brown wrinkled fruit virus.

5. A method for improving plant resistance to ToBRFV, characterized in that, Includes the following steps: plants Tm-2 2 The 643rd amino acid of the gene-encoded protein is mutated to arginine, and the mutated amino acid sequence is shown in SEQ ID NO.2; or, the plant... Tm-2 2 The 643rd amino acid of the gene-encoded protein is mutated to arginine, and the 767th amino acid is mutated to phenylalanine. The mutated amino acid sequence is shown in SEQ ID NO.

4. The plant in question is either a tomato or a tobacco plant.

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