Tomato disease-resistant gene mutant and application thereof in prevention and treatment of ToBRFV

Abstract

The invention discloses a tomato disease-resistant gene mutant and application thereof in prevention and treatment of ToBRFV, and belongs to the technical field of biological prevention and treatment of viral diseases. According to the invention, the 1927th nucleotide of the coding region sequence of the tomato Tm-22 gene is mutated from G to A; or the 643th amino acid of the LRR structural domain of the tomato Tm-22 gene encoding protein is mutated from glycine to arginine, so that the accumulation level of ToBRFV capsid protein (CP) can be obviously reduced. The gene (named as Tm-22-Mut5) not only can be used as a new ToBRFV resistance gene, but also retains the resistance to TMV, ToMV and ToMMV. The method comprises the following steps: carrying out combined mutation on Tm-22-Mut5 and a Tm-22-Mut3-1 mutant (tyrosine at the 767th site in an LRR structural domain is mutated into phenylalanine) screened in an earlier stage, so as to obtain a mutant Tm-22-Mut6. According to the present invention, the analysis results show that the accumulation level of the ToBRFV capsid protein can be significantly reduced by using the Tm-22-Mut6; compared with a mutant pFGCTm-22-Mut3-1 obtained through early research and screening and a newly obtained mutant Tm-22-Mut5, the resistance of the Tm-22-Mut6 to the ToBRFV is further improved, and the resistance of the Tm-22-Mut6 to the ToBRFV is further improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of viral diseases, and particularly relates to a tomato disease-resistant gene mutant and its application in controlling ToBRFV. Background Art

[0002] Tomato brown rugose fruit virus (ToBRFV) is a newly emerging virus. The main symptoms of the virus infecting tomato leaves are mosaic, dark green protrusions, narrow leaves, yellowing and necrosis of leaf veins, reduction in the number of flowers and fruits, yellow or brown patches on the fruits, smaller fruits, wrinkling, and in severe cases, necrosis of the fruit stalk, resulting in a significant decrease in fruit yield and marketability. ToBRFV has extremely strong infectivity, and currently, there is no effective measure to control the infection of ToBRFV to plants.

[0003] According to the gene-for-gene hypothesis, only when the plant's disease-resistant gene R encounters the avirulence gene Avr of the pathogen can the plant exhibit disease resistance (Van der Biezen & Jones, 1998). The Tm-2 2 gene of tomato is a disease-resistant gene belonging to the NBS-LRR family. The Tm-2 2 gene has certain resistance to tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and tomato mottled mosaic virus (ToMMV) in the genus Tobamovirus that infect tomatoes. However, ToBRFV in the genus Tobamovirus completely breaks the Tm-2 2 resistance in tomatoes. Currently, tomato varieties containing the Tm-2 2 gene are susceptible to ToBRFV.

[0004] By mutating and modifying the Tm-2 2 gene, it is possible to obtain mutants with ToBRFV resistance. However, since the full length of the ORF of the Tm-2 2 gene is 2586 bp and encodes 861 amino acids, there are countless possibilities for the mutation direction of its nucleotides or amino acids. Moreover, the performance after mutation is unpredictable and may even change the original resistance of the Tm-2 2 gene. Therefore, screening Tm-2 2 gene mutants with ToBRFV resistance and capable of maintaining the original virus resistance is still the current technical difficulty. Summary of the Invention

[0005] Aiming at the above-mentioned existing technologies, the purpose of the present invention is to provide a tomato disease-resistant gene mutant and its application in controlling ToBRFV.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a tomato disease-resistant gene mutant is provided. The tomato disease-resistant gene mutant is obtained by mutating the nucleotide sequence of the coding region of the Tm-2 2 gene at the nucleotide positions shown in the following (1) or (2):

[0008] (1) The 1927th nucleotide of the coding region sequence of the Tm-2 2 gene is mutated;

[0009] (2) The 1927th nucleotide, the 2300th nucleotide and the 2301st nucleotide of the coding region sequence of the Tm-2 2 gene are mutated simultaneously.

[0010] Preferably, the nucleotide sequence of the tomato disease-resistant gene mutant is as shown in SEQ ID NO.1 or SEQ ID NO.3. Specifically, if the mutation shown in (1) occurs, the 1927th nucleotide of the coding region sequence of the Tm-2 2 gene is mutated from G to A, and the tomato disease-resistant gene mutant is named Tm-2 2 -Mut5, and its nucleotide sequence is as shown in SEQ ID NO.1; if the mutation shown in (2) occurs, the 1927th nucleotide of the coding region sequence of the Tm-2 2 gene is mutated from G to A, the 2300th nucleotide is mutated from A to G, and the 2301st nucleotide is mutated from C to G, and the tomato disease-resistant gene mutant is named Tm-2 2 -Mut6, and its nucleotide sequence is as shown in SEQ ID NO.3.

[0011] In the second aspect of the present invention, a protein encoded by the above tomato disease-resistant gene mutant is provided.

[0012] Compared with the protein encoded by the wild-type Tm-2 2 gene, the 643rd amino acid of the protein encoded by the above tomato disease-resistant gene mutant is mutated; or, the 643rd amino acid and the 767th amino acid are mutated simultaneously.

[0013] Specifically, the 643rd glycine 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, etc.

[0014] Or, the 643rd glycine is mutated to arginine or to other amino acids, and the 767th tyrosine is mutated to phenylalanine or to other amino acids.

[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 -Mut5 has the amino acid sequence as shown in SEQ ID NO.2; the Tm-2 2 -Mut6 has the amino acid sequence as shown in SEQ ID NO.4.

[0016] The above mutant Tm-2 2 -Mut5 is obtained by mutating the glycine (G) at the 643rd position of the protein encoded by the tomato Tm-2 2 gene into arginine (R). The above mutant Tm-2 2 -Mut6 is obtained by mutating the glycine (G) at the 643rd position of the protein encoded by the tomato Tm-2 2 gene into arginine (R), and simultaneously mutating the tyrosine (Y) at the 767th position into tryptophan (W).

[0017] In the third aspect of the present invention, there is provided the use of the above anti-tomato disease-resistant gene mutant in the following (1) or (2):

[0018] (1) Controlling tomato brown rugose fruit virus;

[0019] (2) Cultivating plant varieties resistant to tomato brown rugose fruit virus.

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

[0021] In the fourth aspect of the present invention, there is provided the use of the protein encoded by the above tomato disease-resistant gene mutant in the following (1) or (2):

[0022] (1) Controlling tomato brown rugose fruit virus;

[0023] (2) Preparing a product for controlling tomato brown rugose fruit virus.

[0024] In the fifth aspect of the present invention, there is provided a method for improving the resistance of plants to ToBRFV, comprising the following steps:

[0025] Mutating the amino acid at the 643rd position of the protein encoded by the plant Tm-2 2 gene into an amino acid other than glycine;

[0026] Or, mutating the amino acid at the 643rd position of the protein encoded by the plant Tm-2 2 gene into an amino acid other than glycine, and simultaneously mutating the amino acid at the 767th position into an amino acid other than tyrosine.

[0027] Preferably, mutating the amino acid at the 643rd position of the protein encoded by the plant Tm-22 The 643rd amino acid of the gene-encoded protein is mutated to arginine; or, the plant Tm-2 2 The 643rd amino acid of the gene-encoded protein is mutated to arginine, and at the same time the 767th amino acid is mutated to phenylalanine.

[0028] Advantages of the present invention:

[0029] (1) The present invention discovers for the first time that in the coding region sequence of the tomato Tm-2 2 gene, the 1927th nucleotide is mutated from G to A; or, the 643rd amino acid of the protein encoded by the tomato Tm-2 2 gene is mutated from glycine to arginine, which can significantly reduce the accumulation of the ToBRFV coat protein (CP), and can be used as a new ToBRFV resistance gene, while retaining the resistance to TMV, ToMV, and ToMMV.

[0030] (2) The present invention combines mutations at different sites in the coding region sequence of the Tm-2 2 gene to obtain the mutant Tm-2 2 -Mut6. It is found that Tm-2 2 -Mut6 can significantly reduce the accumulation of the ToBRFV coat protein (CP). Compared with the mutant pFGCTm-2 2 -Mut3-1 screened in the previous study and the newly obtained mutant pFGCTm-2 2 -Mut5, its resistance to ToBRFV is further improved. Description of the Drawings

[0031] Figure 1 : Screening mutants that can react with ToBRFV MP to trigger cell necrosis and determining their action sites.

[0032] Figure 2 : Tm-2 2 -Mut5 reacts with ToBRFV MP and TMV, ToMV, and ToMMV MP to trigger cell necrosis; among them, the left figure: compared with the wild-type Tm-2 2 , Tm-2 2 -Mut5 can produce a hypersensitive necrosis reaction with ToBRFV MP; the right figure: Tm-2 2 -Mut5 can also recognize the MPs of TMV, ToMV, and ToMMV. Western blot detection was performed 4 days after inoculation.

[0033] Figure 3 : Tm-2 2-Mut5 significantly reduced 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 mutants and ToBRFV MP reactions.

[0035] Figure 5 :Tm-2 2 And different Tm-2 2 Effect of mutants on ToBRFV accumulation. Western blot analysis was performed 4 days after inoculation. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0037] As mentioned earlier, ToBRFV completely breaks down Tm-2 in tomatoes 2 Resistance, currently contains Tm-2 2 Tomato varieties with the gene are susceptible to ToBRFV. 2 Gene mutation modification may result in the acquisition of mutants with ToBRFV resistance. However, due to the 2 The ORF of the gene is 2586 bp in length, and the nucleotides at each site have four different mutation directions. In addition, the coding relationship between the nucleotide sequence and the amino acid sequence, therefore, Tm-2 2 There are countless possible mutations in genes. It is necessary to mine meaningful Tm-2 from these numerous mutation possibilities. 2 Genetic mutants are extremely difficult.

[0038] The inventors have proposed that Tm-2 2 Genetic mutants have been studied for many years, and four Tm-2 mutants with ToBRFV resistance have been found. 2 The gene mutant is the same as Tm-2 with the accession number AF536201.1 in NCBI 2 Gene sequence comparison, Tm-2 2 -Tm-2 of Mut1 2 Three nucleotide mutations occurred in the gene coding region, namely A138 G, T418C, and A1425G, and the corresponding amino acid mutations were R46R, C140R, and S475S. 2 -Tm-2 of Mut2 2One nucleotide mutation occurred in the gene coding region, mutated to A1062G, and the corresponding amino acid mutated to I354M. Tm-2 2 -Tm-2 of Mut3-1 2 Two nucleotide mutations occurred in the gene coding region, the mutations were A2300G and C2301G respectively, and the corresponding amino acid mutated to Y767W. Tm-2 2 -Tm-2 of Mut4 2 Three nucleotide mutations occurred in the gene coding region, the mutations were A1062G, A2300G, and C2301G respectively, and the corresponding amino acids mutated to I354M and Y767W.

[0039] However, the quantity of the above mutants is difficult to meet the actual production requirements, and Tm-2 2 The anti-ToBRFV effect of the gene mutants also needs to be further improved.

[0040] In view of this, the present invention further screened and investigated the Tm-2 2 gene mutants, and found a new mutant plasmid resistant to ToBRFV. Sequencing it, comparing with the Tm-2 2 gene sequence with the accession number AF536201.1 in NCBI, it was found that this mutant had three nucleotide mutations, which were A521G, G924A, and G1927A respectively, and the corresponding amino acids mutated to Q174R, L308L, and G643R. After verification, it was found that the nucleotide mutation capable of recognizing the hypersensitive response generated by ToBRFV MP was G1927A, and the corresponding amino acid mutated to G643R.

[0041] Subsequently, G1927A was used as the mutation site to construct a new mutant plasmid, which was named pFGCTm-2 2 -Mut5. Tm-2 in pFGCTm-2 2 -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 - Combine these two mutation sites in Mut5 to obtain Tm-2 2 Mutant plasmid pFGCTm-2 2 - Mut6

[0044] Compare with the Tm-2 gene sequence with the accession number AF536201.1 in NCBI 2 The Tm-2 2 - Mut6 has 3 nucleotide mutations in the coding region. The mutation G1927A results in an amino acid mutation to G643R; the mutations A2300G and C2301G result in an amino acid mutation to Y767W

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

[0046] atggctgaaattcttcttacatcagtaatcaataaatctgtagaaatagctggaaatttactgattcaagaaggaaagcgtttatattggttgaaa

[0047] gaggatatcgattggctccagagagaaatgagacacattcgatcttatgttgacaacgcaaaggccaaggaagctggaggtgattcaagg

[0048] gtcaaaaacttattgaaagatattcaagaattggcaggtgatgtggaggatctcttagatgacttccttccaaaaattcaacaatccaataagtt

[0049] caattattgccttaagaggagttcttttgcagatgagtttgctatggagattgagaagataaagagaagggttgttgacattgaccgaataagg

[0050] aaaacttacaacatcatagatacagataacaataatgatgattgtgttctgctggatcggagaagattattcctacatgctgatgaaacagaga

[0051] tcatcggtttggatgatgacttcaatatgctacaagccaaattacttaatcaagatttgcattatggagttgtttccatagttggcatgcccggtct

[0052] ggggaaaacaactcttgccaagaaactttataggctcattcgtgatcaatttgagtgttctggactggtctacgtttcacaacagccaagagc

[0053] gagtgaaatcttacttgacattgccaaacaaattggactgacggaacagaaaatgaaggaaaatttggaggacaacctgcgatcactcttga

[0054] aaataaaaaggtatgttatcctcctagatgacatttgggatgtggaaatttgggatgatctgaaacttgtccttcctgaatgtgattcaaaagtcg

[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] HYGVVSIVGMPGLGKTTLAKKLYRLIRDQFECSGLVYVSQQPRASEILLDIAKQIGLTEQ

[0079] KMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKVGSRMIITSRNSNV

[0080] GRYIGGESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASPDLVNIGRNIVGRCGGIPL

[0081] AIVVTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASRPCFLYFG

[0082] LYPEDHEIRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLNDLVSRNLIQLAKRTYNG

[0083] RISSCRIHDLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMIEFFRSNP

[0084] KLEKLRVLFCFAKDPSIFSHMAYFDFKLLHTLVVVMSQSFQAYVTIPSKFGNMTCLRYL

[0085] RLEGNICGKLPNSIVKLTRLETIDIDRRSLIQPPSGVWESKHLRHLCYRDYRQACNSCFSI

[0086] SSFYPNIYSLHPNNLQTLMWIPDKFFEPRLLHRLINLRKLGILGVSNSTVKMLSIFSPVLK

[0087] ALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNSQSFPPNLIKLTLAWFSVDRYI

[0088] LAVLKTFPKLRKLKMFICKYNEEKMDLSGEANGYSFPQLEVLHIHSPNGLSEVTCTDDV

[0089] SMPKLKKLLLTGFHCRISLSERLKKLSK

[0090] Co-express the mutant plasmid pFGCTm-2 2 -Mut6 with ToBRFV, and it was found that Tm-2 2 -Mut6 could significantly reduce the accumulation of the coat protein (CP) of ToBRFV. Compared with the mutant pFGCTm-2 2 -Mut3-1 screened in the previous study and the newly obtained mutant pFGCTm-2 2 -Mut5, its resistance to ToBRFV was further improved, showing a synergistic effect.

[0091] In order to enable those skilled in the art to more clearly understand the technical solutions of this application, the technical solutions of this application will be described in detail below in combination with specific embodiments.

[0092] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. The experimental methods without specifying detailed conditions are carried out according to conventional test methods or according to the operation manuals recommended by the suppliers. The Tm-2 2 gene has a GenBank accession number of: AF536201.1. The ToBRFV-SD isolate used in the present invention has a GenBank accession number of MT018320; TMV has a GenBank accession number of MH595920; ToMMV has a GenBank accession number of MW37351; ToMV has a GenBank accession number of KY967221.1.

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

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

[0095] Construct the Tm-2 2 gene mutant library by error-prone PCR as follows:

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

[0097] Subsequently, perform the reaction. The reaction program is as follows: pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 15 s, annealing at 55 °C for 8 s, extension at 72 °C for 2 min 35 s, 35 cycles, and final extension at 72 °C for 2 min. Clone the amplified fragment into the binary expression vector pFGC5941 by homologous recombination, then transfer it into competent Escherichia coli DH5α, extract the plasmid from it, screen the plasmids with the correct size, and obtain 1800 Tm-2 2 mutant plasmids.

[0098] 2. Screen for Tm-2 2 mutants that produce hypersensitive cell necrosis in response to ToBRFV MP

[0099] Use ordinary PCR to amplify MP in the infectious clone pCBToBRFV (for the construction of the infectious clone pCBToBRFV, refer to the literature 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 resistance gene. Molecular Plant Pathology. 2021, 11, (22): 1347–1357). Clone the amplified product into the pCam-35S binary expression vector between the 35S promoter and the NOS terminator by homologous recombination to obtain pCamToBRFV-MP. The sequence of MP is the sequence encoded by the ToBRFV-SD isolate (accession number MT018320 in NCBI).

[0100] The 1800 obtained Tm-2 2 mutant plasmids and the pCamToBRFV-MP plasmid expressing ToBRFV MP were respectively transformed into the competent cells of Agrobacterium tumefaciens GV3101. The OD of the Agrobacterium carrying the Tm-2 2 mutant plasmid and the Agrobacterium of pCamToBRFV-MP was 600 adjusted to 0.5, and then the two were mixed according to a volume ratio of 1:1. The mixed Agrobacterium was injected into the leaves of Nicotiana benthamiana. Two days after inoculation, the necrosis of the leaves in the inoculation area was observed.

[0101] As a result, it was found that among the 1800 obtained Tm-2 2 mutant plasmids, 1 mutant could react with ToBRFV MP to induce cell necrosis. The mutant plasmid that caused cell necrosis was sequenced. The sequencing results showed that 3 nucleotide mutations occurred in the coding region of this mutant, and the mutations were A521G, G924A, and G1927A respectively, and the corresponding amino acid mutations were Q174R, L308L, and G643R. Analysis determined the action site G643R that enabled the mutant to recognize ToBRFV MP and trigger the hypersensitive response ( Figure 1 ).

[0102] Example 2: Construction and disease resistance study of the Tm-2 2 -Mut5 mutant

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

[0104] The amplified fragment with the 1927th nucleotide mutation of the Tm-2 2 gene was amplified by site-directed mutagenesis using the following primers.

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

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

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

[0108] Subsequently, a mutant PCR reaction was carried out. The reaction program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 2 min 35 s, for 15 cycles, annealing at 48°C for 30 s, and final extension at 72°C for 5 min. The amplified product was transferred into Escherichia coli DH5α competent cells, and the plasmid was extracted from them. The plasmid with the correct size was screened to construct a single-site mutant plasmid (Tm-2 2 -Mut5 mutant plasmid).

[0109] The nucleotide mutations occurring in the mutant Tm-2 2 -Mut5 were G1927A respectively, and the corresponding amino acid mutation was G643R.

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

[0111] Co-express pFGCTm-2 2 and pFGCTm-2 2 -Mut5 with ToBRFV MP. Specifically, the Agrobacterium carrying the Tm-2 2 mutant plasmid (or pFGCTm-2 2 ) and the Agrobacterium of pCamToBRFV-MP were adjusted to an OD 600 of 0.5, and then the two were mixed at a volume ratio of 1:1. The mixed Agrobacterium was injected into the leaves of Nicotiana benthamiana. Two days after inoculation, the necrosis of the leaves in the inoculated area was observed.

[0112] Using the same method as above, the obtained pFGCTm-2 2 -Mut5 was co-expressed with the plasmids expressing TMV MP, ToMVMP, and ToMMV MP respectively.

[0113] The phenotypic observation results showed that Tm-2 2 did not react with ToBRFV MP, while Tm-2 2 -Mut5 reacted with ToBRFV MP to induce cell necrosis ( Figure 2 A). In addition, the co-expression experiment results showed that Tm-2 2 -Mut5 could also react with ToMV and ToMMV MP to induce cell necrosis ( Figure 2 B). The above results indicated that Tm-2 2 -Mut5 could mediate resistance to ToBRFV and at the same time retained resistance to TMV, ToMV, and ToMMV.

[0114] The obtained pFGCTm-2 2-Mut5, empty vector pFGC5941, wild-type pFGCTm-2 2 and the infectious clone pCBToBRFV were respectively transformed into competent Agrobacterium tumefaciens GV3101. The Agrobacterium tumefaciens carrying pFGCTm-2 2 -Mut5, the Agrobacterium tumefaciens carrying pFGC5941, pFGCTm-2 2 of the Agrobacterium tumefaciens and the Agrobacterium tumefaciens carrying pCBToBRFV were adjusted to an OD 600 of 0.5, and then the Agrobacterium tumefaciens carrying pFGCTm-2 2 -Mut5, pFGCTm-2 2 of the Agrobacterium tumefaciens and the Agrobacterium tumefaciens carrying pFGC5941 were respectively mixed with the Agrobacterium tumefaciens carrying pCBToBRFV at a volume ratio of 1:1. The mixed Agrobacterium tumefaciens was injected into the leaves of Nicotiana benthamiana. Four days after inoculation, the total protein of the leaves in the inoculated area was extracted. Western blot was used to detect the coat protein (CP)( Figure 3 ).

[0115] Using the same method as above, pFGCTm-2 2 -Mut5, empty vector pFGC5941, pFGCTm-2 2 were respectively co-expressed with the infectious clones of TMV, ToMV, and ToMMV. Among them, the construction of the TMV infectious clone refers to 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 harbouring the Tm-2 2resistance gene. Molecular Plant Pathology. 2021, 11, (22): 1347–1357; The construction of the ToMV infectious clone was carried out by using the primers PCB301-ToMV-F: TTTCATTTGGAGAGGGTATTTATTATTACAACAATTACC and PCB301-ToMV-R: GATATAAGTACAGACTGGGCCCCTACCGGGGGTTCCGG to amplify the full length of ToMV, which was cloned between the 35S promoter and the NOS terminator of the binary expression vector pCB301; 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 the coat protein (CP) was detected by Western blot.

[0116] The results are as Figure 3 shown, and the results indicate that: compared with the controls (empty vector pFGC5941, wild-type pFGCTm-2 2 ), Tm-2 2 -Mut5 can significantly reduce the accumulation of the ToBRFV coat protein (CP). In addition, the results of the co-expression experiment show that Tm-2 2 -Mut5 can also significantly reduce the accumulation of TMV, ToMV and ToMMV.

[0117] Example 3: Construction and disease resistance study of the Tm-2 2 -Mut6 mutant

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

[0119] The amplified fragment of the 767th amino acid mutation of the mutant Tm-2 2 -Mut5 obtained in Example 2 was amplified by site-directed mutagenesis using the following primers.

[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 system, which contains: 25 μL of buffer, 1 μL of 10 mM dNTP, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 1 μL of 1 U / μL high-fidelity DNA polymerase, 0.5 μL of 30 ng / μL pFGCTm - 2, and 20.5 μL of double-distilled water. 2 0.5 μL, 20.5 μL of double-distilled water.

[0123] Subsequently, perform the mutant PCR reaction. The reaction program is as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 2 min 35 s, for 15 cycles, annealing at 48 °C for 30 s, and final extension at 72 °C for 5 min. Transfer the amplified product into Escherichia coli DH5α competent cells, extract the plasmid from them, screen for plasmids with the correct size, and construct Tm - 2 2 -Mut6 mutant.

[0124] Amplify the amplified fragment with nucleotide mutations at positions 2300 and 2301 of the Tm - 2 2 gene by the above primer-directed mutagenesis method, and construct the single-site mutant plasmid (Tm - 2 2 -Mut3 - 1 mutant plasmid) using the same method.

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

[0126] Co-express pFGCTm - 2 2 and pFGCTm - 2 2 -Mut6, pFGCTm - 2 2 -Mut5, pFGCTm - 2 2 -Mut3 - 1 with ToBRFV MP. Specifically, adjust the OD 2 of the Agrobacterium carrying the Tm - 2 2 mutant plasmid (or pFGCTm - 2 600 ) and the Agrobacterium of pCamToBRFV - MP to 0.5, and then mix the two in a volume ratio of 1:1. Inject the mixed Agrobacterium into the leaves of Nicotiana benthamiana. Two days after inoculation, observe the necrosis of the leaves in the inoculated area.

[0127] The phenotypic observation results are asFigure 4 The indicated Tm-2 2 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 -Mut6 produces a necrotic reaction with ToBRFV MP that is stronger than that of Tm-2 2 -Mut5, Tm-2 2 -Mut3-1 is more obvious.

[0128] The obtained pFGCTm-2 2 -Mut6, pFGCTm-2 2 -Mut5, pFGCTm-2 2 -Mut3-1 wild-type pFGCTm-2 2 and the infectious clone pCBToBRFV were respectively transformed into competent Agrobacterium tumefaciens GV3101. The Agrobacterium carrying pFGCTm-2 2 -Mut6, pFGCTm-2 2 -Mut5, pFGCTm-2 2 -Mut3-1, pFGCTm-2 2 and the Agrobacterium carrying pCBToBRFV were adjusted to an OD600 of 0.5, and then the Agrobacterium carrying pFGCTm-2 2 mutants and pFGCTm-2 2 were respectively mixed with the Agrobacterium carrying pCBToBRFV at a volume ratio of 1:1. The mixed Agrobacterium was injected into the leaves of Nicotiana benthamiana. Four days after inoculation, total protein was extracted from the leaves in the inoculated area. Western blot was used to detect the accumulation level of coat protein (CP).

[0129] The results are as Figure 5 shown. The results indicate that: on the 4th day after inoculation, compared with the control (wild-type pFGCTm-2 2 ), Tm-2 2 -Mut3-1, Tm-2 2 -Mut5, Tm-2 2 -Mut6 can significantly reduce the accumulation of ToBRFV coat protein (CP). Tm-2 2 -Mut6 has less virus accumulation than Tm-2 2 -Mut5, Tm-2 2 -Mut3-1. We believe that Tm-22 - Mut6 compared with Tm-2 2 - Mut5, Tm-2 2 - Mut3-1 enhances the recognition of ToBRFV MP. In actual production, Tm-2 2 - Mut6 is more beneficial for the plant to quickly recognize ToBRFV at the initial stage of virus infection and inhibit virus infection.

[0130] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A tomato disease-resistant gene mutant, characterized in that, The tomato disease-resistant gene mutant is obtained by introducing nucleotide mutations at the positions shown in (1) or (2) below in the nucleotide sequence of the coding region of the Tm-2 2 gene: (1)Tm-2 2 The 1927th nucleotide of the coding region sequence of the gene is mutated; (2)Tm-2 2 The 1927th nucleotide, the 2300th nucleotide, and the 2301st nucleotide of the coding region sequence of the gene are mutated simultaneously.

2. The tomato disease-resistant gene mutant according to claim 1, characterized in that, The nucleotide sequence of the tomato disease-resistant gene mutant is shown in SEQ ID NO.1 or SEQ ID NO.

3.

3. The protein encoded by the tomato disease-resistant gene mutant according to claim 1 or 2.

4. The protein according to claim 3, wherein The 643rd amino acid of the protein is mutated; alternatively, the 643rd amino acid and the 767th amino acid are mutated simultaneously.

5. The protein according to claim 4, wherein The amino acid sequence of the protein is shown in SEQ ID NO.2 or SEQ ID NO.

4.

6. The application of the anti-tomato disease-resistant gene mutant according to claim 1 or 2 in the following (1) or (2): (1) Preventing and controlling tomato brown rugose fruit virus; (2) Cultivating plant varieties resistant to tomato brown rugose fruit virus.

7. The application according to claim 6, characterized in that The plant variety is tomato, pepper or tobacco.

8. The application of the protein encoded by the tomato disease-resistant gene mutant according to any one of claims 3-5 in the following (1) or (2): (1) Preventing and controlling tomato brown rugose fruit virus; (2) Preparing a product for preventing and controlling tomato brown rugose fruit virus.

9. A method for improving the resistance of plants to ToBRFV, characterized in that, Comprising the following steps: Mutate the 643rd amino acid of the protein encoded by the plant Tm-2 2 gene into an amino acid other than glycine; Alternatively, mutate the 643rd amino acid of the protein encoded by the plant Tm-2 2 gene to an amino acid other than glycine, and simultaneously mutate the 767th amino acid to an amino acid other than tyrosine.

10. The method according to claim 9, wherein Mutate the 643rd amino acid of the protein encoded by the plant Tm-2 2 gene into arginine; or, mutate the 643rd amino acid of the protein encoded by the plant Tm-2 2 gene into arginine and simultaneously mutate the 767th amino acid into phenylalanine.

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