Tomato Tm-2 2 Disease resistance protein mutants and their application in the prevention and control of ToBRFV

Abstract

The invention discloses a tomato Tm-2 2 Disease resistance protein mutants and their application in the prevention and treatment of ToBRFV belong to the technical field of biological control of viral diseases. The present invention first discovered that tomato Tm-2 2 The 1062nd nucleotide of the coding region sequence of the gene was mutated from A to G; or the tomato Tm‑2 2 The 354th amino acid of the gene-encoded protein mutates from isoleucine to methionine, which can significantly reduce the accumulation of ToBRFV capsid protein (CP), and can be used as a new ToBRFV resistance gene, which is of great significance for the prevention and treatment of ToBRFV. 2 While achieving resistance to ToBRFV, the disease-resistant protein mutant can also maintain its original resistance to TMV, ToMV and ToMMV, providing a new source of disease-resistant genes for the breeding of disease-resistant plant varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control of viral diseases, and in particular to a tomato Tm-2 2 Disease-resistant protein mutants and their application in preventing and controlling ToBRFV. Background Art

[0002] Tomato Tm-2 2 The gene belongs to the NBS-LRR family, which is located on the long arm of chromosome 9 and can mediate resistance to tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). 2 The gene is an allele of Tm-2 and its susceptibility gene tm-2. The proteins encoded by them have different amino acid sequences in the NBS domain and LRR domain, respectively. This difference determines the Tm-2 2 The disease resistance or susceptibility characteristics of genes and their alleles. 2 The amino acid sequences of genes and the proteins they encode can help us understand the disease resistance of tomato varieties at the molecular level, deepen our understanding of the recognition and response mechanisms of disease-resistant gene-encoded proteins to viruses, and lay the foundation for the application of disease-resistant genes.

[0003] Tomato brown rugose fruit virus (ToBRFV) belongs to the genus Tobamovirus. It was first reported in Israel in 2014 and has gradually spread to many countries including my country. In April 2021, ToBRFV was listed as a quarantine virus by my country. ToBRFV has strong transmission ability and harmfulness, which seriously threatens the safe production of tomatoes.

[0004] Planting disease-resistant varieties is the most effective way to prevent and control viral diseases, but ToBRFV can infect plants containing Tm-2 2 Tomato varieties with disease resistance genes are therefore urgently needed. 2 Disease resistance genes are modified to breed plant varieties that are resistant to tomato brown fruit virus. Summary of the invention

[0005] In view of the above prior art, the present invention aims to provide a tomato Tm-2 2 Disease resistance protein mutant and its application in preventing and controlling ToBRFV. The present invention utilizes random mutation method to treat disease resistance gene Tm-2 2 Artificial evolution was performed to screen out a tomato Tm-2 that is resistant to ToBRFV from a large number of artificial mutants. 2Disease-resistant protein mutants provide a new source of disease-resistant genes for the breeding of ToBRFV-resistant plant varieties.

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

[0007] In a first aspect of the present invention, a tomato Tm-2 2 Disease resistance protein mutant, the tomato Tm-2 2 Tomato Tm-2 2 The 354th amino acid of the gene-encoded protein was mutated.

[0008] Preferably, the tomato Tm-2 2 The amino acid sequence of the disease-resistant protein mutant is shown in SEQ ID NO.2.

[0009] Tomato Tm-2 of the present invention 2 Tomato Tm-2 2 The 354th amino acid of the gene-encoded protein mutated from isoleucine (I) to methionine (M).

[0010] Furthermore, the tomato Tm-2 of the present invention 2 The disease resistance protein mutants also include tomato Tm-2 2 The 354th amino acid of the gene-encoded protein is mutated to a mutation direction other than methionine (M), for example: 2 The 354th amino acid of the gene-encoded protein mutated from isoleucine (I) to alanine (A), valine (V), leucine (L), proline (P), glycine (G), serine (S), tyrosine (Y), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), tryptophan (W), histidine (H), threonine (T), phenylalanine (F), cysteine ​​(C), asparagine (N) or glutamine (Q). As long as the mutated tomato Tm-2 2 The disease-resistant protein mutants have anti-ToBRFV activity and fall within the protection scope of the present invention.

[0011] In a second aspect of the present invention, the tomato Tm-2 2 Gene encoding disease resistance protein mutant.

[0012] Preferably, the coding gene is a nucleic acid molecule as shown in i) or ii) below:

[0013] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0014] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0015] The third aspect of the present invention provides a recombinant expression vector or an engineered bacterium comprising the above encoding gene.

[0016] In a fourth aspect of the present invention, the tomato Tm-2 2 Application of disease resistance protein mutants in the following (1) or (2):

[0017] (1) Prevent and control tomato brown fruit virus;

[0018] (2) Prepare products for preventing and controlling tomato brown wrinkled fruit virus.

[0019] In a fifth aspect of the present invention, the tomato Tm-2 2 Application of the coding gene of the disease-resistant protein mutant or the recombinant expression vector or engineered bacteria containing the coding gene in the following (1) or (2):

[0020] (1) Prevent and control tomato brown fruit virus;

[0021] (2) Cultivate plant varieties that are resistant to tomato brown fruit virus.

[0022] Preferably, the plant variety is tomato, tobacco or pepper.

[0023] A sixth aspect of the present invention provides a method for improving the resistance of tomato plants to ToBRFV, comprising the following steps:

[0024] Tomato plant Tm-2 2 In the coding region sequence of the gene, the nucleotide 1062 mutated from A to G;

[0025] Alternatively, tomato plants are exogenously introduced with tomato Tm-2 2 Gene encoding disease resistance protein mutant.

[0026] A seventh aspect of the present invention provides a method for cultivating a tomato variety resistant to ToBRFV, comprising the following steps:

[0027] (1) The tomato Tm-2 2 Tomato plants with a mutant gene encoding a disease resistance protein are self-pollinated or crossed with another tomato plant to obtain the next generation population;

[0028] (2) Selecting tomato Tm-2 from the next generation population 2 Tomato plants containing a disease resistance protein mutant encoding gene, when the tomato Tm-2 2 When the gene encoding the disease resistance protein mutant is homozygous, the tomato plant is resistant to ToBRFV.

[0029] Beneficial effects of the present invention:

[0030] The present invention first discovered that tomato Tm-2 2 In the coding region sequence of the gene, the nucleotide at position 1062 was mutated from A to G; or, the tomato Tm-2 2 The 354th amino acid of the gene-encoded protein mutates from isoleucine to methionine, which can significantly reduce the accumulation of ToBRFV capsid protein (CP), and can be used as a new ToBRFV resistance gene, which is of great significance for the prevention and treatment of ToBRFV. 2 The disease-resistant protein mutants not only achieve resistance to ToBRFV, but also maintain their original resistance to TMV, ToMV and tomato mottle mosaic virus (ToMMV), providing a new source of disease-resistant genes for the breeding of disease-resistant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 :Tm-2 2 With Tm-2 2 -Mut2 nucleotide sequence alignment.

[0032] Figure 2 :Tm-2 2 With Tm-2 2 -Mut2 amino acid sequence alignment.

[0033] Figure 3 :Tm-2 2 -Mut2 reacts with ToBRFV MP and TMV, ToMV and ToMMV MP to induce cell necrosis; in the figure, A: with wild-type Tm-2 2 In comparison, Tm-2 2 -Mut2 can produce allergic necrosis reaction with ToBRFV MP; B: Tm-2 2 -Mut2 can also recognize MPs of TMV, ToMV and ToMMV.

[0034] Figure 4 :Tm-2 2 -Mut2 significantly reduced the accumulation of ToBRFV as well as TMV, ToMV, and ToMMV. DETAILED DESCRIPTION

[0035] 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.

[0036] As mentioned above, the disease resistance gene Tm-2 2 The gene can effectively resist the infection of tobacco mosaic virus TMV, ToMV and ToMMV, but not ToBRFV. Therefore, it is very urgent to discover new resistance resources for the prevention and control of ToBRFV.

[0037] Tm-2 2 The ORF of the gene is 2586 bp in length and encodes 861 amino acids. Due to the long amino acid sequence of the encoded protein, amino acid mutations at different sites have an impact on the Tm-2 2 The effect of gene function is unpredictable, so the effect of Tm-2 2 It is difficult to modify disease-resistant genes.

[0038] The inventor has been committed to the research of plant antiviral for a long time. In the previous research, the inventor constructed Tm-2 by error-prone PCR. 2 The gene mutant library was then constructed into Tm-2 2 The mutant plasmid was co-expressed with the pCamToBRFV-MP plasmid expressing ToBRFV MP to screen for mutant plasmids that induce cell necrosis. The results showed that 1200 Tm-2 2 A mutant pFGCTm-2 resistant to ToBRFV was screened out from the mutant plasmids 2 -Mut1, sequencing revealed that this mutant had three nucleotide mutations.

[0039] To explore more Tm-2 that can resist ToBRFV 2 The present invention further constructs a new Tm-2 gene mutant by error-prone PCR. 2 The mutant library was then generated from 1800 Tm-2 2 A mutant plasmid resistant to ToBRFV was screened out from the mutant plasmids and named pFGCTm-2 2 -Mut2.

[0040] Mutant plasmid pFGCTm-2 2 -Mut2 was sequenced, pFGCTm-2 2 -Tm-2 in Mut2 2 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1, and is as follows:

[0041]

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

[0043] MAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMRHIRSYVDNAKAKEAGGDSRVKNLL

[0044] KDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRSSFADEFAMEIEKIKRRVVDIDRIRKTYNIIDTDNN

[0045] NDDCVLLDRRRLFLHADETEIIGLDDDFNMLQAKLLNQDLHYGVVSIVGMPGLGKTTLAKKLYRLIR

[0046] DQFECSGLVYVSQQPRASEILLDIAKQIGLTEQKMKENLEDNLRSLLKIKRYVILLDDIWDVEIWDDLK

[0047] LVLPECDSKVGSRMIITSRNSNVGRYIGGESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASPDLV

[0048] NIGRNIVGRCGGMPLAIVVTAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASRP

[0049] CFLYFGLYPEDHEIRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLNDLVSRNLIQLAKRTYNGRISS

[0050] CRIHDLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMIEFFRSNPKLEKLRVLFCFAK

[0051] DPSIFSHMAYFDFKLLHTLVVVMSQSFQAYVTIPSKFGNMTCLRYLRLEGNICGKLPNSIVKLTRLETI

[0052] DIDRRSLIQPPSGVWESKHLRHLCYRDYGQACNSCFSISSFYPNIYSLHPNNLQTLMWIPDKFFEPRLL

[0053] HRLINLRKLGILGVSNSTVKMLSIFSPVLKALEVLKLSFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNS

[0054] QSFPPPNLIKLTLAYFSVDRYILAVLKTFPKLRKLKMFICKYNEEKMDLSGEANGYSFPQLEVLHIHSPNGLSEVTCTDDVSMPKLKKLLLTGFHCRISLSERLKKLSK*

[0055] Tm-2 with NCBI accession number: AF536201.1 2 By comparing the gene sequences, it was found that Tm-2 2 -One nucleotide mutation occurred in the coding region of Mut2, namely A1062G, and the corresponding amino acid mutation was I354M.

[0056] The mutant plasmid pFGCTm-2 2 -Mut2 was co-expressed with ToBRFV and Tm-2 was found 2 -Mut2 can significantly reduce the accumulation of ToBRFV capsid protein (CP) without affecting the accumulation of TMV, ToMV and ToMMV CP. 2 -Mut1, its resistance to ToBRFV, TMV, ToMV and ToMMV was further improved.

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0058] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out in accordance with conventional test methods or the operating instructions recommended by the supplier. 2 The accession number of the gene in NCBI is: AF536201.1. The accession number of the ToBRFV-SD isolate used in the present invention in NCBI is MT018320; the accession number of TMV in NCBI is MH595920; the accession number of ToMMV in NCBI is MW37351; and the accession number of ToMV in GenBank is KY967221.1.

[0059] Example 1: Tm-2 2 Construction and screening of gene mutant library

[0060] 1. Tm-2 2 Construction of gene mutant library:

[0061] Construction of Tm-2 by error-prone PCR 2 The gene mutant library is as follows:

[0062] First, a 50 μL reaction system was prepared, which included: 15 μL of buffer, 5 μL of 2.5 mM dNTP for error-prone, 1 μL of 10 μM forward primer (5'-ttacaattaccatggatggctgaaattcttcttacatc-3', SEQ ID NO.3), 1 μL of 10 μM reverse primer (5'-actcacctaggatcctcatttactcagctttttaagccg-3', SEQ ID NO.4), 2.5 μL of 20 mM NCl2, 0.3 μL of 5 U / μL DNA polymerase, and 30 ng / μL template (wild-type plasmid pFGCTm-2 2 )0.5μL, double distilled water 24.7μL.

[0063] The reaction was then carried out, and the reaction procedure was 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 post-extension at 72°C for 2 min. The amplified fragment was cloned into the binary expression vector pFGC5941 by homologous recombination, and then transformed into Escherichia coli DH5α competent cells, from which plasmids were extracted and screened for plasmids with the correct plasmid size. A total of 1800 Tm-2 2 Mutant plasmids.

[0064] 2. Screening of Tm-2 that reacts with ToBRFV MP to produce allergic cell necrosis 2 Mutants

[0065] The infectious clone pCBToBRFV was constructed by 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 2resistance gene.Molecular Plant Pathology.2021,11,(22):1347–1357) and cloned 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 (NCBI accession number MT018320). Its nucleotide sequence is shown in SEQ ID NO.5; its amino acid sequence is shown in SEQ ID NO.6.

[0066] The 1800 Tm-2 2 The mutant plasmid and the pCamToBRFV-MP plasmid expressing ToBRFV MP were transformed into Agrobacterium GV3101 competent cells. 2 OD of Agrobacterium containing mutant plasmid and pCamToBRFV-MP 600 Adjust 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, and observe the necrosis of the leaves in the inoculated area 2 days after inoculation.

[0067] The results showed that the 1800 Tm-2 2 There is only one mutant Tm-2 in the mutant plasmid 2 -Mut2 can react with ToBRFV MP to induce cell necrosis. The mutant plasmid pFGCTm-2 that induces cell necrosis 2 -Mut2 was sequenced. The sequencing results showed that pFGCTm-2 2 -Mut2 had one nucleotide mutation, A1062G ( Figure 1 ), the corresponding amino acid mutation is I354M ( Figure 2 ).

[0068] Example 2: Tm-2 2 Study on resistance of gene mutants to ToBRFV

[0069] 1. Tm-2 2 - Construction of Mut2 mutant plasmid:

[0070] The following primers were used to amplify Tm-2 by directed mutagenesis. 2 Amplified fragment of the gene with mutation at nucleotide position 1062.

[0071] Primer pair 1-Tm-2 2-A1062G-F:gccgctagccatagtggtgactgcaggcatgttaag; (SEQ IDNO.7)

[0072] Primer pair 1-Tm-2 2 -A1062G-R: actatggctagcggcatacctccacatctcccaactata. (SEQ ID NO.8)

[0073] First, prepare 50 μL of mutation PCR reaction system, which includes: 25 μL buffer, 1 μL 10 mM dNTP, 1 μL 10 μM forward primer, 1 μL 10 μM reverse primer, 1 μL 1U / μL DNA high-fidelity polymerase, 30 ng / μL pFGCTm-2 2 0.5μL, double distilled water 20.5μL.

[0074] Then, the mutation PCR reaction was carried out, and the reaction procedure was as follows: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s, 55℃ annealing for 15s, 72℃ extension for 2min 35s, 15 cycles, 48℃ annealing for 30s, and 72℃ post-extension for 5min. The amplified product was transferred into Escherichia coli DH5α competent cells, and plasmids were extracted from them. Plasmids with the correct plasmid size were screened, and a single site mutation plasmid (Tm-2 2 -Mut2 mutant plasmid).

[0075] 2. Tm-2 2 -Mut2 mutant plasmid and ToBRFV MP co-expression test:

[0076] pFGCTm-2 2 and pFGCTm-2 2 -Mut2 was co-expressed with TMV and ToBRFV MP. 2 Mutant plasmid (or pFGCTm-2 2 OD of Agrobacterium tumefaciens with pCamToBRFV-MP 600 Adjust 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, and observe the necrosis of the leaves in the inoculated area 2 days after inoculation.

[0077] The obtained pFGCTm-2 2 -Mut2 was co-expressed with plasmids expressing TMV MP, ToMV MP, and ToMMV MP, respectively.

[0078] Phenotypic observations showed that Tm-2 2Can react with TMV MP to induce cell necrosis, but not with ToBRFV MP; Tm-2 2 -Mut2 not only reacts with TMV MP, but also reacts with ToBRFV MP to induce cell necrosis ( Figure 3 A). In addition, the co-expression experiment results showed that Tm-2 2 -Mut2 can also react with ToMV and ToMMV MP to induce cell necrosis ( Figure 3 B). The above results show that Tm-2 2 -Mut2 can mediate resistance to ToBRFV while retaining resistance to TMV, ToMV and ToMMV.

[0079] 3. Tm-2 2 - Mut2 mutant plasmid and ToBRFV co-expression test:

[0080] The obtained pFGCTm-2 2 -Mut2, empty vector pFGC5941 and infectious clone pCBToBRFV were transformed into competent Agrobacterium GV3101. 2 OD of Agrobacterium tumefaciens containing -Mut2, Agrobacterium tumefaciens containing pFGC5941, and Agrobacterium tumefaciens containing pCBToBRFV 600 Adjust to 0.5, then carry pFGCTm-2 2 Agrobacterium carrying -Mut2 and Agrobacterium carrying pFGC5941 were mixed with Agrobacterium carrying pCBToBRFV at a volume ratio of 1:1. The mixed Agrobacterium was injected into Nicotiana benthamiana leaves. Three days after inoculation, the total protein of the leaves in the inoculated area was extracted. The accumulation of coat protein (CP) was detected by Western blot.

[0081] Using the same method as above, pFGCTm-2 2 -Mut2 and empty vector pFGC5941 were co-expressed with infectious clones of TMV, ToMV, and ToMMV, respectively. The construction of TMV infectious clone was referenced to Yan ZY, MaHY, WangL, 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 har bouring the Tm-2 2resistance gene.Molecular Plant Pathology.2021,11,(22):1347–1357; The construction of ToMV infectious clone was carried out by amplifying the full length of ToMV using primers PCB301-ToMV-F: TTTCATTTGGAGAGGGTATT TATTATTACAACAATTACC (SEQ ID NO.9), PCB301-ToMV-R: GATATAAGTACAG ACTGGGCCCCTACCGGGGGTTCCGG (SEQ ID NO.10), and cloning it into the binary expression vector pCB301 between the 35S promoter and the NOS terminator; The construction of ToMMV infectious clone was based on the reference Tettey Carlos, Yan, ZY, Ma, HY, Zhao MS, Geng C, Tian YP, Li XD. Tomato mottlemosaicvirus: 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 by Western blot.

[0082] The results are as follows Figure 4 As shown, the results showed that compared with the control (empty vector pFGC5941), Tm-2 2 -Mut2 can significantly reduce the accumulation of ToBRFV capsid protein (CP). In addition, the co-expression experiment results showed that Tm-2 2 -Mut2 also significantly reduced the accumulation of TMV, ToMV, and ToMMV CPs.

[0083] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A tomato Tm-2 2 A disease resistance protein mutant, characterized in that Tomato Tm-2 2 The amino acid sequence of the disease-resistant protein mutant is shown in SEQ ID NO.

2.

2. The tomato Tm-2 according to claim 1 2 Gene encoding disease resistance protein mutant.

3. A recombinant expression vector or engineered bacteria comprising the coding gene according to claim 2.

4. The tomato Tm-2 according to claim 1 2 Application of disease resistance protein mutants in the following (1) or (2): (1) Prevent and control tomato brown wrinkled fruit virus disease; (2) preparing products for preventing and treating tomato brown wrinkled fruit virus disease; The plant is tomato or tobacco.

5. The tomato Tm-2 according to claim 2 2 Use of the gene encoding the disease-resistant protein mutant or the recombinant expression vector or engineered bacteria described in claim 3 in the following (1) or (2): (1) Prevent and control tomato brown wrinkled fruit virus disease; (2) Cultivating plant varieties resistant to tomato brown fruit virus; The plant is tomato or tobacco.

6. A method for improving the resistance of tomato plants to ToBRFV, characterized in that: The following steps are involved: Tomato Tm-2 2 The nucleotide at position 1062 in the coding region sequence of the gene is mutated from A to G, thereby encoding the amino acid sequence shown in SEQ ID NO.2; Alternatively, the tomato Tm-2 of claim 2 is exogenously introduced into the tomato plant. 2 Gene encoding disease resistance protein mutant.

7. A method for breeding a tomato variety resistant to ToBRFV, characterized in that: The following steps are involved: (1) The tomato Tm-2 2 Tomato plants with a mutant gene encoding a disease resistance protein are self-pollinated or crossed with another tomato plant to obtain the next generation population; (2) Selecting from the next generation population the tomato Tm-2 containing the tomato Tm-2 described in claim 2 2 Tomato plants containing a disease resistance protein mutant encoding gene, when the tomato Tm-2 2 When the gene encoding the disease resistance protein mutant is homozygous, the tomato plant is resistant to ToBRFV.

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