Disease-resistant gene mutants and their application in controlling tomato brown fruit virus disease

Abstract

The present invention discloses a disease-resistant gene mutant and its application in preventing and treating tomato brown wrinkled fruit virus disease, belonging to the field of biotechnology. 2 In the coding region sequence of the gene, the nucleotide at position 138 mutates from A to G, the nucleotide at position 418 mutates from T to C, and the nucleotide at position 1425 mutates from A to G; or 2 The 140th amino acid of the gene-encoded protein mutates from cysteine ​​to arginine, which can significantly reduce the accumulation of ToBRFV capsid protein (CP). It can serve as a new ToBRFV resistance gene and is of great significance for the prevention and control of ToBRFV.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a disease-resistant gene mutant and application thereof in preventing and treating tomato brown wrinkled fruit virus disease. Background Art

[0002] Tomato brown rugose fruit virus (ToBRFV) is a newly emerging virus belonging to the genus Tobacco mosaic virus. It has become the most serious virus that harms tomatoes. The virus causes brown or yellow spots on tomato fruits, completely depriving the fruits of their commercial value. ToBRFV can be transmitted through contact and bear hives (Bombus terrestris), as well as through seeds. In 2014, ToBRFV first appeared in Israel and has occurred in 33 countries to date. In order to prevent the spread of the virus, many countries have listed it as a quarantine object. On April 9, 2021, my country added it to the "List of Quarantine Pests for Import of Plants of the People's Republic of China".

[0003] Planting disease-resistant varieties is the most economical and effective means of preventing and controlling viral diseases. 2 The genes are the three disease resistance genes that have been identified for tomato mosaic virus disease. 2 The gene is located near the centromere of the long arm of tomato chromosome 9 and is an allele of the Tm-2 gene and the tm-2 gene. 2 The gene encodes a coiled-coil domain / nucleotide binding-ARC domain / leucine-rich repeat domain (CC-NB-ARC-LRR) resistance protein. This protein can recognize the movement protein (MP) of tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV) of the tobacco mosaic virus genus, thereby stimulating the host's immune response. However, ToBRFV can infect Tm-2 2 To date, no resistance genes against ToBRFV have been found in tomato varieties with the same gene. Summary of the invention

[0004] In view of the above-mentioned prior art, the object of the present invention is to provide a disease-resistant gene mutant and its application in preventing and controlling tomato brown wrinkled fruit virus disease.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a disease-resistant gene mutant, wherein the disease-resistant gene mutant is a Tm-2 2 The nucleotide sequence of the gene coding region undergoes at least one of the following nucleotide mutations (1)-(3):

[0007] (1) A138 G;

[0008] (2) T418C;

[0009] (3)A1425 G.

[0010] Preferably, the nucleotide sequence of the disease-resistant gene mutant is as shown in SEQ ID NO.1.

[0011] The second aspect of the present invention provides a protein encoded by the above-mentioned disease-resistant gene mutant.

[0012] Preferably, the amino acid sequence of the protein is shown as SEQ ID NO.2.

[0013] The third aspect of the present invention provides a recombinant expression vector or an engineered bacterium comprising the above-mentioned disease-resistant gene mutant.

[0014] The fourth aspect of the present invention provides the use of the above-mentioned disease resistance gene mutant in the following (1) or (2):

[0015] (1) Prevent and control tomato brown wrinkled fruit virus disease;

[0016] (2) Cultivate tomato varieties resistant to tomato brown fruit virus disease.

[0017] The fifth aspect of the present invention provides the use of the protein encoded by the above disease resistance gene mutant in the following (1) or (2):

[0018] (1) Prevent and control tomato brown wrinkled fruit virus disease;

[0019] (2) Prepare products for preventing and treating tomato brown wrinkled fruit virus disease.

[0020] The sixth aspect of the present invention provides the use of a recombinant expression vector, a transgenic cell line or an engineered bacterium comprising the above-mentioned disease-resistant gene mutant in the following (1) or (2):

[0021] (1) Prevent and control tomato brown wrinkled fruit virus disease;

[0022] (2) Cultivate tomato varieties resistant to tomato brown fruit virus disease.

[0023] A seventh aspect of the present invention provides a method for breeding a tomato variety resistant to tomato brown wrinkled fruit virus disease, comprising the following steps:

[0024] Tomato Tm-2 2In the coding region sequence of the gene, the nucleotide at position 138 mutated from A to G, the nucleotide at position 418 mutated from T to C, and the nucleotide at position 1425 mutated from A to G.

[0025] Beneficial effects of the present invention:

[0026] The present invention first discovered that tomato Tm-2 2 In the coding region sequence of the gene, the nucleotide at position 138 mutates from A to G, the nucleotide at position 418 mutates from T to C, and the nucleotide at position 1425 mutates from A to G; or, 2 The 140th amino acid of the gene-encoded protein mutates from cysteine ​​to arginine, which can significantly reduce the accumulation of ToBRFV capsid protein (CP). It can serve as a new ToBRFV resistance gene and is of great significance for the prevention and control of ToBRFV. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 :Tm-2 2 With Tm-2 2 -Mut1 nucleotide sequence alignment.

[0028] Figure 2 :Tm-2 2 With Tm-2 2 -Mut1 amino acid sequence alignment.

[0029] Figure 3 :Tm-2 2 -Mut1 reacts with ToBRFV MP as well as TMV, ToMV and ToMMV MP to induce cell necrosis.

[0030] Figure 4 :Tm-2 2 -Mut1 significantly reduced the accumulation of ToBRFV as well as TMV, ToMV, and ToMMV. DETAILED DESCRIPTION

[0031] 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.

[0032] As mentioned above, Tm-2 2The gene is a disease-resistant gene that can effectively resist infection by tobacco mosaic virus TMV, ToMV and tomato mottle mosaic virus (ToMMV), but not ToBRFV. Since no ToBRFV resistance gene that can be used for breeding has been reported, it is urgent to find new resistance resources for the prevention and control of ToBRFV. 2 Artificial evolution was performed to screen for the disease-resistant gene Tm-2 with a broader disease resistance spectrum among a large number of mutants. 2 Mutants are an effective way to obtain new disease-resistant materials.

[0033] Based on this, the present invention constructs Tm-2 by error-prone PCR 2 The mutant library was then constructed into the Tm-2 2 The mutant plasmid was co-expressed with the pCamToBRFV-MP plasmid expressing ToBRFV MP, and the mutant plasmid that caused cell necrosis was screened and named pFGCTm-2 2 -Mut1. Mutant plasmid pFGCTm-2 2 -Mut1 was sequenced, Tm-2 2 -The nucleotide sequence of the coding region of Mut1 is shown in SEQ ID NO.1, and is as follows:

[0034]

[0035] The encoded amino acid sequence is shown in SEQ ID NO.2 as follows:

[0036] MAEILLTSVINKSVEIAGNLLIQEGKRLYWLKEDIDWLQREMRHIRSYVDNAKAKEAGGD

[0037] SRVKNLLKDIQELAGDVEDLLDDFLPKIQQSNKFNYCLKRSSFADEFAMEIEKIKRRVVD

[0038] IDRIRKTYNIIDTDNNNDDRVLLDRRRLFLHADETEIIGLDDDFNMLQAKLLNQDLHYGV

[0039] VSIVGMPGLGKTTLAKKLYRLIRDQFECSGLVYVSQQPRASEILLDIAKQIGLTEQKMKE

[0040] NLEDNLRSLLKIKRYVILLDDIWDVEIWDDLKLVLPECDSKVGSRMIITSRNSNVGRYIG

[0041] GESSLHALQPLESEKSFELFTKKIFNFDDNNSWANASPDLVNIGRNIVGRCGGIPLAIVV

[0042] TAGMLRARERTEHAWNRVLESMGHKVQDGCAKVLALSYNDLPIASRPCFLYFGLYPEDHE

[0043] IRAFDLINMWIAEKFIVVNSGNRREAEDLAEDVLNDLVSRNLIQLAKRTYNGRISSCRIH

[0044] DLLHSLCVDLAKESNFFHTAHDAFGDPGNVARLRRITFYSDNVMIEFFRSNPKLEKLRVL

[0045] FCFAKDPSIFSHMAYFDFKLLHTLVVVMSQSFQAYVTIPSKFGNMTCLRYLRLEGNICGK

[0046] LPNSIVKLTRLETIDIDRRSLIQPPSGVWESKHLRHLCYRDYGQACNSCFSISSFYPNIY

[0047] SLHPNNLQTLMWIPDKFFEPRLLHRLINLRKLGILGVSNSTVKMLSIFSPVLKALEVLKL

[0048] SFSSDPSEQIKLSSYPHIAKLHLNVNRTMALNSQSFPPPNLIKLTLAYFSVDRYILAVLKT

[0049] FPKLRKLKMFICKYNEEKMDLSGEANGYSFPQLEVLHIHSPNGLSEVTCTDDVSMPKLKKLLLTGFHCRISLSERLKKLSK*

[0050] Tm-2 with NCBI accession number: AF536201.1 2 By comparing the gene sequences, it was found that Tm-2 2 -Mut1 had three nucleotide mutations in its coding region, namely A138 G, T418C, and A1425 G, and the corresponding amino acid mutations were R46R, C140R, and S475S.

[0051] The mutant plasmid pFGCTm-2 2 -Mut1 was co-expressed with ToBRFV and Tm-2 was found 2 -Mut1 can significantly reduce the accumulation of ToBRFV capsid protein (CP) without affecting the accumulation of TMV, ToMV and ToMMV CP. 2 Gene, Tm-2 2 -Mut1 has achieved unexpected technical effects in resistance to ToBRFV and can be used as a new ToBRFV resistance gene, thus the present invention was proposed.

[0052] 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.

[0053] 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. 2The 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.

[0054] Example 1: Tm-2 2 Construction of gene mutant library

[0055] Construction of Tm-2 by error-prone PCR 2 The gene mutant library is as follows:

[0056] First, a 50 μL reaction system was prepared, which included: 15 μL buffer, 5 μL of 2.5 mM dNTPs for error-prone, 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, and 30 ng / μL template (wild-type plasmid pFGCTm-2 2 )0.5μL, double distilled water 24.7μL.

[0057] 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, and a total of 1200 Tm-2 2 Mutant plasmids.

[0058] Example 2: Screening for Tm-2 that produces allergic cell necrosis in response to ToBRFV MP 2 Mutants

[0059] 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 2 resistance gene.Mol.Plant Pathol.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 (accession number MT018320 in NCBI). Its nucleotide sequence is shown in SEQ ID NO.3; the amino acid sequence is shown in SEQ ID NO.4. The details are as follows:

[0060] MP Nucleotide Sequence:

[0061] ATGGCTCTTGTTAAGGGTAAAGTTAATATTAATGAGTTCATAGACTTGTCAAAATCAGAAAAATTTCTTCCGTCTATGTTCACACCTGTTAAGAGTGTCATGATCTCCAAGGTTGATAAGATATTGGTTCATGAAGATGAATCTTTGTCCGAAGTCAATTTACTTAAAGGTGTAAAACTCATTGATGGTGGCTATGTACATCTTGCTGGTCTTGTGGTGACAGGTGAATGGAATTTGCCAGATAATTGTCGTGGTGGTGTCAGTGTCTGTTTGGTCGATAAGAGAATGGAGAGAGCGGACGAGGCAACTCTTGCTTCATACTATACCGCAGCGGCTAAGAAAAGGTTTCAGTTCAAAGTCGTTCCAAATTACAACATCACTACCAAGGACGCAGAAAAGGCAGTTTGGCAAGTACTAGTTAATATTAGAAATGTTAAAATTGCTGCGGGTTACTGTCCGCTGTCATTAGAATTTGTGTCAGTGTGTATTGTTTATAAAAATATTATAAAACTCGGTTTGAGAGAGAAAATTACGAGCGTCACGGATGGAGGGCCCATGGAACTATCAGAAGAAGTTGTTGATGAGTTCATGGAAGAAGTCCCGATGTCTGTAAGGCTTGCAAAATTTCGTTCGAAGACCGGAAAAAAGTTTAGTAGTAAAAGTGAGAATAATAGTGGTAATAATAGGCCGAAACCAAACAAAAACCAAAGGAAGGAAAAGGGTTTAAAAGTTAGGGTTGAGAAGGATAATTTAATTGATAATGAATTGGAGACTTACGTCGCCGATTCAGATTCGTATTAA。

[0062] MP amino acid sequence

[0063] MALVKGKVNINEFIDLSKSEKFLPSMFTPVKSVMISKVDKILVHEDESLSEVNLLKGVKLIDGGYVHLAGLVVTGEWNLPDNCRGGVSVCLVDKRMERADEATLASYYTAAAKKRFQFKVVPNYNITTKDAEK AVWQVLVNIRNVKIAAGYCPLSLEFVSVCIVYKNIIKLGLREKITSVTDGGPMELSEEVVDEFMEEVPMSVRLAKFRSKTGKKFSSKSENNSGNNRPKPNKNQRKEKGLKVRVEKDNLIDNELETYVADSDSY*

[0064] The 1200 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.

[0065] The results showed that the 1200 Tm-2 2 There is only one mutant Tm-2 in the mutant plasmid 2 -Mut1 can react with ToBRFV MP to induce cell necrosis. The mutant plasmid pFGCTm-2 that induces cell necrosis 2 -Mut1 was sequenced. The sequencing results showed that pFGCTm-2 2 -Mut1 had three nucleotide mutations, namely A138 G, T418C, A1425G ( Figure 1 ), the corresponding amino acid mutations are R46R, C140R, S475S ( Figure 2 ), where A138G and A1425G are synonymous mutations, and T418C is a non-synonymous mutation.

[0066] Example 3: Tm-2 2 Study on resistance of gene mutants to ToBRFV

[0067] 1. Tm-2 2 - Construction of Mut1 mutant plasmid:

[0068] The following primers were used to amplify Tm-2 by directed mutagenesis. 2Amplified fragments of nucleotide mutations at positions 138, 418, and 1425 of the gene.

[0069] Primer pair 1-Tm-2 2 -A138G-F:CACATTCGGTCTTATGTTGACAACGCAAAGG; (SEQ ID NO.5)

[0070] Primer pair 1-Tm-2 2 -A138G-R:ATAAGACCGAATGTGTCTCATTTCTCTCTGGAG. (SEQ ID NO.6)

[0071] Primer pair 2-Tm-2 2 -T418C-F:GATGATCGTGTTCTGCTGGATCGGAGAAGATTAT; (SEQ IDNO.7)

[0072] Primer pair 2-Tm-2 2 -T418C-R:CAGAACACGATCATCATTATTGTTATCTGTATCTATG ATG. (SEQ ID NO.8)

[0073] Primer pair 3-Tm-2 2 -A1425G-F:AGAATTTCGAGTTGTCGCATACATGACTTGTTAC;

[0074] (SEQ ID NO.9)

[0075] Primer pair 3-Tm-2 2 -A1425G-R:ACAACTCGAAATTCTTCCATTATGTCCTTTTTGG. (SEQ IDNO.10)

[0076] 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.

[0077] Then, the mutation PCR reaction was carried out, and the reaction procedure was as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 2 min 35 s, 15 cycles, 48°C annealing for 30 s, and 72°C post-extension for 5 min. The amplified product was transferred into Escherichia coli DH5α competent cells, plasmids were extracted from them, plasmids with the correct plasmid size were screened, and single-site mutation plasmids were constructed.

[0078] The single-site mutation plasmid was used as a template for mutation PCR to obtain a double-site mutation plasmid, and then the double-site mutation plasmid was subjected to mutation PCR to obtain a triple-site mutation plasmid (Tm-2 2 -Mut1 mutant plasmid).

[0079] 2. Tm-2 2 -Mut1 mutant plasmid and ToBRFV MP co-expression test:

[0080] pFGCTm-2 2 and pFGCTm-2 2 -Mut1 was co-expressed with TMV and ToBRFV MP. 2 Mutant plasmid (or pFGCTm-2 2 OD of Agrobacterium tumefaciens with pCamToBRFV-MP (or pCamTMV-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.

[0081] The obtained pFGCTm-2 2 -Mut1 was co-expressed with plasmids expressing ToMV MP and ToMMV MP, respectively.

[0082] Phenotypic observations showed that Tm-2 2 Can react with TMV MP to induce cell necrosis, but not with ToBRFV MP; Tm-2 2 -Mut1 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 -Mut1 can also react with ToMV and ToMMV MP to induce cell necrosis ( Figure 3 B)

[0083] 3. Tm-2 2 -Mut1 mutant plasmid and ToBRFV co-expression test:

[0084] The obtained pFGCTm-2 2 -Mut1, empty vector pFGC5941 and infectious clone pCBToBRFV were transformed into competent Agrobacterium GV3101. 2 OD of Agrobacterium tumefaciens containing -Mut1, pFGC5941 and pCBToBRFV 600 Adjust to 0.5, then carry pFGCTm-2 2 Agrobacterium carrying -Mut1 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.

[0085] Using the same method as above, pFGCTm-2 2 -Mut1 and empty vector pFGC5941 were co-expressed with the infectious clones of TMV, ToMV, and ToMMV, respectively, and the accumulation of coat protein (CP) was detected by Western blot.

[0086] The results are as follows Figure 4 As shown, the results showed that compared with the control (empty vector pFGC5941), Tm-2 2 -Mut1 can significantly reduce the accumulation of ToBRFV capsid protein (CP). In addition, the co-expression experiment results showed that Tm-2 2 -Mut1 also significantly reduced the accumulation of TMV, ToMV, and ToMMV CPs.

[0087] 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 disease-resistant gene mutant, characterized in that: The nucleotide sequence of the disease-resistant gene mutant is shown in SEQ ID NO.

1.

2. The protein encoded by the 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.

3. A recombinant expression vector or engineered bacteria comprising the disease-resistant gene mutant according to claim 1.

4. Use of the disease-resistant gene mutant according to claim 1 in the following (1) or (2): (1) Prevent and control tomato brown wrinkled fruit virus disease; (2) Cultivate tomato varieties resistant to tomato brown fruit virus; The plant is tomato or tobacco.

5. Use of the protein according to claim 2 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.

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

7. A method for breeding a tomato variety resistant to tomato brown fruit virus disease, characterized in that: The following steps are involved: Tomato Tm-2 2 In the coding region sequence of the gene, the nucleotide at position 138 mutated from A to G, the nucleotide at position 418 mutated from T to C, and the nucleotide at position 1425 mutated from A to G, thereby encoding the amino acid sequence shown in SEQ ID NO.2.

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