Application of tbr2 protein and its encoding gene and molecular marker in resistance to tomato brown rugose fruit virus disease

Abstract

The application discloses TBR2 protein and an application of an encoding gene and a molecular marker thereof in resisting tomato brown rugose fruit virus disease, and belongs to the technical field of genetic engineering. TBR2 A new disease-resistant gene of the tomato brown rugose fruit virus disease is identified on the 4th chromosome of the tomato TBR2 ; the disease-resistant gene has stable disease resistance; backcrossing and transformation of the disease-resistant gene into a sensitive cultivar can obviously improve the resistance of the cultivar to the tomato brown rugose fruit virus disease. Based on the disease-resistant gene TBR2 , the application also develops molecular markers respectively related to the resistance of the tomato brown rugose fruit virus disease; the molecular markers of the application can be used for identifying plant materials resistant to the tomato brown rugose fruit virus disease and cultivating plant varieties resistant to the tomato brown rugose fruit virus disease, and have important significance for accelerating the breeding process of the disease-resistant tomato.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to the application of a TBR2 protein and its encoding gene, and molecular markers in resistance to tomato brown wrinkle virus disease. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) is a newly discovered plant virus belonging to the family Cladoviridae and the genus Tobacco Mosaic Virus. ToBRFV primarily infects tomatoes (…). Solanum lycopersicon ),chili( Capsicum annuum ) and Ben's tobacco ( Nicotiana benthamiana ), sticky tobacco ( Nicotiana glutinosa ),tobacco( Nicotiana tabacum In severe cases, economic losses can reach 100%.

[0003] For a long time, Tm-1, Tm-2, Tm-2 2 Disease-resistant genes are the core resistance resources for tomatoes against Tobacco Mosaic Virus (TMV) viruses, among which... Tm-2 2 The gene encodes NLR-type immune receptors, which specifically recognize viral motility proteins (MPs) and activate immune responses. It is widely used in disease resistance breeding and exhibits stable resistance.

[0004] However, ToBRFV, through sequence variations and adaptive mutations in its motor protein (MP), can overcome all known tomato antiviral gene-mediated resistance and systematically infect viruses carrying Tm-2. 2 The presence of broad-spectrum resistance genes in tomato varieties has rendered traditional disease-resistant materials completely ineffective. Existing commercially available disease-resistant tomato varieties generally lack effective resistance to ToBRFV, and there is a lack of safe, stable, and efficient antiviral solutions in production. Therefore, breeding and creating new tomato materials resistant to ToBRFV has become an urgent problem to be solved in current tomato disease-resistant breeding. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of TBR2 protein and its encoding gene and molecular markers in resistance to tomato brown wrinkle virus disease.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of TBR2 protein in resistance to tomato brown wrinkle virus disease, said TBR2 protein being the protein shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.1; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0007] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0008] In the above applications, plant resistance to tomato brown wrinkled fruit virus can be improved by overexpressing TBR2 protein in plants or by applying TBR2 protein exogenously. The concentration of exogenous TBR2 protein can be adjusted according to factors such as plant species and disease severity.

[0009] A second aspect of the invention provides a gene encoding the TBR2 protein ( TBR2 The application of the TBR2 protein encoding gene in resistance to tomato brown wrinkle virus disease, wherein the gene encoding the TBR2 protein is a nucleic acid molecule as shown in (i), (ii), or (iii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.3; (iii) Nucleic acid molecules other than (i) or (ii) that encode the amino acid sequence shown in SEQ ID NO.1.

[0010] In the above applications, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA or mRNA.

[0011] In the above applications, the resistance of plants to tomato brown wrinkle fruit virus disease was improved by overexpressing the gene encoding the TBR2 protein in plants.

[0012] In some preferred embodiments of the present invention, overexpression of the gene encoding the TBR2 protein in plants is achieved by the following substances: C1) Expression cassette containing the gene encoding the TBR2 protein; C2) A recombinant vector containing the gene encoding the TBR2 protein, or a recombinant vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing the gene encoding the TBR2 protein, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) A transgenic plant cell line containing the gene encoding the TBR2 protein, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing the gene encoding the TBR2 protein, or transgenic plant tissue containing the expression cassette described in C1); C6) Transgenic plant organs containing the gene encoding the TBR2 protein, or transgenic plant organs containing the expression cassette described in C1).

[0013] In the above applications, the plant is selected from one or more of tomatoes, peppers, tobacco, or tung oil.

[0014] A third aspect of the present invention provides a molecular marker associated with resistance to tomato brown wrinkle fruit virus disease, said molecular marker comprising: InDel- TBR2- 1 and InDel- TBR2- 2; The InDel- TBR2- The nucleotide sequence of 1 is shown in SEQ ID NO.4, and the InDel- TBR2- The nucleotide sequence of 2 is shown in SEQ ID NO.5.

[0015] Molecular marker InDel- TBR2- 1 is a 175bp disease resistance-specific fragment, which differs in length by 39bp from the disease susceptibility-specific fragment (SEQ ID NO. 6). Molecular marker InDel- TBR2- 2 is a 500bp disease resistance-specific fragment, which differs from the disease susceptibility-specific fragment (SEQ ID NO.7) by 295bp in length.

[0016] The molecular markers of this invention are significantly associated with resistance to tomato brown wrinkled fruit virus (TBR). Using the molecular markers of this invention, the encoding gene of the TBR2 protein and plant materials resistant to TBR can be identified, which is of great significance for breeding resistance to TBR.

[0017] In a fourth aspect, the present invention provides the application of the above-mentioned molecular markers in resistance to tomato brown wrinkle virus disease.

[0018] In the above applications, primer pairs for detecting the molecular markers are used to identify plant materials resistant to tomato brown wrinkled fruit virus disease, or to cultivate plant varieties resistant to tomato brown wrinkled fruit virus disease.

[0019] In some preferred embodiments of the present invention, the primer pair for detecting the molecular marker includes: detecting InDel- TBR2- Primer pair A and detection InDel- 1 TBR2- Primer pair B for 2; The nucleotide sequences of primer pair A are shown in SEQ ID NO.8 and SEQ ID NO.9; the nucleotide sequences of primer pair B are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0020] The beneficial effects of this invention are: This invention identified a novel resistance gene against tomato brown wrinkle fruit virus disease on chromosome 4 of tomato. TBR2 It has stable disease resistance. Backcrossing and transferring this disease-resistant gene into sensitive cultivars can significantly improve the resistance of the variety to tomato brown wrinkled fruit virus.

[0021] Based on disease resistance genes TBR2 This invention also developed molecular markers related to resistance to tomato brown wrinkled fruit virus disease. Using these molecular markers, plant materials resistant to tomato brown wrinkled fruit virus disease can be identified, and plant varieties resistant to tomato brown wrinkled fruit virus disease can be bred, which is of great significance for accelerating the breeding process of disease-resistant tomatoes. Attached Figure Description

[0022] Figure 1 Transgenic tomato strains TBR2 Gene expression level detection results.

[0023] Figure 2 Overexpression TBR2 Fruits of the ToBRFV-infected transgenic tomato line (TBR2-OE) and background material.

[0024] Figure 3 Overexpression TBR2 Results of viral load determination in fruits of the tomato transgenic line (TBR2-OE) and background materials after ToBRFV infection.

[0025] Figure 4 The molecular marker InDel- of this invention TBR2- 1 and InDel- TBR2- 2. Electrophoretic patterns of PCR amplification products in BC5F1:BR1002×TB0249; M is the marker, with the size of the marker bands from bottom to top being 100 bp, 250 bp, 500 bp, and 750 bp; A is the amplification band pattern of resistance to tomato brown wrinkle fruit virus, with product size of 175 bp or 500 bp alone; B is the amplification band pattern of susceptibility to tomato brown wrinkle fruit virus, with product size of 136 bp or 795 bp alone; H is the heterozygous genotype.

[0026] Figure 5Leaf phenotypes of plants identified by molecular markers as resistant to tomato brown fruit wrinkle virus (TBV) and plants identified by molecular markers as susceptible to TBV after inoculation with ToBRFV; In the figure, A represents plants identified by molecular markers as resistant to TBV; B represents plants identified by molecular markers as susceptible to TBV.

[0027] Figure 6 The results of ToBRFV inoculation of plants identified by molecular markers as resistant to Tomato Brown Fruit Curl Virus (TBV) and plants identified by molecular markers as susceptible to TBV show the viral load in their leaves. In the figure, A represents plants identified by molecular markers as resistant to TBV, and B represents plants identified by molecular markers as susceptible to TBV. Detailed Implementation

[0028] 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.

[0029] As mentioned earlier, ToBRFV can overcome the aforementioned Tm-1, Tm-2, and Tm-2. 2 Resistance mediated by disease-resistant genes can systemically infect Tm-2 carriers. 2 Tomato varieties with broad-spectrum resistance genes have rendered traditional disease-resistant materials completely ineffective. Existing commercially available disease-resistant tomato varieties generally lack effective resistance to ToBRFV, and there is a lack of safe, stable, and efficient antiviral solutions in production.

[0030] The putative late blight resistance protein homolog R1B-14 (XP_015072398.1) is a putative late blight resistance protein homolog, and its function has not yet been reported. In this invention, R1B-14 is named the TBR2 protein, and its amino acid sequence is shown in SEQ ID NO.1. TBR2 The nucleotide sequence of the gene is shown in SEQ ID NO.2, and the nucleotide sequence of the CDS region is shown in SEQ ID NO.3.

[0031] To study the function of the TBR2 protein, this invention constructs an overexpression... TBR2 Research on the resistance of transgenic tomato lines to tomato brown wrinkle virus disease revealed that overexpression of the gene in tomatoes... TBR2 This gene can effectively improve the resistance of tomato plants to ToBRFV. Therefore, TBR2 This gene could serve as a novel ToBRFV resistance gene.

[0032] 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.

[0033] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: Tomato material TB0249, described in the literature "A biotechnology-based male-sterility system for hybrid seed production in tomato" (Du M. et al., The Plant Journal, 2020), is susceptible to ToBRFV. Tomato material BR1002 is an existing introgression line for tomato; it is based on cultivated tomato (… Solanum lycopersicum Using ) as the genetic background, wild tomatoes () were backcrossed to introduce ) Solanum pennellii The disease resistance gene was obtained by introducing a gene fragment into the tomato material BR1002. TBR2 The material exhibits resistance to ToBRFV. The public can obtain the relevant biological materials from the applicant; however, these materials can only be used for verifying the patented technology and cannot be used for other purposes.

[0034] Example 1: Overexpression TBR2 Construction of transgenic tomato lines 1. Construction of the recombinant expression vector pK7FWG2-TBR2: Will TBR2 The CDS sequence (SEQ ID NO.3) of the gene was ligated into the pK7FWG2 vector after double digestion with Xho I and EcoRI to construct the recombinant expression vector pK7FWG2-TBR2.

[0035] 2. Preparation of recombinant positive Agrobacterium bacterial suspension: The successfully constructed recombinant expression vector pK7FWG2-TBR2 was transformed into Agrobacterium LBA4404 to obtain recombinant positive Agrobacterium.

[0036] Recombinant positive Agrobacterium was inoculated onto YEB solid medium (containing 50 μg / ml Rif and 50 μg / ml Kan) and cultured at 28°C for 2 days. Single colonies were picked and cultured in 5 ml of the same YEB liquid medium (containing 50 μg / ml Rif and 50 μg / ml Kan) at 28°C and 200 rpm in the dark for 1.5 days. 500 μl of bacterial culture was added to 50 ml of YEB liquid medium and cultured until OD reached the target value.600 =1.8~2.0; centrifuge at 4000 rpm for 10 min, resuspend in YEB; centrifuge again for 8 min, resuspend in MS salt medium (100 ml MS salt medium contains 0.433 g MSSalts Powder and 3 g sucrose) to the bacterial concentration OD. 600 = 1.5.

[0037] 3. Obtaining transgenic plants: Plump seeds of the TB0249 strain, a susceptible material in the laboratory, were selected and sterilized in a clean bench: soaked in 75% alcohol for 2 min and rinsed 3 times with sterile water; soaked in saturated sodium phosphate for 20 min and rinsed 3 times with sterile water; soaked in 1% sodium hypochlorite for 10 min and rinsed 7 times with sterile water; and soaked in sterile water for 4–6 h. The seeds were then sown on 1 / 2 MS medium and germinated in a light incubator (cotyledons fully expanded in 6–8 days). Cotyledons were cut off, soaked in MS liquid medium (containing 0.2 mg / L 2,4-D and 0.1 mg / L KT) for 1 h, blotted dry with sterile filter paper, and pre-cultured in A1 solid medium (MS + 1 mg / L IAA + 1.75 mg / L ZT) for 1 day; The pre-cultured cotyledons were immersed in recombinant positive Agrobacterium tumefaciens bacterial suspension for 15 min, blotted dry with sterile filter paper, and returned to A1 solid medium for co-culture for 2 days (conditions: 26℃, 16 h light / 8 h dark, 600 μmol / L). -2 s -1 illumination); Transfer to A2 resistant medium (MS + 1.0 mg / L IAA + 1.75 mg / L ZT + 75 mg / L Kan + 200 mg / L Tim) and culture under the same conditions. Change the medium every 3 weeks until callus formation, then transfer to A3 medium (MS + 1.0 mg / L IAA + 1.75 mg / L ZT + 50 mg / L Kan + 200 mg / L Tim) to induce shoot growth. Cut off the growing points and transfer to A4 medium (MS + 50 mg / L Kan + 200 mg / L Tim) for rooting. Screening for T0 generation positive seedlings: After DNA testing showed positive results, seedlings were transplanted into soil and harvested as usual. T1 generation seedlings were planted; individual seeds were disinfected and sown on 1 / 2 MS medium containing 50 mg / L kanamycin. If all T1 generation seeds were resistant, they were homozygous and used for subsequent experiments.

[0038] After resistance screening and molecular identification, the T2 generation overexpression was finally obtained. TBR2 The expression level of the gene in the tomato transgenic line (TBR2-OE) was detected by quantitative real-time PCR. The results showed that the TBR2-OE transgenic line was significantly different from the control TB0249. TBR2 Gene expression levels were significantly increased. Figure 1 ).

[0039] Example 2: Overexpression TBR2 Resistance of transgenic tomato lines to tomato brown wrinkle virus disease. 1. Test method: ToBRFV (MT018320.1) was inoculated into the tomato transgenic line (TBR2-OE) constructed in the control TB0249 and Example 1. The infectious Agrobacterium was inoculated into one true leaf, with 0.1 ml injected into each plant. After three months, the disease status of the fruit was investigated, and the viral load of ToBRFV was measured.

[0040] 2. Test Results: Photos of tomato fruits after ToBRFV inoculation are shown below. Figure 2 As shown, the results indicated that the TB0249 tomato fruit was significantly diseased, with brown wrinkled spots on the fruit surface; while the fruit of the TBR2-OE transgenic plant was healthy and disease-free.

[0041] The results of the determination of ToBRFV viral load in the fruit are as follows: Figure 3 As shown, the results indicate that ToBRFV viral load is almost undetectable in TBR2-OE, while the viral load is higher in TB0249.

[0042] In summary, overexpression in tomatoes TBR2 The gene can effectively improve the resistance of tomato plants to ToBRFV.

[0043] Example 3: Development of molecular markers and design of detection primers related to resistance to tomato brown wrinkled fruit virus. 1. Molecular marker development: The genome sequences of tomato materials resistant to and susceptible to tomato brown wrinkle fruit virus (TWRV) were analyzed. Genomic differential sequences were identified through comparison, and samples were screened for those related to TWRV. TBR2 InDel markers that are tightly linked to genes.

[0044] Two molecular markers associated with resistance to tomato brown wrinkle fruit virus were ultimately identified: InDel- TBR2- 1 and InDel- TBR2- 2. Among them: InDel- TBR2- 1 is a 175bp disease-resistant specific fragment (nucleotide sequence as shown in SEQ ID NO.4), which corresponds to a 136bp disease-resistant specific fragment (nucleotide sequence as shown in SEQ ID NO.6) in the susceptible material.

[0045] InDel- TBR2- 2 is a 500bp disease-resistant specific fragment (nucleotide sequence as shown in SEQ ID NO.5), which corresponds to a 795bp disease-resistant specific fragment (nucleotide sequence as shown in SEQ ID NO.7) in the susceptible material.

[0046] 2. Design of detection primers: According to InDel- TBR2- Based on the sequence of 1 and the corresponding disease-specific fragment, detection primer pair A was designed, and its nucleotide sequence is shown below: InDel- TBR2- 1-F: 5'- ACCAGCATAGGCCTCGGATA-3'; (SEQ ID NO. 8) InDel- TBR2- 1-R: 5'-CTGATGCAGCTTCGGTCTCA-3'. (SEQ ID NO.9) According to InDel- TBR2- Based on the sequence of 2 and the corresponding disease-specific fragment, detection primer pair B was designed, and its nucleotide sequence is shown below: InDel- TBR2- 2-F: 5'-TCATTTATGCACAACCTCAAG-3'; (SEQ ID NO. 10) InDel- TBR2- 2-R: 5'-AGTCCTCATACGATAAAGTCA-3'. (SEQ ID NO.11) Example 4: Functional validation of molecular markers associated with resistance to tomato brown wrinkled fruit virus disease In protected areas, tomato variety TB0249 is used as a recurrent parent to carry disease-resistant genes. TBR2 The tomato material BR1002 was backcrossed and transformed into TB0249, and each generation was screened for carrying the molecular markers developed in Example 3. TBR2 The offspring plants of the gene. Through five generations of backcrossing, we obtained BC5:BR1002×TB0249, and after self-crossing, we obtained BC5F1:BR1002×TB0249.

[0047] We used molecular markers to genotype the BC5F1:BR1002×TB0249 population, screening for homozygous BR1002 and TB0249 genotypes. Individual DNA was extracted from the BC5F1:BR1002×TB0249 population. Extraction methods are detailed in the instructions for the All-Purpose Genotyping Plant Genomic DNA Extraction Kit. DNA was extracted from individual plants using two pairs of molecular markers (InDel-...). TBR2- 1 and InDel- TBR2- 2) PCR amplification and agarose gel electrophoresis were performed using the primer pairs to determine the genotype of the tomato sample to be tested, based on the molecular weight of the PCR amplification products. If the tomato sample to be tested utilizes the InDel- TBR2- The PCR amplification product of primer pair A was only a single 175 bp band, which was detected using InDel- TBR2- If the PCR amplification product of primer pair B is only a single 500 bp band, then the tomato to be tested is identified as genotype A; and is determined to be resistant to tomato brown wrinkle virus disease.

[0048] If the tomato sample to be tested utilizes the InDel- TBR2- The PCR amplification product of primer pair A was only a single 136bp band, which was detected using InDel- TBR2- If the PCR amplification product of primer pair B is only a single 795bp band, then the tomato to be tested is identified as genotype B; and it is determined to be susceptible to tomato brown wrinkle virus disease.

[0049] If the PCR amplification product of the tomato sample shows other band patterns, then the tomato sample is a heterozygous genotype (denoted as H).

[0050] The test results of some tomato samples are as follows: Figure 4 As shown. Homozygous A and B genotype plants were selected and inoculated with ToBRFV when they had one true leaf. Ten days after inoculation, the viral load of each plant was measured and the leaves were observed.

[0051] Ten days after inoculation, genotype B plants showed significant virus accumulation, and when transplanted into protected environments, leaf curling and wrinkling were observed. In contrast, genotype A plants showed very little virus accumulation at the growing point of the leaves ten days after inoculation, and the leaves appeared normal. Figure 5 6).

[0052] 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. The application of TBR2 protein overexpression in the resistance of tomato to brown wrinkle virus, characterized in that, The TBR2 protein is as shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.1; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The application according to claim 1, characterized in that, The resistance of plants to tomato brown wrinkle fruit virus can be improved by overexpressing TBR2 protein in plants or by applying TBR2 protein exogenously. The plant in question is a tomato.

3. The application of the gene encoding TBR2 protein overexpression in the resistance of tomato to brown wrinkle virus, characterized in that, The gene encoding the TBR2 protein is a nucleic acid molecule as shown in (i), (ii), or (iii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.3; (iii) Nucleic acid molecules other than (i) or (ii) that encode the amino acid sequence shown in SEQ ID NO.

1.

4. The application according to claim 3, characterized in that, Improve plant resistance to tomato brown wrinkle fruit virus by overexpressing the gene encoding the TBR2 protein in plants; The plant in question is a tomato.

5. The application according to claim 4, characterized in that, Overexpression of the gene encoding the TBR2 protein in plants is achieved through the following substances: C1) Expression cassette containing the gene encoding the TBR2 protein; C2) A recombinant vector containing the gene encoding the TBR2 protein, or a recombinant vector containing the expression cassette described in C1); C3) Recombinant microorganisms containing the gene encoding the TBR2 protein, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) A transgenic plant cell line containing the gene encoding the TBR2 protein, or a transgenic plant cell line containing the expression cassette described in C1); C5) Transgenic plant tissue containing the gene encoding the TBR2 protein, or transgenic plant tissue containing the expression cassette described in C1); C6) Transgenic plant organs containing the gene encoding the TBR2 protein, or transgenic plant organs containing the expression cassette described in C1).

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