A TBT1 protein, resistance gene, molecular marker, and its application for enhancing resistance to ToBRFV.

Abstract

This invention discloses a TBT1 protein, a resistance gene, a molecular marker, and their applications for enhancing resistance to ToBRFV, belonging to the field of plant resistance gene technology. This invention also identified a novel gene on chromosome 11 of tomato that exhibits resistance to ToBRFV. TBT1 Overexpression in tomatoes TBT1 It can significantly enhance resistance to brown wrinkled fruit virus. Compared to disease-resistant genes, the disease-resistant gene of this invention... TBT1 This invention does not exert selective pressure on the virus, effectively preventing viral gene evolution from overcoming resistance and achieving long-term stable disease resistance. It addresses the technical pain points of existing disease resistance genes being prone to loss of resistance and requiring frequent variety updates. Furthermore, this invention has developed two related to disease resistance genes. TBT1 The tightly linked molecular markers of this invention can be used to detect and identify resistance genes for tomato brown wrinkle fruit virus disease. This method can be used in a simple, fast, and high-throughput manner in breeding practice, thus accelerating the process of disease-resistant tomato breeding.

Description

Technical Field

[0001] This invention relates to the field of plant disease resistance gene technology, specifically to a TBT1 protein, a disease resistance gene, a molecular marker, and their applications for improving resistance to ToBRFV. Background Technology

[0002] Tomato brown rugose fruit virus (ToBRFV) belongs to the genus Tobacco Mosaic Virus in the family Tobacco Mosaicviraceae. It infects tomatoes, causing symptoms such as mosaic patterns and wrinkling of leaves, brown spots on fruits, roughness and wrinkling, and reduced marketability. It is characterized by high mechanical transmission efficiency and significant risks of seed-borne and contact transmission, making it one of the most important quarantine pests in global tomato production, posing a persistent threat to both greenhouse and fresh tomato industries. Currently, control measures for ToBRFV are limited. Chemical agents cannot effectively inhibit virus replication and spread, and agricultural control measures can only delay the spread of the virus, failing to fundamentally solve the disease problem. Developing resistant or disease-tolerant tomato varieties has become the most economical and sustainable control strategy.

[0003] Current research on tomato resistance to ToBRFV primarily focuses on resistance genes. However, ToBRFV can penetrate tomato materials carrying traditional resistance genes such as Tm-2², rendering these genes ineffective or providing insufficient protection. Therefore, relying solely on traditional resistance sites is insufficient to meet the needs of ToBRFV control. Newly discovered resistance genes often achieve defense by inhibiting viral replication and eliminating the virus, exerting strong selective pressure on ToBRFV. This can lead to rapid viral evolution and the generation of new variants, thereby overcoming resistance and causing it to quickly become ineffective, making long-term stable control impossible.

[0004] Compared with disease-resistant genes, there is a lack of research on disease-resistant genes that have strong adaptability, stable disease resistance, do not inhibit virus replication, and only alleviate disease symptoms. There are no reports of disease-resistant genes that can be stably applied to breeding tomatoes for resistance to ToBRFV. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a TBT1 protein, resistance gene, molecular marker, and its application for improving resistance to ToBRFV. This invention identifies a marker on tomato chromosome 11 that enhances resistance to Tomato Brown Ruffle Virus. TBT1 Genes, and in TBT1 Two genes related to disease resistance genes were developed upstream and downstream. TBT1 Tightly linked molecular markers can be used easily, quickly, and in high-throughput breeding practices, accelerating the process of resistance breeding in tomatoes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a TBT1 protein that enhances resistance to ToBRFV, said TBT1 protein being any of the proteins shown in (A1)-(A3) 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); (A3) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

[0007] In the aforementioned proteins, the protein tag refers to a polypeptide or protein sequence fused with the target protein through in vitro DNA recombination technology for expression, used to improve the expression effect of the target protein and / or facilitate the detection, tracking, and purification of the target protein. The protein tag includes, but is not limited to, one or more of the following: Flag tag, His tag, MBP tag, HA tag, Myc tag, GST tag, and SUMO tag.

[0008] A second aspect of the present invention provides the use of the above-described TBT1 protein in either (1) or (2) below: (1) Improve the plant's resistance to tomato brown wrinkled fruit virus disease; (2) Prepare drugs for the prevention and control of brown wrinkled fruit virus disease in tomatoes.

[0009] In the above applications, the resistance of plants to tomato brown wrinkle fruit virus disease was improved by overexpressing TBT1 protein in plants or by applying TBT1 protein exogenously.

[0010] In the above applications, the drug for preventing and controlling tomato brown wrinkled fruit virus disease can use the TBT1 protein as the sole active ingredient; or the TBT1 protein can be used in combination with other substances that are effective against tomato brown wrinkled fruit virus disease.

[0011] A third aspect of the present invention provides a resistance gene that enhances resistance to ToBRFV, said resistance gene being... TBT1 A gene is a nucleic acid molecule as shown in (i), (ii), (iii), or (iv) 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 that encode the amino acid sequence shown in SEQ ID NO.1, other than (i) or (ii); (iv) A nucleic acid molecule that has 90% or more identity with the nucleic acid molecule defined in (i) or (ii) and whose encoded protein is functionally equivalent to the protein shown in SEQ ID NO.1.

[0012] This invention identified a novel resistance gene to tomato brown wrinkle virus in chromosome 11 of tomato using map-based cloning, and named it... TBT1 Genes. This invention TBT1 While the gene does not inhibit viral replication, it can significantly alleviate symptoms, thus ensuring crop yield and quality in the presence of the virus. Given the rapid mutation of viruses, resistance genes have unique application value.

[0013] In a fourth aspect, the present invention provides the use of the above-mentioned disease resistance gene in the following (1) or (2): (1) Improve the plant's resistance to tomato brown wrinkled fruit virus disease; (2) Cultivate plant varieties that are resistant to tomato brown wrinkled fruit virus disease.

[0014] In the above applications, the disease resistance genes are overexpressed in plants to improve the plant's resistance to tomato brown wrinkled fruit virus (TWRV) or to cultivate plant varieties resistant to TWRV.

[0015] In some preferred embodiments of the present invention, overexpression of disease resistance genes in plants is achieved using the following substances: C1) contains TBT1 Gene expression cassettes; C2) contains TBT1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TBT1 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TBT1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TBT1 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TBT1 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

[0016] In the above applications, the plant is preferably a plant of the Solanaceae family, including but not limited to: tomato, pepper, eggplant, potato, tobacco, etc.

[0017] A fifth aspect of the present invention provides a molecular marker for specific identification. TBT1 Genes; the molecular markers include: InDel- TBT1- 1 and InDel- TBT1- 2; The InDel- TBT1- 1 is composed of the nucleotide fragment shown in SEQ ID NO.4 and the nucleotide fragment shown in SEQ ID NO.5; the InDel- TBT1- 2 consists of the nucleotide fragment shown in SEQ ID NO.6 and the nucleotide fragment shown in SEQ ID NO.7.

[0018] Among the molecular markers mentioned above, InDel- TBT1- 1 and InDel- TBT1- 2 belongs to the disease resistance gene TBT1 Closely linked InDel molecular markers; where: InDel- TBT1- The nucleotide fragment shown in SEQ ID NO.4 of SEQ ID NO.1 was co-separated from the disease resistance gene, with a fragment size of 179 bp; the nucleotide sequence fragment shown in SEQ ID NO.5 was co-separated from the disease susceptibility gene, with a fragment size of 143 bp.

[0019] InDel- TBT1- The nucleotide fragment shown in SEQ ID NO.6 of 2 was co-separated from the disease resistance gene, with a fragment size of 152 bp; the nucleotide sequence fragment shown in SEQ ID NO.7 was co-separated from the disease susceptibility gene, with a fragment size of 110 bp.

[0020] This invention has obtained the same as TBT1 Molecular markers tightly linked to disease resistance genes enable the identification of... TBT1 The precise identification of genes and disease-resistant germplasm has laid an important foundation for accelerating the breeding process of disease-resistant tomato brown wrinkle virus.

[0021] In a sixth aspect, the present invention provides the use of the above-mentioned molecular marker as a target in at least one of the following (1)-(3): (1) Identification of disease resistance genes TBT1 ; (2) Identification of plant materials resistant to tomato brown wrinkled fruit virus disease; (3) Cultivate plant varieties resistant to tomato brown wrinkled fruit virus disease.

[0022] In the above applications, primer pairs for detecting the aforementioned molecular markers are used to identify disease resistance genes. TBT1 Or to identify plant materials resistant to tomato brown wrinkled fruit virus disease.

[0023] In some preferred embodiments of the present invention, the primer pairs for detecting molecular markers include primer pair A and primer pair B; The nucleotide sequences of primer pair A are shown in SEQ ID NO. 8 and SEQ ID NO. 9, and are used to detect InDel- TBT1- 1; The nucleotide sequences of primer pair B are shown in SEQ ID NO.10 and SEQ ID NO.11, and are used to detect InDel- TBT1- 2.

[0024] The beneficial effects of this invention are: (1) This invention is the first to identify a new gene on chromosome 11 of tomato that has resistance to brown wrinkled fruit virus. TBT1 Overexpression of disease resistance genes in tomatoes TBT1 It can significantly enhance resistance to brown wrinkled fruit virus. Compared to disease-resistant genes, the disease-resistant gene of this invention... TBT1 It does not exert selective pressure on the virus, which can effectively prevent the virus gene from evolving and breaking through resistance, thus achieving long-term and stable disease resistance. It solves the technical pain point that the resistance of existing disease resistance genes is easy to become ineffective and that varieties need to be frequently updated.

[0025] (2) This invention relates to disease resistance genes TBT1 Two upstream and downstream companies were developed for disease resistance genes. TBT1 The tightly linked molecular markers of this invention can be used to detect and identify resistance genes for tomato brown wrinkle fruit virus disease. This method can be used in a simple, fast, and high-throughput manner in breeding practice, thus accelerating the process of disease-resistant tomato breeding. Attached Figure Description

[0026] Picture 1 Overexpression TBT1 In the genetically modified tomato lines TBT1 Results of relative gene expression level detection; in the figure, TB0249 is the background material; TBT1-OE For overexpression TBT1 Transgenic tomato strains with genetically modified genes.

[0027] Picture 2 Overexpression TBT1 Photographs of tomato transgenic lines and background materials infected with Brown Ruffled Fruit Virus (BRV); in the figure, A is the background material (WT) in fruit infected with BRV; B is the overexpressing tomato transgenic line. TBT1 Transgenic tomato lines with genetically modified genes ( TBT1-OE (The fruit after being infected with the brown wrinkled fruit virus.)

[0028] Picture 3 Overexpression TBT1The results of virus load determination in leaves of transgenic tomato lines and background materials after infection with Brown Wrinkle Fruit Virus (TB0249) are shown in the figure. TBT1-OE For overexpression TBT1 Transgenic tomato strains with genetically modified genes.

[0029] Picture 4 Using the molecular marker InDel- TBT1- 1 and InDel- TBT1- 2. Electrophoretic patterns of some individual plants in the BC5F1:TG100×TB0249 population were detected; in the figure, A is the disease-resistant genotype, B is the disease-susceptible genotype, and H is the heterozygous genotype.

[0030] Picture 5 Leaf 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.

[0031] Picture 6 The results of ToBRFV inoculation of plants identified by molecular markers as resistant to Tomato Brown Fruit 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

[0032] 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.

[0033] 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.

[0034] 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: TG100, a material tolerant to ToBRFV, is an existing cultivated tomato material. The tomato susceptible material "MoneyMaker" is documented in the journal article "A Transcriptional Network Promotes Anthocyanin Biosynthesis in Tomato Flesh". Molecular Plant The material TB0249, which is susceptible to ToBRFV, is described in the literature “A biotechnology-based male-sterility system for hybrid seed production in tomato” (Du M. et al., The Plant Journal, 2020).

[0035] Tomato brown rugose fruit virus (ToBRFV) is an isolate from Shandong (ToBRFV-SD), isolated in 2019 from a diseased tomato sample in Yucheng City, Shandong Province. The complete genome sequence of this isolate has been submitted to NCBI GenBank, accession number MT018320. Its biological characteristics, host range, and molecular features have been reported in detail (Yan et al., 2021, Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection). Journal of Integrative Agriculture ).

[0036] The public can obtain the aforementioned biological materials from the applicant. The obtained biological materials can only be used for the verification of the patented technical solution and cannot be used for other purposes.

[0037] Example 1: Map-based cloning of the resistance gene for tomato brown wrinkle virus During the breeding process in our laboratory, we discovered a material TG100 that showed resistance to the tomato brown wrinkle virus in a protected environment. We then crossed TG100 as the resistant parent with MoneyMaker as the susceptible parent to obtain F1. The F1 generation was then self-crossed to obtain the segregating F2 generation, which was used for map-based cloning of the tomato brown wrinkle virus resistance gene carried by TG100.

[0038] We constructed disease-resistant and disease-susceptible pools using the aforementioned F2 generation segregating population, with 50 plants in each pool. Linkage identification was performed using primers covering the entire genome, initially locating the disease resistance gene to a region of 2.1 Mb–7.4 Mb on chromosome 11. Using encrypted molecular markers, recombinant single plants were screened from a population of 3900 F2 plants, ultimately finely locating the disease resistance gene to a region of 3.5 Mb–5.1 Mb. Sequence analysis of the two materials within this finely located region ultimately revealed that… TBT1 As our candidate disease resistance gene TBT1 The gDNA sequence of the gene is shown in SEQ ID NO.2, and the CDS region sequence is shown in SEQ ID NO.3; the amino acid sequence of the encoded TBT1 protein is shown in SEQ ID NO.1.

[0039] Example 2: Disease resistance gene TBT1 Functional verification 1. Overexpression TBT1 Construction of transgenic tomato lines (1) Construction of recombinant expression vector pK7FWG2- TBT1 : Will TBT1 The CDS sequence (SEQ ID NO.3) of the gene was ligated into the pK7FWG2 vector, which had been double-digested with Xho I and EcoRI, to construct the recombinant expression vector pK7FWG2- TBT1 .

[0040] (3) Preparation of recombinant positive Agrobacterium bacterial suspension: The successfully constructed recombinant expression vector pK7FWG2- TBT1 Recombinant positive Agrobacterium was obtained by transferring it into Agrobacterium LBA4404.

[0041] 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 YEB liquid medium (containing 50 μg / ml Rif and 50 μg / ml Kan) in the dark at 28°C and 200 rpm for 1.5 days. 500 μl of bacterial culture was added to 50 ml of YEB liquid medium (containing 50 μg / ml Rif and 50 μg / ml Kan) and cultured until OD500 was reached. 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 MS Salts Powder and 3 g sucrose) to the bacterial concentration OD. 600 = 1.5; (3) Obtaining transgenic plants: Select plump seeds of the susceptible material TB0249 and sterilize them in a clean bench: soak in 75% alcohol for 2 min, rinse 3 times with sterile water; soak in saturated sodium phosphate for 20 min, rinse 3 times with sterile water; soak in 1% sodium hypochlorite for 10 min, rinse 7 times with sterile water; soak in sterile water for 4-6 h. Sow on 1 / 2 MS medium and germinate in a light incubator (cotyledons fully expanded in 6-8 days). Cotyledons were excised and soaked in MS liquid medium (containing 0.2 mg / L 2,4-D and 0.1 mg / L KT) for 1 h. After being blotted dry with sterile filter paper, they were pre-cultured in A1 solid medium (MS + 1 mg / L IAA + 1.75 mg / L ZT) for 1 day. They were then transferred to A2 resistant medium (MS + 1.0 mg / L IAA + 1.75 mg / L ZT + 75 mg / L Kan + 200 mg / L Tim) and cultured under the same conditions. The medium was changed every 3 weeks until callus formation, then transferred 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. The growing points were excised and transferred 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 sterilized and sown on 1 / 2 MS solid medium containing 75 mg / L kanamycin. If all T1 generation seeds were resistant, they were homozygous and used for subsequent experiments.

[0042] After resistance screening and molecular identification, the T2 generation overexpression was finally obtained. TBT1 Transgenic tomato lines with genetically modified genes ( TBT1-OE After quantitative real-time PCR was used to detect the expression level, it was found that... TBT1-OE Compared to the control TB0249, the transgenic line... TBT1 The relative expression level of the gene was significantly increased. Picture 1 ).

[0043] 2. Overexpression TBT1 Investigation of the resistance of transgenic tomato lines to ToBRFV In comparison with TB0249 and TBT1-OE Transgenic tomatoes were inoculated with Tomato Brown Wrinkle Fruit Virus (TCBV) to investigate whether the fruits and leaves developed the disease during the ripening period, and to measure the viral load 14 days after inoculation with TCBV.

[0044] The results showed that TB0249 tomato fruits exhibited significant disease, with brown, wrinkled spots appearing on the fruit surface; and TBT1-OEThe transgenic plant produces healthy, disease-free fruit. Picture 2 The viral load of brown wrinkled tomato fruit was... TBT1-OE There was no significant difference in viral load compared to TB0249. Picture 3 ).

[0045] Based on the above results, TBT1 The gene played a significant role in the infection of tomatoes by Tomato Brown Ruffle Virus (TRUV). Although it could not inhibit the replication of TRUV, it effectively enhanced the disease resistance of tomatoes. TBT1 The gene belongs to the disease resistance gene category. Overexpression. TBT1 Genes can significantly improve the disease resistance of tomatoes.

[0046] Example 3: With disease resistance genes TBT1 Development of tightly linked molecular markers and detection primers 1. Related to disease resistance genes TBT1 Development of tightly linked InDel molecular markers: Disease resistance gene based on map-based cloning in Example 1 TBT1 Two more genes related to disease resistance were developed. TBT1 The tightly linked InDel molecular markers are named InDel- TBT1- 1 and InDel- TBT1- 2. Among them: The InDel- TBT1- 1 is composed of the nucleotide fragment shown in SEQ ID NO.4 and the nucleotide fragment shown in SEQ ID NO.5; the nucleotide fragment shown in SEQ ID NO.4 is co-separated with the disease resistance gene, and the fragment size is 179 bp; the nucleotide sequence fragment shown in SEQ ID NO.5 is co-separated with the disease susceptibility gene, and the separated fragment size is 143 bp.

[0047] The InDel- TBT1- 2 consists of the nucleotide fragment shown in SEQ ID NO.6 and the nucleotide fragment shown in SEQ ID NO.7; the nucleotide fragment shown in SEQ ID NO.6 is co-separated with the disease resistance gene, and the fragment size is 152 bp; the nucleotide sequence fragment shown in SEQ ID NO.7 is co-separated with the disease susceptibility gene, and the separated fragment size is 110 bp.

[0048] 2. Primer design for detecting molecular markers: Based on the above InDel- TBT1- 1 and InDel- TBT1- 2. Further primer pairs for detecting the above molecular markers were designed, including primer pair A and primer pair B; The nucleotide sequences of primer pair A are shown in SEQ ID NO. 8 and SEQ ID NO. 9, and are used to detect InDel- TBT1- 1; The nucleotide sequences of primer pair B are shown in SEQ ID NO.10 and SEQ ID NO.11, and are used to detect InDel- TBT1- 2. The specific sequence is as follows: InDel- TBT1- 1-F:5'-TGGGATTGTAAGGGGCTACA-3'; (SEQ ID NO.8) InDel- TBT1- 1-R:5'-GCCCGTTAACTTCATCTTTGC-3'. (SEQ ID NO.9) InDel- TBT1- 2-F:5'-CCCCAACCTTGTGCGGAT-3'; (SEQ ID NO.10) InDel- TBT1- 2-R:5'-TCTTTCTCTTGCGCACCAA-3'. (SEQ ID NO.11) The above-mentioned detection molecular marker InDel- TBT1- 1 and InDel- TBT1- Primer pair A and primer pair B were used to detect the DNA of the tomato sample. If the PCR product of primer pair A showed a specific band of 179 bp, it indicated that the sample contained a disease resistance gene. TBT1 It exhibits resistance to tomato brown wrinkle virus disease; if the PCR product of primer pair B shows a specific band of 152 bp, it indicates that the sample contains the resistance gene. TBT1 It exhibits tolerance to tomato brown wrinkle fruit virus disease.

[0049] Example 4: With disease resistance genes TBT1 Functional validation of tightly linked molecular markers In the protected area, TB0249 was used as the recurrent parent to transmit the disease resistance gene carried by TG100. TBT1 In TB0249, after backcrossing, each generation was screened for the tightly linked molecular markers developed in Example 3. TBT1 The offspring plants of the gene. Through five generations of backcrossing, we obtained BC5:TG100×TB0249, and after self-crossing, we obtained BC5F1:TG100×TB0249.

[0050] Using the BC5F1:TG100×TB0249 population to target the molecular marker InDel- designed in Example 3 TBT1- 1 and InDel- TBT1- 2. Perform functional verification.

[0051] DNA was extracted from a subset of individual plants in the BC5F1:TG100×TB0249 population. The extraction method is detailed in the instructions for the TransGen Universal Plant Genomic DNA Extraction Kit. The InDel- molecular marker described in Example 3 was used for detection. TBT1- 1 and InDel- TBT1- PCR amplification was performed using primer pair A and primer pair B, and the PCR amplification products were then analyzed by gel electrophoresis.

[0052] If the tomato sample to be tested utilizes the InDel- TBT1- The PCR amplification product of primer pair A was only a single 179 bp band; using InDel- TBT1- If the PCR amplification product of primer pair B is only a 152bp band, then the tomato being tested is a disease-resistant genotype (denoted as A). If the tomato sample to be tested utilizes the InDel- TBT1- The PCR amplification product of primer pair A was only a single 143 bp band; using InDel- TBT1- If the PCR amplification product of primer pair B is only a single 110 bp band, then the tomato being tested has a susceptible genotype (denoted as B). If the PCR amplification product of the tomato sample shows other band patterns, then the tomato sample is a heterozygous genotype (denoted as H).

[0053] The test results of some tomato samples are as follows: Picture 4 As shown.

[0054] Select individual plants identified by molecular markers as either disease-resistant genotype (denoted as A) or disease-susceptible genotype (denoted as B), and inoculate them with ToBRFV when they have one true leaf. Fourteen days after inoculation, the viral load of each individual plant was measured and the leaves were observed.

[0055] Test results 14 days after vaccination showed that TBT1-OE There was no significant difference in viral load between the strain and the control TB0249, but they showed obvious phenotypic differences: TBT1-OE The leaves and fruits of the strain showed no obvious symptoms, while the control TB0249 exhibited typical leaf wrinkling, mosaic patterns, and brown wrinkled spots on the fruit. This result confirms that... TBT1 The gene does not inhibit viral replication, but rather confers resistance to the tomato brown wrinkle virus, meaning that it can maintain normal growth and reduce disease symptoms even under high viral load conditions. Picture 5 , Picture 6 ).

[0056] The results of the molecular markers of the present invention in detecting resistance to tomato brown wrinkled fruit virus are consistent with the actual results, proving that the molecular markers of the present invention can be used for the detection of resistance to tomato brown wrinkled fruit virus, and the results are accurate and reliable.

[0057] 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. Applications of TBT1 protein in the following (1) or (2): (1) Improve the plant's resistance to tomato brown wrinkled fruit virus disease; (2) Preparation of drugs for the prevention and control of tomato brown wrinkled fruit virus disease; The TBT1 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); The plant in question is a tomato.

2. The application according to claim 1, characterized in that, By overexpressing the TBT1 protein in plants, the plant's resistance to tomato brown wrinkle fruit virus disease was improved.

3. Application of disease resistance genes in the following (1) or (2): (1) Improve the plant's resistance to tomato brown wrinkled fruit virus disease; (2) To cultivate plant varieties resistant to tomato brown wrinkled fruit virus disease; The disease resistance gene is TBT1 A gene 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 that encode the amino acid sequence shown in SEQ ID NO.1, other than (i) or (ii); The plant in question is a tomato.

4. The application according to claim 3, characterized in that, By overexpressing disease resistance genes in plants, the resistance of plants to tomato brown wrinkled fruit virus (TWRV) can be improved, or plant varieties resistant to TWRV can be bred.

5. The application according to claim 4, characterized in that, Overexpression of disease resistance genes in plants is achieved using the following substances: C1) contains TBT1 Gene expression cassettes; C2) contains TBT1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains TBT1 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains TBT1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains TBT1 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1); C6) contains TBT1 Transgenic plant organs containing genes, or transgenic plant organs containing the expression cassette described in C1).

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