Plant antiviral gene xian and application in antiviral

Abstract

The application provides a plant antiviral gene XIAN and application thereof in antiviral, and belongs to the field of functional gene technology. The application provides a plant antiviral gene, wherein the plant antiviral gene comprises a conservative domain, the conservative domain is a MAM33 motif, or a conservative domain with more than 80% homology with the MAM33 motif. The application discloses Atxian, NbXIAN, SlXIAN, GmXIAN and GhXIAN genes all containing the MAM33 motif, in corresponding plants, the virus resistance of the mutants after silencing of the genes is weakened, and the virus resistance is improved after overexpression, which proves that the plant antiviral gene can significantly regulate the antiviral phenotype of the plant, so that germplasm with different antiviral phenotypes is created.

Description

Technical Field

[0001] This invention belongs to the field of functional gene technology, specifically relating to the plant antiviral gene XIAN and its application in antiviral therapy. Background Technology

[0002] Plant viral diseases are known as "plant cancer," severely impacting crop yield and quality, causing at least $30 billion in losses globally each year. Among these, diseases caused by viruses of the genera *Begomovirus* (transmitted by the whitefly *Bemisia tabaci*), *Tospovirus* (transmitted by the thrips), and *Potyvirus* (transmitted by the aphid) are the most prominent. Tomlinson (1987) conducted a questionnaire survey on the relative importance of various viral diseases on vegetables in 28 countries and regions, finding that cucumber mosaic virus (CMV) caused the greatest damage to vegetable crops, followed by turnip mosaic virus (TuMV).

[0003] TuMV belongs to the family Potyviridae and the genus *Potamovirus*, and is widely distributed in North America, Europe, South Africa, Asia, and Australia. It is the only virus in the Potyviridae family capable of infecting *Brassica* plants, causing severe damage to *Brassica* crops in Europe, Asia, and North America. TuMV has a wide host range, infecting 318 species of dicotyledonous plants from 156 genera and 43 families, including Asteraceae, Amaranthaceae, Fabaceae, and Brassicaceae. Furthermore, TuMV can infect monocotyledonous plants from at least seven families, including Amaryllidaceae, Araceae, Commelinaceae, Iridaceae, Liliaceae, Liliaceae, and Orchidaceae. In the early stages of TuMV infection of Brassica plants, the leaves show symptoms such as ring spots, wrinkling, and mottling; in the later stages of infection, the plants are severely stunted, exhibiting symptoms such as chlorosis, necrosis, and even death of the entire plant, causing serious economic losses.

[0004] In the long-term interaction between plants and viruses, plants have evolved a variety of antiviral mechanisms, the most well-known of which are RNA silencing and resistance (R) gene-mediated plant antiviral immune responses. Since the first R gene in plants was cloned in 1992, more and more plant innate immune receptors and signaling pathways have been discovered. However, related research has mainly focused on plant resistance to pathogenic fungi, bacteria, and oomycetes. Until the last 20 years, studies have shown that plant innate immunity (PAMP-triggered immunity, PTI) is involved in plant antiviral immune responses. For example, turnip crinkle virus (TCV) infection can induce the expression of important co-receptor kinases BAK1 and BKK1 in PTI, and compared with wild-type Arabidopsis, the bak1-4 and bkk1-1 mutants are more susceptible to TCV and have more severe necrotic spots. The capsid protein CP of plumpox virus (PPV) can inhibit flg22-induced expression of defense-related genes such as FRK1 (Flg22-induced receptor kinase 1) and NHL10 (NDR1 / HIN1-like 10) and ROS burst; and the bak1-5bkk1-1 double mutant and bik1 mutant are also more susceptible to PPV. In addition, the movement protein MP of cucumber mosaic virus (CMV) can also act as an effector to inhibit flg22, elf18, and chitin-induced ROS burst and expression of defense-related genes. Plant innate immune responses are the first line of defense against pathogens, inducing ROS bursts, stomatal closure, and callose deposition to resist pathogen infection and prematurely activating effector-induced specific immune responses. Therefore, the discovery of antiviral genes in plant innate immune signaling pathways is beneficial for the future breeding of antiviral crop varieties.

[0005] The XAIN mitochondrial glycoprotein family of proteins possesses the typical MAM33 (Mitochondrial acidic matrix 33) domain and is conserved in eukaryotes. XIAN is a multilocalized protein capable of localizing in both mitochondria and cytoplasm. Currently, this gene family has not been reported in plants. In animals, the homolog of XIAN, C1qbp, was initially co-purified with the nuclear splicing factor SF-2. It was later found to bind to mtRNA, regulating the translation of mitochondrial gene-encoded proteins, influencing mitochondrial oxidative phosphorylation and mROS (mitochondrial ROS) bursts, and thus playing an important role in the immune system. Current research on XIAN focuses on animals; the identification of plant XIAN and its function in plant immunity have not been reported. Summary of the Invention

[0006] This invention provides the plant antiviral gene XIAN and its application in antiviral treatment. The plant antiviral gene XIAN can be applied to plant immunity. Overexpression of the plant antiviral gene can improve the plant's resistance to viruses, thereby achieving the purpose of controlling viral diseases.

[0007] This invention provides a plant antiviral gene, which includes a conserved domain, wherein the conserved domain is a MAM33 motif, or a conserved domain having more than 80% homology with the MAM33 motif.

[0008] The amino acid sequence encoded by the MAM33 motif is shown in SEQ ID No. 1.

[0009] In a preferred embodiment of the present invention, when the plant antiviral gene is derived from Arabidopsis thaliana, the nucleotide sequence of the MAM33 motif is as shown in SEQ ID No. 2;

[0010] When the plant antiviral gene is derived from tobacco, the nucleotide sequence of the MAM33 motif is shown in SEQ ID No. 3;

[0011] When the plant antiviral gene is derived from tomato, the nucleotide sequence of the MAM33 motif is shown in SEQ ID No. 4;

[0012] When the plant antiviral gene is derived from soybean, the nucleotide sequence of the MAM33 motif is shown in SEQ ID No. 5;

[0013] When the plant antiviral gene is derived from cotton, the nucleotide sequence of the MAM33 motif is shown in SEQ ID No. 6.

[0014] In a preferred embodiment of the present invention, when the plant antiviral gene is derived from Arabidopsis thaliana, the nucleotide sequence is as shown in SEQ ID No. 7;

[0015] When the plant antiviral gene is derived from tobacco, its nucleotide sequence is shown in SEQ ID No. 8;

[0016] When the plant antiviral gene is derived from tomato, its nucleotide sequence is shown in SEQ ID No. 9;

[0017] When the plant antiviral gene is derived from soybean, its nucleotide sequence is shown in SEQ ID No. 10;

[0018] When the plant antiviral gene is derived from cotton, its nucleotide sequence is shown in SEQ ID No. 11.

[0019] The present invention also provides a set of primer pairs for amplifying the above-mentioned plant antiviral genes.

[0020] This invention also provides the application of the above-mentioned plant antiviral genes in regulating plant antiviral traits.

[0021] In a preferred embodiment of the present invention, the regulation of plant antiviral traits includes overexpressing the plant antiviral gene or a homologous gene of the plant antiviral gene to enhance the plant's resistance to viruses; and inhibiting the expression of the plant antiviral gene or a homologous gene of the plant antiviral gene to reduce the plant's resistance to viruses.

[0022] In a preferred embodiment of the present invention, the virus comprises a positive-strand single-stranded RNA genome virus.

[0023] The present invention also provides the application of the above-mentioned plant antiviral genes in the creation of plant germplasm with different antiviral phenotypes.

[0024] The present invention also provides a method for improving plant virus resistance, comprising overexpressing the above-mentioned plant antiviral gene or a homolog of the antiviral gene in the genome of the target plant.

[0025] The present invention also provides an overexpression vector for the above-mentioned plant antiviral genes.

[0026] Beneficial Effects: This invention provides a plant antiviral gene containing a conserved domain, which is a MAM33 motif or a conserved domain with more than 80% homology to the MAM33 motif. This invention discloses five plant antiviral genes—AtXIAN, NbXIAN, SlXIAN, GmXIAN, and GhXIAN—derived from Arabidopsis thaliana, tobacco, tomato, soybean, and cotton, all of which contain the conserved amino acid sequence of the MAM33 motif.

[0027] This invention utilizes mutants, complemented and overexpressed plants to discover that mutants with silenced AtXIAN, NbXIAN and SlXIAN genes exhibit weakened resistance to viruses, while overexpression enhances resistance. This demonstrates that the plant antiviral genes described in this invention can significantly regulate the antiviral phenotype of plants, thereby creating germplasm with different antiviral phenotypes. Attached Figure Description

[0028] Figure 1 Information on mutation sites in Atxian mutant plants (A) and triprim homozygosity identification (B), and a comparison of AtXIAN protein expression levels in AtXIAN-replaced plants (C) and AtXIAN-overexpressing plants (D).

[0029] Figure 2 The graph shows the differences in viral load at the mRNA level (A) and protein level (B) after Atxian mutant, Col-0 wild type, AtXIAN reintroduced plants, and AtXIAN overexpressing plants infected with turnip mosaic virus.

[0030] Figure 3 A comparison of NbXIAN gene expression levels in control tobacco Benedictine and tobacco with silenced NbXIAN gene;

[0031] Figure 4 A graph showing the difference in viral load after turnip mosaic virus infection of control tobacco (Tobacco Benedictine burmannii) and tobacco with silenced NbXIAN gene;

[0032] Figure 5 A comparison of NbXIAN gene expression levels in control tobacco and tobacco overexpressing the NbXIAN gene;

[0033] Figure 6 A graph showing the difference in viral load after turnip mosaic virus infection of control tobacco (Nicotiana benthamiana) and tobacco overexpressing the NbXIAN gene;

[0034] Figure 7 A comparison of SlXIAN gene expression levels in control tomatoes and tomatoes with silenced SlXIAN genes;

[0035] Figure 8 A graph showing the difference in viral load between cucumber mosaic virus-infected control tomatoes and tomatoes with silenced SlXIAN genes.

[0036] Figure 9 The image shows the pPR001-FLAG vector. Detailed Implementation

[0037] This invention provides a plant antiviral gene, which includes a conserved domain, wherein the conserved domain is a MAM33 motif, or a conserved domain having more than 80% homology with the MAM33 motif.

[0038] The amino acid sequence encoded by the MAM33 motif is shown in SEQ ID No. 1.

[0039] The MAM33 (Mitochondrial acidic matrix 33) domain described in this invention is known to be conserved in eukaryotes, but has not been reported in plants. The MAM33 motif described in this invention is a conserved domain of a plant-derived gene, specifically a relatively conserved sequence in mitochondrial glycoproteins, having the amino acid sequence shown in SEQ ID No. 1; when derived from Arabidopsis thaliana, the nucleotide sequence of this conserved domain is shown in SEQ ID No. 2; when derived from tobacco, the nucleotide sequence of this conserved domain is shown in SEQ ID No. 3; when derived from tomato, the nucleotide sequence of this conserved domain is shown in SEQ ID No. 4; when derived from soybean, the nucleotide sequence of this conserved domain is shown in SEQ ID No. 5; and when derived from cotton, the nucleotide sequence of this conserved domain is shown in SEQ ID No. 6.

[0040] In one embodiment of the present invention, the plant antiviral gene is derived from Arabidopsis thaliana, named AtXIAN, and its encoded protein amino acid sequence is shown in SEQ ID No. 12; the nucleotide sequence is shown in SEQ ID No. 7. In one embodiment of the present invention, the plant antiviral gene is derived from tobacco, named NbXIAN, and its nucleotide sequence is shown in SEQ ID No. 8. In one embodiment of the present invention, the plant antiviral gene is derived from tomato, named SlXIAN, and its nucleotide sequence is shown in SEQ ID No. 9. In one embodiment of the present invention, the plant antiviral gene is derived from soybean, named GmXIAN, and its nucleotide sequence is shown in SEQ ID No. 10. In one embodiment of the present invention, the plant antiviral gene is derived from cotton, named GhXIAN, and its nucleotide sequence is shown in SEQ ID No. 11.

[0041] The present invention also provides a set of primer pairs for amplifying the above-mentioned plant antiviral genes.

[0042] In this invention, because the XIAN protein sequences from different plant sources have low sequence similarity, different primer pairs are required when using different genomes as templates for amplification. This invention provides primer information for five sets of primers for amplifying the XIAN gene:

[0043] AtXIAN-F (SEQ ID No. 13): 5'-ATGGCTTTAGCTTGGTGCGT-3';

[0044] AtXIAN-R (SEQ ID No. 14): 5'-TCACTGTTCCATGAACTCCTTA-3';

[0045] NbXIAN-F (SEQ ID No. 15): 5'-ATGGCAGTCACTAATATAATTAGAC-3';

[0046] NbXIAN-R (SEQ ID No. 16): 5'-CTATGCTTCAATGAATTTCTTCAGA-3';

[0047] SlXIAN-F (SEQ ID No. 17): 5'-ATGGCTGTCACTAACATCATC-3';

[0048] SlXIAN-R (SEQ ID No. 18): 5'-CTATGCCTCGATGAATTTCT-3';

[0049] GmXIAN-F (SEQ ID No. 19): 5'-ATGGCCATGTACGCGATGCT-3';

[0050] GmXIAN-R (SEQ ID No. 20): 5'-GAAGAACTTCGTCGAGAAATGA-3';

[0051] GhXIAN-F (SEQ ID No. 21): 5'-ATGGCTTCACTTCCATTCTCC-3';

[0052] GhXIAN-R (SEQ ID No. 22): 5'-TCATTCTTCAATGAACTTCTTGAGG-3'.

[0053] This invention also provides the application of the above-mentioned plant antiviral genes in regulating plant antiviral traits.

[0054] In a preferred embodiment of the present invention, the regulation of plant antiviral traits includes overexpressing the plant antiviral gene or a homologous gene of the plant antiviral gene to enhance the plant's resistance to viruses; and inhibiting the expression of the plant antiviral gene or a homologous gene of the plant antiviral gene to reduce the plant's resistance to viruses.

[0055] The viruses verified in the embodiments of the present invention include positive-strand single-stranded RNA genome (+ssRNA) viruses, such as turnip mosaic virus (TuMV) and cucumber mosaic virus (CMV).

[0056] The present invention also provides the application of the above-mentioned plant antiviral genes in the creation of plant germplasm with different antiviral phenotypes.

[0057] This invention, using mutant, complemented, and overexpression plants, found that the Atxian mutant exhibited decreased resistance to plant viruses, while the AtXIAN complemented plants showed similar resistance to plant viruses to the control Col-0. AtXIAN overexpression significantly improved Arabidopsis resistance to plant viruses. Furthermore, this invention, through sequence alignment, identified the homologous genes NbXIAN and SlXIAN of AtXIAN in Nicotiana benthamiana and Tomato Cooperative 903. Silencing NbXIAN and SlXIAN genes in Nicotiana benthamiana and Tomato using virus-induced gene silencing technology significantly reduced resistance to plant viruses in both species. Simultaneously, overexpression of the NbXIAN gene using Agrobacterium-mediated transient transformation significantly increased resistance to plant viruses in Nicotiana benthamiana. Therefore, the plant antiviral genes described in this invention can significantly regulate plant antiviral phenotypes, thereby creating germplasm with different antiviral phenotypes.

[0058] The present invention also provides a method for improving plant virus resistance, comprising overexpressing the above-mentioned plant antiviral gene or a homolog of the antiviral gene in the genome of the target plant.

[0059] The present invention does not specifically limit the overexpression method; overexpression can be performed using conventional methods in the art.

[0060] The present invention also provides an overexpression vector for the above-mentioned plant antiviral genes.

[0061] The present invention does not have any particular limitation on the type of basic backbone vector of the overexpression vector. Common plant overexpression vectors in the art can be used, such as pCAMBIA1305-GFP and pPR001-FLAG selected in the examples.

[0062] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of the plant antiviral gene XIAN provided by the present invention and its application in antiviral activity, should not be construed as limiting the scope of protection of the present invention.

[0063] Example 1: Screening and Cloning of the AtXIAN Gene

[0064] 1. Obtaining the AtXIAN gene

[0065] By screening a yeast two-hybrid library, proteins that potentially interact with AtCERES were identified. NCBI-Blast sequence alignment revealed that the interacting region was a portion of the AtXIAN sequence. The full-length AtXIAN sequence was then located using the gene number from NCBI, as shown in SEQ ID No. 7.

[0066] 2. Obtaining the AtXIAN gene sequence

[0067] (1) Using cDNA from wild-type Arabidopsis thaliana Col-0 (from Professor Liang Yan of Zhejiang University) as a template, PCR amplification was performed. The full-length AtXIAN gene fragment was ligated from the restriction sites XbaI and BamHI to the vectors pCAMBIA1305 and pPR001-FLAG (vector map is shown in [link]). Figure 9 The 753 bp nucleotide sequence was transformed into E. coli using the heat shock method and confirmed by sequencing. The plasmids were named pCAMBIA1305-AtXIAN-GFP and pPR001-AtXIAN-FLAG, respectively.

[0068] (2) Primer design: AtXIAN-F (SEQ ID No. 13) and AtXIAN-R (SEQ ID No. 14).

[0069] Example 2: Effects of Arabidopsis thaliana Atxian mutant, AtXIAN reintroduced plants, and AtXIAN overexpressing plants on resistance to turnip mosaic virus.

[0070] 1. Obtaining homozygous mutants of the Atxian gene

[0071] (1) On the Arashare website (http: / / www.arashare.cn / index / ), find the Atxian mutant by searching for the gene number and order WiscDsLox449A04;

[0072] (2) Extract gDNA from Atxian mutant and determine whether the mutant is homozygous by using a three-primer identification method.

[0073] (3) Primer design:

[0074] AtXIAN-LP (SEQ ID No. 23): 5'-AAACCCCAATAGAATGGCATC-3';

[0075] AtXIAN-RP (SEQ ID No. 24): 5'-TGGGTGTGTTTGCAAAATAGTC-3';

[0076] AtXIAN-BP (SEQ ID No. 25): 5'-AACGTCCGCAATGTGTTATTAAGTTGTC-3'.

[0077] The results are as follows Figure 1 As shown, the Atxian mutant has a mutation in the 5'UTR region (A), and three-primer identification revealed that Atxian is a homozygous mutant (B).

[0078] 2. Obtaining Arabidopsis plants with AtXIAN gene complementation and overexpression

[0079] (1) The recombinant plasmids pCAMBIA1305-AtXIAN-GFP and pPR001-AtXIAN-FLAG shown in Example 1 were transformed into EHA105 Agrobacterium tumefaciens by electroporation.

[0080] (2) Wild-type Col-0 and Atxian mutants in full bloom were infected by Agrobacterium-mediated flower immersion method to obtain transgenic Arabidopsis thaliana overexpressing AtXIAN-FLAG with Col-0 background and transgenic Arabidopsis thaliana replenished with AtXIAN-GFP with Atxian mutant background. Transgenic positive Arabidopsis thaliana was obtained through resistance screening.

[0081] The results are as follows Figure 1 As shown, AtXIAN-GFP(C) and AtXIAN-FLAG(D) can be correctly expressed in both the Atxian mutant and the wild-type Col-0 plant.

[0082] 3. Effects of Arabidopsis thaliana Atxian mutant, AtXIAN reintroduced plants, and AtXIAN overexpressing plants on resistance to turnip mosaic virus.

[0083] (1) The tobacco containing turnip mosaic virus was ground with a homogenizer to obtain a crude extract containing turnip mosaic virus. Three-week-old Arabidopsis Atxian mutant, AtXIAN replacement plant and AtXIAN overexpressing plant were inoculated by friction inoculation.

[0084] (2) After 14 days, RNA and protein were extracted from the leaves of Arabidopsis thaliana Atxian mutant, AtXIAN reintroduced plants and AtXIAN overexpressing plants. The levels of turnip mosaic virus in mRNA and protein were detected by qRT-PCR and Western blot.

[0085] (3) Primer design

[0086] qRT-TuMV-F (SEQ ID No. 26): 5'-CAGGTTTGACAGACGAGCAA-3';

[0087] qRT-TuMV-R (SEQ ID No. 27): 5'-CCAGAGGTTCCAGCGTTTAC-3';

[0088] qRT-UBQ10-F (SEQ ID No. 28): 5'-CCTTGTATAATCCCTGATGA-3';

[0089] qRT-UBQ10-R (SEQ ID No. 29): 5'-AACAGGAACGGAAACATAGT-3'.

[0090] The results are as follows Figure 2 As shown, the Atxian mutant exhibited decreased resistance to turnip mosaic virus, while the AtXIAN-replenished plants showed similar resistance to turnip mosaic virus as the control Col-0. Overexpression of AtXIAN significantly improved the resistance of Arabidopsis thaliana to turnip mosaic virus.

[0091] Example 3: Effect of silencing NbXIAN in Tobacco Benedict on plant resistance to turnip mosaic virus.

[0092] 1. Obtaining the NbXIAN gene

[0093] (1) The homolog of AtXIAN, NbXIAN, was obtained by NCBI-Blast, and its nucleotide sequence is shown in SEQ ID No. 8.

[0094] (2) Using wild-type Nicotiana benthamiana cDNA as a template, PCR amplification was performed, and the full-length fragment of the NbXIAN gene was ligated into the vector pCAMBIA1305. The gene was then transformed into E. coli by heat shock, and the sequence was confirmed to be correct. The 774 bp nucleotide sequence is shown in SEQ ID No. 8;

[0095] (3) Primer design: NbXIAN-F (SEQ ID No. 15) and NbXIAN-R (SEQ ID No. 16).

[0096] 2. Obtaining Tobacco Benedictine plants with silenced NbXIAN gene

[0097] (1) The NbXIAN gene sequence was analyzed using the VIGS design website, and the optimal silencing fragment of 401 bp was selected. The cDNA of Nicotiana benthamiana was used as a template for PCR amplification. The silencing fragment was ligated into the vector pTRV2 and transformed into Escherichia coli by heat shock. The sequence was verified to be correct. The preferred nucleotide sequence of the 401 bp is shown in SEQ ID No. 30. The recombinant plasmid pTRV2-NbXIAN was transformed into Agrobacterium tumefaciens EHA105 by electroporation.

[0098] (2) Tobacco rattle virus (TRV) was used to assist in the infection of tobacco. The recombinant plasmid TRV-PDS (which can inhibit the expression of the endogenous gene PDS and produce a phenotypic mutation of leaf whitening) was used as a positive control, and silencing GFP was used as a negative control (Reference: Ratcliff, F., Martin-Hernandez, A., and Baulcombe, D. (2001) Technical Advance: Tobacco Rattle Virus as a Vector for Analysis of Gene Function by Silencing. The Plant Journal, 25, 237-245. https: / / doi.org / 10.1046 / j.0960-7412.2000.00942.x).

[0099] When *Nicotiana benthamiana* reached the two true leaf stage, a suspension of the TRV2-NbXIAN infectious clonal virus was inoculated into the leaves using the Agrobacterium-mediated transformation method. After 14 days of growth, the positive control showed a distinct phenotype of leaf whitening. Total RNA was extracted from the plant leaves using the Trizol method and analyzed using PrimeScript. TM After cDNA was synthesized by reverse transcription using the RT reagent kit, the silencing efficiency of the NbXIAN gene in common tobacco was detected by real-time PCR.

[0100] (3) Primer design:

[0101] vigs-NbXIAN-F (SEQ ID No. 31): 5'-CCTTAAAATCATTCAGTCCGA-3';

[0102] vigs-NbXIAN-R (SEQ ID No. 32): 5'-CCGAGAAATCAGGTCCTTCA-3';

[0103] qRT-NbXIAN-F (SEQ ID No. 33): 5'-CCACCGTAGTAAAACCGTGGC-3';

[0104] qRT-NbXIAN-R (SEQ ID No. 34): 5'-GTTCGTCAGATTCCTCGGCA-3';

[0105] qRT-ACTIN-F (SEQ ID No. 35): 5'-CAATCCAGACACTGTACTTTCTCTC-3';

[0106] qRT-ACTIN-R (SEQ ID No. 36): 5'-AAGCTGCAGGTATCCATGAGACTA-3'.

[0107] The results are as follows Figure 3 As shown, after silencing the NbXIAN gene in Nicotiana benthamiana using VIGS technology, the expression level of the NbXIAN gene was significantly lower than that of the control group.

[0108] 2. Resistance of Tobacco Benzoinii to Turnip Mosaic Virus after Silencing the NbXIAN Gene in Common Tobacco

[0109] The crude extract containing turnip mosaic virus was obtained by grinding the *Nicotiana benthamiana* containing turnip mosaic virus using a homogenizer. Wild-type *Nicotiana benthamiana* and NbXIAN gene-silenced tobacco were then inoculated by friction inoculation. After 3 days, RNA and protein were extracted from the leaves of wild-type *Nicotiana benthamiana* and NbXIAN gene-silenced tobacco systems, and the amount of turnip mosaic virus was detected by qRT-PCR.

[0110] The results are as follows Figure 4 As shown, after silencing the NbXIAN gene in Nicotiana benthamiana using VIGS technology, the viral load of turnip mosaic virus was significantly higher than that in the control group.

[0111] Example 4: Effect of NbXAIN overexpression on resistance to turnip mosaic virus in Nicotiana benthamiana

[0112] 1. Obtaining Nicotiana benthamiana plants overexpressing the NbXIAN gene

[0113] (1) Using the cDNA of Nicotiana benthamiana as a template, PCR amplification was performed. The ORF fragment encoding the mature protein of the NbXIAN gene was ligated into the plant expression vector pCAMBIA1305-GFP through the XbaI and BamHI restriction sites. The vector was then transformed into E. coli by heat shock. After the sequence was confirmed to be correct, the recombinant plasmid pCAMBIA1305-NbXIAN-GFP was transformed into Agrobacterium tumefaciens EHA105 by electroporation.

[0114] (2) When *N. benthamiana* reached the 4-5 true leaf stage, an infectious clone suspension of pCAMBIA1305-NbXIAN-GFP was inoculated into *N. benthamiana* leaves using Agrobacterium-mediated transient transformation. Gene expression peaked 72 hours after inoculation. Total RNA was extracted from plant leaves using the Trizol method and analyzed using PrimeScript. TM After cDNA was synthesized by reverse transcription using the RT reagent Kit, the expression level of the NbXIAN gene in Nicotiana benthamiana was detected by real-time PCR.

[0115] The results are as follows Figure 5 As shown, after transient expression of the NbXIAN gene in Nicotiana benthamiana using Agrobacterium-mediated transformation, the expression level of the NbXIAN gene was significantly higher than that of the control group.

[0116] 2. Effects of NbXIAN gene overexpression on turnip mosaic virus resistance in Tobacco Benzoenta.

[0117] The crude extract containing turnip mosaic virus was obtained by homogenizing the tobacco containing turnip mosaic virus using a homogenizer. Wild-type tobacco and tobacco overexpressing the NbXIAN gene were then inoculated by friction inoculation. After 3 days, RNA and protein were extracted from the leaves of wild-type tobacco and tobacco overexpressing the NbXIAN gene system, and the amount of turnip mosaic virus was detected by qRT-PCR.

[0118] The results are as follows Figure 6 As shown, after overexpressing the NbXIAN gene in Nicotiana benthamiana using Agrobacterium-mediated transformation, the viral load of turnip mosaic virus was significantly lower than that in the control group.

[0119] Example 5: Effect of silencing SlXAIN on resistance to cucumber mosaic virus in tomatoes

[0120] 1. Obtaining the SlXIAN gene

[0121] (1) The homolog of AtXIAN, SlXIAN, was obtained by NCBI-Blast, and its nucleotide sequence is shown in SEQ ID No. 9.

[0122] (2) The SlXIAN gene sequence was analyzed by the VIGS design website, and the optimal silencing fragment of 450bp was selected. The cDNA of Tomato Cooperation 903 purchased from Taobao was used as a template for PCR amplification. The silencing fragment was ligated into the vector pTRV2 through the XbaI and BamHI restriction sites. It was then transformed into E. coli by heat shock method. The sequencing confirmed that the 450bp nucleotide sequence was correct. The preferred nucleotide sequence is shown in SEQ ID No. 37. The recombinant plasmid pTRV2-SlXIAN was transformed into Agrobacterium tumefaciens EHA105 by electroporation method.

[0123] (3) Primer design

[0124] vigs-SlXIAN-F (SEQ ID No. 38): 5'-AAGGACTGCTTCTCAAGTTG-3';

[0125] vigs-SlXIAN-R (SEQ ID No. 39): 5'-GAGGAATATTGGACTGATTG-3';

[0126] 2. Obtaining Tomato Plants with Silent 903SlXIAN Gene

[0127] (2) Tobacco rattle virus (TRV) was used to assist in the infection of tobacco. The recombinant plasmid TRV-PDS (which can inhibit the expression of the endogenous gene PDS and produce a phenotypic mutation that causes leaves to turn white) was used as a positive control, and silencing GFP was used as a negative control.

[0128] When the tomato seedlings reached approximately 0.5 cm in length, the TRV2-SlXIAN infectious clonal virus suspension was vacuum-impregnated into the tomato seeds using the Agrobacterium-mediated transformation method. When the seedlings developed 2-3 true leaves, the positive control showed a distinct phenotype of leaf whitening. Total RNA was extracted from the plant leaves using the Trizol method and analyzed using PrimeScript. TM After the cDNA was synthesized by reverse transcription using the RT reagent kit, the silencing efficiency of the SlXIAN gene in tomato 903 was detected by real-time PCR.

[0129] (3) Primer design:

[0130] qRT-CMV-F (SEQ ID No. 40): 5'-CGGCGGAAGACCATGATTT-3';

[0131] vigs-CMV-R (SEQ ID No. 41): 5'-CCTTCCGCCCATTCGTTAC-3';

[0132] qRT-SlXIAN-F (SEQ ID No. 42): 5'-TGTTGCTGAGGATCAGATTGCT-3';

[0133] qRT-SlXIAN-R (SEQ ID No. 43): 5'-TTGTGCTGGGCTTGATACCC-3';

[0134] qRT-SlACTIN-F (SEQ ID No. 44): 5'-AGGCAGGATTTGCTGGTGATGATGCT-3';

[0135] qRT-SlACTIN-R (SEQ ID No. 45): 5'-ATACGCATCCTTCTGTCCCATTCCGA-3'.

[0136] The results are as follows Figure 7 As shown, after silencing the SlXIAN gene in Nicotiana benthamiana using VIGS technology, the expression level of the SlXIAN gene was significantly lower than that in the control group.

[0137] 3. Resistance to Cucumber Mosaic Virus after Silencing the SlXIAN Gene in Tomato Cooperative 903

[0138] The tobacco plant containing cucumber mosaic virus was ground using a homogenizer to obtain a crude extract containing cucumber mosaic virus. Wild-type tomato cooperative 903 and SlXIAN gene-silenced tomato cooperative 903 were inoculated by friction inoculation. After 14 days, RNA and protein were extracted from the leaves of wild-type tomato cooperative 903 and SlXIAN gene-silenced tomato cooperative 903 systems. The viral load of cucumber mosaic virus was detected by qRT-PCR.

[0139] The results are as follows Figure 8 As shown, after silencing the SlXIAN gene in tomato 903 using VIGS technology, the viral load of cucumber mosaic virus was significantly higher than that in the control group.

[0140] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of plant antiviral genes in regulating plant antiviral traits, characterized in that, The regulation of plant antiviral traits involves overexpressing the plant antiviral gene to enhance the resistance of Nicotiana benthamiana to turnip mosaic virus; and inhibiting the expression of the plant antiviral gene to reduce the resistance of Nicotiana benthamiana to turnip mosaic virus. The nucleotide sequence of the plant antiviral gene is shown in SEQ ID No.

8.

2. The application of plant antiviral genes in creating plant germplasm with different antiviral phenotypes, characterized in that, The nucleotide sequence of the plant antiviral gene is shown in SEQ ID No.

8. The antiviral gene is against turnip mosaic virus, and the plant is Nicotiana benthamiana.

3. A method for improving plant virus resistance, characterized in that, To overexpress a plant antiviral gene in the genome of a plant, the nucleotide sequence of which is shown in SEQ ID No. 8, wherein the plant is Nicotiana benthamiana and the virus is turnip mosaic virus.

Images