KR20060111205A - An antiviral agent comprising nttlp1 isolated from nicotiana tabacum to cmv infection - Google Patents

Abstract

An antiviral agent against CMV(cucumber mosaic virus) infection comprising NtTLP1(Nicotiana tabacum Thaumatin-like protein 1) isolated from Nicotiana tabacum is provided to increase expression of NtTLP1 by interacting with CMV1a proteins, and inhibit sensitivity to the CMV infection, thereby reducing damage caused by CMV. The antiviral agent against CMV infection comprises NtTLP1 isolated from Nicotiana tabacum that is encoded by the gene having the nucleotide sequence of SEQ ID NO:1 and has antiviral activity against CMV, wherein the NtTLP1 belongs to the PR-5 group isolated from Nicotiana tabacum. A transgenic tobacco plant having resistance against CMV is produced by transforming the tobacco plant with a recombinant vector pMBP2 containing the gene of SEQ ID NO:1.

Description

Antiviral agent comprising NtTLP1 isolated from Nicotiana tabacum to CMV infection}

1 is a diagram subjected to gel blot analysis of tobacco genomic DNA,

2 is a diagram showing an in vitro interaction test of recombinant NtTLP1 and CMV 1a protein. Purification (a) of His-NtTLP1 protein through affinity chromatography and far-western analysis of the binding of NtTLP1 and CMV 1a protein were performed. is a diagram showing the results of the assay (b),

3 and 4 show the interaction of NtTLP1 :: GFP with CMV 1a :: 2xHA in planta , FIG. 3 shows immunoprecipitated proteins using anti-GFP, and FIG. 4 shows anti-HA. Figure showing the results of the immunoblotting analysis using,

5 is a diagram showing the intracellular location of NtTLP1 :: GFP in Arabidopsis protoplasts, where GFP and NtTLP1 :: GFP were transformed into Arabidopsis protoplasts and then expressed these introduced genes. The image was taken with a fluorescent microscope after 24 hours.

6 is a diagram showing the adjacent position of NtTLP1 :: GFP and CMV 1a :: RFP in Arabidopsis protoplasts,

7 and 8 are diagrams showing the intracellular positions of GFP (FIG. 7) and NtTLP1 :: GFP (FIG. 8), which are controls in onion cells,

9 is a diagram showing the interaction of NtTLP1 and CMV migration-related proteins (Coat Protein and MP) in yeast cells, pVA3-1, pTD1-1 plasmid positive control group (a), pACT2, pAS2-1 plasmid negative control group (b), Y187 main yeast intertransformation is shown in (c) to (f), blue dots show positive β-galactosidase activity,

10 is a diagram showing the results of RT-PCR expression pattern of three different TLP (thaumatin-like proteins) genes for CMV-Kor inoculation,

11 to 14 are diagrams showing the expression of NtTLP1 gene in NtTLP1 transgenic T1 tobacco plants and the extent of virus proliferation after CMV treatment, and FIG. 11 is the RT of transgenic T1 tobacco plants compared to control plants that were not transformed. -PCR and Western blot analysis. FIG. 12 shows transgenic T1 tobacco through ELISA analysis using anti-CMV lgG (3 μg / ml) 1, 2, 3, 4, 6 days after inoculation. Figure showing the accumulation amount of CMV-Kor CP in the inoculation leaves of plants,

Fig. 13 shows the accumulation of CMV-Kor CP in the upper lobe of the inoculation lobe of the transgenic T1 tobacco plant,

14 shows the accumulation of CMV RNA in transgenic and control plants.

The present invention relates to Nicotiana tabacum Thaumatin-like protein 1 (NtTLP1) isolated from Nicotiana tabacum having antiviral activity against Cucumber mosaic virus (CMV), an antiviral agent comprising the same, and NtTLP1. The present invention relates to a transgenic tobacco plant having resistance to the oimosaic virus by transforming with a pMBP2 recombinant vector containing full-length cDNA and overexpressing NtTLP1.

CMV is distributed throughout the world and covers the widest range of plant infectious viruses and crops (weeds of 1,000 plant species) and has a globally important economy. Symptoms are extremely diverse, and one of the most common symptoms is that the growth of the plant is severely inhibited, resulting in loss of productivity, with apparent mosaic symptoms on the leaves. Yellow spots or gangrene spots appear on the fruit, sometimes the skin is rough and crumbled with an unclear color.

CMV belongs to the cucumovirus group and is an RNA plant virus with a tripartite genome of positive single-strand. The goenome consists of three types of RNA and often contains satellite RNAs with 330 to 380 sequences.

CMV is a virus that causes diseases in major crops such as cucumbers, pumpkins, strawberries, cabbages, and watermelons. The host's response to CMV infection is resistance, general pathological, and the presence or activity of certain viral genetic products. And the like. These reactions are observed by the molecular interactions that occur between the host and viral factors. These interactions between plants and viruses directly affect viral replication, intercellular migration, tissue migration, symptomatic development, as well as induction of host defense responses (Carrington and Whitham, 1998). Although much information is available regarding the role of viral agents involved in this process, little is known about the host factors involved. Among them, only a few examples of resistance to CMV infection have been reported. A single dominant gene Cry with CMV resistance in cowpea has been identified (Nasu et al., 1996) and two single, independent recessive CMV resistance genes, cum-1 , in Arabidopsis thaliana. And cum2-1 have been identified (Yoshii et al., 2004). However, resistance genes that interact directly with CMV viral proteins have not been reported.

There are three capped messenger-sense RNAs, RNAs 1, 2 and 3, in the genome of CMV (Palukaitis et al., 1992). RNAs 1 and 2 encode proteins 1a and 2a. 1a and 2a proteins are essential for viral RNA replication by forming viral replication complexes in cells (Hayes and Buck, 1990; Nitta et al., 1988), and RNA 2 also encodes 2b protein (Ding et al., 1994), which is involved in host-specific long-distance migration of CMV viruses (Ding et al., 1995), and also serves as a suppressor for virus-induced gene silencing (Brigneti et al., 1998). RNA 3 encodes protein 3a and the coat protein (CP), whereas CP is translated from subgenomic RNA 4, whereas 3a protein is translated directly from RNA 2 (Schwinghamer and Symons, 1975). 3a protein and CP are essential for efficient migration between cells of CMV (Boccard and Baulcombe, 1993; Canto et al., 1997; Ding et al., 1995; Suzuki et al., 1991).

PR proteins are plant proteins derived during pathogenic infection or related situations (van Loon et al., 1994) and expressed as plant resistance inducing components more quickly in non-friendly interactions where nontoxic pathogens attack resistant plants. (Dong et al., 1991). Recently, other protective genes have been identified, including PR proteins and PAD3, PAD4, PR1, TLP, BG2 and cf-2 resistance gene-like genes, which are induced during friendly interactions with some viruses. (Whitham et al., 2003). Although the biological or biochemical functions of various PR proteins have been studied over the last few decades, only a few PR proteins, including chitinase, β-1 and 3-glucanase, Only those related to their catalytic action have been identified (Mauch et al., 1988), and some thaumatin-like proteins (TLPs) have only been recognized as hydrolyzing β-1 and 3-glucan as antimicrobial factors (Grenier et al., 1999). On the one hand, the function of other PR proteins in plant defense should be fully understood. In addition, little is known about the function of PR proteins derived from the host-virus friendly relationship.

Accordingly, the present inventors, as a factor affecting CMV virus cleavage, the host factor interacting with the CMV 1a protein through the yeast two-hybrid system NtTLP1 (belonging to the PR-5 group of PR protein ( Nicotiana tabacum thaumatin-like protein 1) was isolated and confirmed that NtTLP1 interacts directly with CMV 1a protein in planta as well as in vitro , and NtTLP1 gene expression is the result of CMV attack and overexpresses NtTLP1 gene. NtTLP1 functions as an antiviral protein and inhibits the replication and migration of the virus by confirming that the transgenic plants have reduced susceptibility to CMV infection and that NtTLP1 also interacts with CMV MP and CP involved in viral migration in yeast. The present invention has been completed by confirming the function.

An object of the present invention is NtTLP1 (Nictiana tabacum Thaumatin-like protein 1) isolated from Nicotiana tabacum having antiviral activity against Cucumber mosaic virus (CMV), an antiviral agent comprising the same and full-tightness of NtTLP1. Transfection with pMBP2 recombinant vector containing length cDNA overexpresses NtTLP1 to provide a transgenic tobacco plant having resistance to the cucumber mosaic virus.

The present invention provides NtTLP1 ( Nicotiana tabacum Thaumatin-like protein 1) encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against Cucumber mosaic virus (CMV).

The NtTLP1 refers to a protein belonging to the PR-5 group separated from Nicotiana tabacum .

The present invention also provides an antiviral agent containing NtTLP1 encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against cucumber mosaic virus as an active ingredient.

In addition, transgenic tobacco plants having resistance to the cucumber mosaic virus are provided by being transformed with a pMBP2 recombinant vector containing the gene sequence of SEQ ID NO: 1 to overexpress NtTLP1.

Hereinafter, the present invention will be described in detail.

The present invention provides NtTLP1 ( Nicotiana tabacum Thaumatin-like protein 1) encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against Cucumber mosaic virus (CMV).

The gene encoding NtTLP1 encodes a 226 amino acid residue protein having 21 amino acid secreting signal peptides, and NtTLP1 is a protein belonging to a PR-5 group isolated from Nicotiana tabacum , having a molecular weight of 24.6. kDa with an isoelectric point of 5.3, similar to other TLPs (Thaumatin-like protein).

The NtTLP1 is induced and expressed by transcription of NtTLP1 by inoculation of CMV, and is initially present in the cytoplasm and then gradually released into the cell wall or extracellular space. The NtTLP1 forms an intracellular viral replication complex and interacts with two sites, the helicase site (CMV-Hel) and the methyltransferase site (CMV-MT) of CMV 1a protein and CMV 1a protein, which are essential proteins for viral RNA replication. In vitro , they bind directly to the CMV 1a protein. In planta , NtTLP1 and CMV 1a protein interact by forming complexes. In addition, NtTLP1 interacts with CP, a coat protein of CMV, and MP, a mobile protein, to affect not only proliferation in CMV's host cell but also the rate and extent of its acceleration, thereby preventing antiviral activity against CMV. Has

NtTLP1 interacting with the CMV 1a protein can be identified through yeast two-hybrid screening, and the interaction between NtTLP1 and CMV 1a protein in vitro can be identified by Far-western analysis. The interaction of both proteins in in planta can be observed by performing immunoprecipitation experiments. In addition, in order to identify the NtTLP1 and CMV 1a proteins, in vivo targeting experiments using a green fluorescence microscope (GFP) and a red fluorescence micoscope (RFP) may be performed by fluorescence electron microscopy, respectively.

The present invention also provides an antiviral agent containing NtTLP1 encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against cucumber mosaic virus. Antiviral agent containing NtTLP1 as an active ingredient, which affects the proliferation of CMV and the rate and range of proliferation and shows antiviral activity, can be administered to plants damaged by CMV to increase CM productivity by overcoming CMV. Can be.

The present invention also provides a transgenic tobacco plant having resistance to cucumber mosaic virus by transforming with a pMBP2 recombinant vector containing the gene sequence of SEQ ID NO: 1 to overexpress NtTLP1.

The transgenic tobacco plant clones a cDNA of NtTLP1 into a polylinker site in a pMBP2 vector, which is a binary vector, and uses Hoekema et al. (Hoekema, A., Hirsch, P., Hooykaas, PJJ and Schilperoort, R., A). binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid., Nature , 303 , pp179-180, 1983), by inserting it into tobacco plants using Agrobacterium-mediated transformation. It can manufacture.

In the transgenic tobacco plants overexpressing NtTLP1, the overexpression of NtTLP1 significantly reduces CP accumulation and CMV RNA accumulation, which are coating proteins of CMV-Kor, thereby inhibiting the proliferation of CMV. Therefore, by producing a transgenic plant that overexpresses the NtTLP1 for not only tobacco plants but all plants that can be damaged by the cucumber mosaic virus, damage to the cucumber mosaic virus can be reduced.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples. However, the following Examples and Experimental Examples illustrate the present invention and the scope of the invention is not limited thereto.

Example 1. Extraction of CMV-Kor RNAs (Virus strain and RNA extraction)

The proliferation and purification of CMV-Kor were performed by application of Kwon et al. (Kwon, CS, Paek, KH and Chung WI, Full-length cDNA cloning and nucleotide sequence analysis of Cucumber mosaic virus (Strain Kor) RNA 2., J. Plant Biol ., 39 , pp 265-271, 1996). In order to extract the RNAs of CMV-Kor proliferated and purified above, RNAs of CMV-Kor were extracted from virus particles using TNE (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM Na ₂ -EDTA). Extracted and precipitated with ethanol. The extracted RNAs were dissolved in water treated with diethylpyrocarbonate and quantified by observation at an absorbance of 259 nm.

Example 2. Preparation of Tobacco Plants and Viral Inoculation

Tobacco (Tobacco, Nicotiana tabacum cv. NC82) plants were grown at 25 ° C. with a light and dark cycle of 16 hours of light / 8 hours of darkness. Tobacco plant juice containing CMV-Kor particles obtained in Example 1 was prepared by grinding infected leaves in phosphate buffer (0.25 M Na2HPO4, 5 mM EDTA), and 2-3 tobacco seedlings (8-10 leaves of Step) Rub mixed with carborundum (mesh 500, Hayashi Chemical) on the surface of the young leaves. Falsely inoculated plants as controls were prepared by rubbing only with phosphate buffer. The leaves were harvested 48 hours after inoculation and used as RNA sources for gene library construction. Tobacco ( Nicotiana tabacum cv. Samsun NN) leaf tissues were inoculated with TMV (Tobacco mosaic virus, strain P0) by applying a method such as shin (Shin, R., Park, CJ, An, JM and Paek, KH, A). novel TMV-induced hot pepper cell wall protein gene ( CaTin2 ) is associated with virus-specific hypersensitive response pathway., Plant Mol . Biol ., 51 , pp 687-701, 2003).

Experimental Example 1. NtTLP1 ( Nicotiana tabacum Isolation of Thaumatin-like Protein 1) and Interaction Between NtTLP1 and CMV 1a Proteins

CMV 1a protein is involved in the process of systemic infection as well as replication of viruses (Palukaitis and Garcia-Arenal, 2003). CMV infection is also presumed to be regulated by interactions between other proteins of viral origin and interaction of putative host factors with CMV la proteins. Therefore, the following two-hybrid screening experiments were performed to identify host factors that interact with the CMV 1a protein.

1-1. East two-hybrid screening experiment

The MatchMaker II GAL4 two-hybrid system was purchased from Clontech Laboratories. Tobacco cDNA library was prepared using pACT2 activation domain expression vector as RNA extracted from tobacco leaves infected with CMV obtained in Example 2. The tobacco cDNA library was screened using the pAS2-1 / CMV 1a binding site fusion vector. The two fusion vectors are mutually transformed in Sacromyces cerevisiae at the main Y190 (Saccharomyces cerevisiae strain Y190, MATa, HIS3, lacZ, trp1, leu2, cyhr2) or HF7c (MATa, HIS3, lacZ, trp1, leu2). will be. The transformed yeast cells were selected in minimal synthetic dropout (SD) media lacking Leu, Trp and His. In order to remove false positive clones during the screening process, the SD medium plate was treated with 50 mM (Y190) or 5 mM (3-amino-1, 2, 4-triazole). Supplemented with HF7c). Yeast cells selected above were grown for 8-12 days at 30 ° C., and then 5-bromo-4-chloro-3-indoli-β-d-galacttoside (X-gal) was used as a substrate. Β-galactosidase activity was observed through colony-lift filter assays. Putative positive clones (Leu +, Trp +, His +) identified by this assay were selected for retransformation. Plasmids were isolated from each Leu +, Trp +, His + and LacZ + transformants using the Yeast Plasmid Isolation Kit (Clontech). E. coli XL1-Blue cells (Stratagene) were transformed with the plasmid isolated above by electroporation. PACT2 fusion plasmids propagated to bacteria were identified and analyzed. In addition, for yeast three-hybrid analysis, CMV 1a and CP or CMV 1a and MP were cloned into pBridge vector (Clontech).

1-2. Isolation of NtTLP1

When the tobacco cDNA library prepared in the above experiment was screened using a full-length CMV 1a gene, 368 colonies were initially expressed in Leu, Trp and His among 1.5 × 10 ⁶ transformants. Grown on deficient plates. The 369 colonies were retransformed and subjected to a colony-lift filter assay.

As a result of the colony-lift filter analysis performed in Experimental Example 1-2, β-galactosidase activity was confirmed in 14 clones. Sequence analysis of the 14 clones confirmed that two of these clones were homologous to the TLP plant gene belonging to the PR-5 group of PR proteins. This gene, assumed to be NtTLP1, encodes a 226 amino acid residue protein with 21 amino acid secreting signal peptides and is similar in nature to other TLPs (Malehorn, DE, Borgmeyer, JR, Smith, CE and Shah, DM, Characterization and expression of an antifungal zeamatin-like protein ( ZIP ) gene from Zea mays ., Plant Physiol ., 106 , pp 1471-148, 1994).

As shown in Figure 1, NtTLP1 was confirmed by Southern blot analysis that the member of the small gene family in the tobacco genome. The molecular weight of the expected protein is 24.6 kDa and the calculated isoelectric point is 5.3 (GenBank accession number AY745249).

The NtTLP1 isolated in Experimental Example 1-2 interacted with CMV, as shown in Table 1 below, HIS3 and lazZ reporter genes (HIS3 and lazZ reporter genes) in which GAL 4 binding or direct interaction with active sites were excluded. This was demonstrated by the use of additional plasmid combinations and configurations for experiments for automatic activation of. pACT2 :: NtTLP1 (prey) as well as pAS2-1 :: CMV 1a (bait) did not activate the HIS3 or lazZ reporter gene on its own. Not only does NtTLP1 (expressed from pACT2 :: NtTLP1) interact with the GAL4 DNA-binding domain itself, but also when it is transfected with a parent plasmid that encodes both a GAL4 DNA-binding site or a GAL4 activation site, CMV 1a (expressed from pACT2 :: NtTLP1). from pAS2-1 :: CMV 1a) The plasmid also failed to interact with the GAL4 activation domain without any mixed sequence.

On the other hand, NtTLP1 interacted with CMV-MT having the methyltransferase site of CMV 1a protein as well as CMV-Hel, the helicase site of CMV 1a protein. Therefore, it was confirmed that NtTLP1 interacts with two domains of full-length CMV 1a protein and CMV 1a protein in yeast.

Interaction of CMV 1a with NtTLP1 in Yeast Cells GAL 4 BD ^a Vector GAL 4 AD ^b Vector His β-galactosidase pAS2-1 :: CMV 1a ^-c - - - pACT2 :: NtTLP1 - - pAS2-1 :: CMV 1a pACT2 - - pAS2-1 pACT2 :: NtTLP1 - - pAS2-1 :: CMV 1a pACT2 :: NtTLP1 + + pAS2-1 :: CMV 1a-MT ^d pACT2 :: NtTLP1 + + pAS2-1 :: CMV 1a-Hel ^e pACT2 :: NtTLP1 + +

(Note) ^a BD: DNA-binding site, ^b AD: activation domain, ^c : no coexpressed vector, ^d : methyltransferase domain of CMV 1a protein CMV 1a protein), ^e : Helicase domain of CMV 1a protein

Experimental Example 2. in vitro Direct interaction of NtTLP1 with CMV-1a protein

Far-western assay was performed to determine whether CMt-1a protein and NtTLP1 interact directly in vitro .

2-1. Isolation of Full-length cDNA clones of NtTLP1

In order to isolate full-length cDNA clones of NtTLP1 isolated in Experimental Examples 1-2, a specific primer for NtTLP1, 5'-CCTTAAGCTGGGCTGGTTACCTCCTACAGCATT-3 ', was devised based on a sequence of partial cDNAs. . The cDNA library constructed for yeast two-hybrid screening in Experimental Example 1-1 was used as a template for PCR amplification using a specific primer and a general primer (T7 primer and Reverse primer).

2-2. Far-westhern assay

NtTLP1 cDNA fragments were prepared by PCR and cloned into the BamHI site of the pQE30 vector (Qiagen). Primer sets used for this amplification are NtTLP1 ΔS-5 ′ (5′-GGGATCCATGCATGCTGCCACTTTTG-3 ′) and NtTLP1-3 ′ (5′-GGATCCAAGGGCAGAAGACAACCC-3 ′). The resulting frame fusion plasmid was transformed into E. coli strain BL21 (DE3). Overexpression of NtTLP1 with six histidine residues attached to the N terminus was induced by isopropyl-β-d-thiogalactoside at 30 ° C. for 3 hours. After induction, the bacteria were pelleted, soaked in lysis buffer (50 mM Na2HPO4, pH 8.0, 300 mM NaCl) and sonicated on ice for 1 hour with Vibracell Sonifier (Sonics and Materials). . The lysate was centrifuged for 10 minutes at a rate of 13,000 x g, and then the supernatant was chromatographed on Ni-NTA affinity resin (QIAGEN). Recombinant protein was eluted at pH 6.0-4.0 in elution buffer (50 mM Na 2 HPO 4, 300 mM NaCl, 10% glycerol). Proteins were separated via 12% SDS-PAGE and transported to nitrocellulose membranes. The cell membranes were washed three times in 4 ° C., AC buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 2.5 mM MgCl 2, and 75 mM KCl) containing 5% milk and radiolabeled CMV in the same solution. Incubated for 2 hours in the presence of 1a protein. The cell membrane was then washed three times at 4 ° C. with probe-free AC buffer, briefly dried and visualized by autoradiography.

2-3. in vitro  Transcription / Translation Experiment

For CMV 1a protein, ³⁵ S-Met labeled CMV 1a protein was obtained using a eukaryotic i n vitro translation system capable of synthesizing the protein directly from CMV 1a cDNA downstream of the T7 promoter. In vitro transcription / translation experiments used the pGBKT7 plasmid with the coding sequence of the CMV 1a protein under the control of the T7 promoter as a template for in vitro transcription / translation reactions using the TNT wheat germ system (Promega). The protein was synthesized for 1 hour using ³⁵ S-Met (Amersham Pharmacia Biotech) as radiolabel. The synthesized protein was extracted and purified from E. coli culture transformed with His vector (lane 1) or His-NtTLP1 (lane 2). The proteins were digested by SDS-PAGE and transported to nitrocellulose.

As a result of the above experiment, as shown in FIG. 2, a polypeptide of about 26 kDa, similar in size to the estimated size of recombinant His-NtTLP1, was purified in close homology through affinity chromatography (see a of FIG. 2). . Lane 1 represents the purified protein from E. coli culture transformed with His vector, lane 2 is purified from recombinant His-NtTLP1 protein (5 μg).

In addition, as a result of the Far Western analysis for confirming the binding of the NtTLP1 and CMV 1a protein, the radiation from CMV 1a protein labeled with ³⁵ S-Met was confirmed only in lane 2 in which recombinant His-NtTLP1 was dissolved ( B of FIG. 2). This result clearly shows that NtTLP1 isolated by East-to-hybrid screening in Experimental Example 1-2 can bind directly to CMV in vitro .

Experimental Example 3. in planta Direct interaction of NtTLP1 with CMV-1a protein

Immunprecipitation was performed to confirm the direct interaction of NtTLP1 with CMV 1a protein in planta .

Two repeats of the HA (hemagglutinin) epitope (YPYDVPDYA) were inserted into the SacI site of a modified pCAMBIA2300 vector (CAMBIA) with a 35S promoter and a Nos terminator. HA epitopes were fused on top of CMV la cDNA. Immunoprecipitation was performed by the application of Kraft et al. (Crofts, AJ, Leborgne-Castel, N., Pesca, M., Vitale, A. and Denecke, J., BiP and Calreticulin form an abundant complex that is independent of endoplasmic reticulum stress., Plant Cell , 10 , pp813-824, 1998).

In addition, the following experiment was carried out to confirm the interaction in polyethylene glycol-mediated protoplast transformation and in planta.

For in vivo targeting of NtTLP1, fragments with signal sequences were synthesized by PCR using primers NtTLP1-5 (5'-GGGATCCATGAACTTCCTCAAAAGCTTC-3 ') and NtTLP1 Nonstop (5'-AAGGATCCAAGGGCAGAAGACAACCC-3'). The NtTLP1-green fluorescent protein (NtTLP1-GFP) and CMV 1a-red fluorescent protein (CMV 1a-RFP) mixed constructs template the coding regions of NtTLP1 and CMV 1a cDNAs with the N-terminus of the GFP coding region as a template without a stop codon. Synthesized. The mixed construct was then subjected to a polyethylene glycol mediated transformation process described in Jin et al. (Jin, JB, Kim, YA, Kim, SJ, Lee, SH, Kim, DH, Cheong, GW and Hwang, I., A A new dynamin-like protein, ADL6, is involved from the trans-Golgi network to the central vacuole in Arabidopsis., Plant Cell , 13 , pp1511-1526, 2001) was inserted into Arabidopsis protoplasts prepared from seedlings. The expression of this mixed construct was observed using a Zeiss Axioplan fluorescence microscope (JENA) to observe the diversity of time points after transformation, and the images were captured by Axiocam digital cameras. Jena). To obtain the whole cell extract, protoplasts were harvested 17 hours after transformation and centrifuged at 50 g for 5 minutes, followed by 50 mM HEPES buffer (pH 7.5, 0.1 M NaCl, 2 mM EDTA, 2 mM). Immersed again in 200 L of 2-mercaptoethanol). The protoplasts were dissolved by sonication and centrifuged at 5,000 × g speed. The whole cell extract was then separated into a soluble layer and a membrane portion by ultracentrifugation at a rate of 100,000 × g. The pellets were then immersed back in the original volume of HEPES buffer. This fraction was analyzed by NtTLP1-GFP or CMV 1a by protein gel blot analysis using polyclonal anti-GFP (Clontech) or anti-HA antibodies (Clontech). It was used as a probe to confirm the presence of -2XHA. In the protein gel blot analysis, Shin et al. (Shin, R., Park, CJ, An, JM and Paek, KH, A novel TMV-induced hot pepper cell wall protein gene ( CaTin2 ) is associated with virus -specific hypersensitive response pathway., Plant Mol . Biol ., 51 , pp 687-701, 2003). The transported membranes were immunoblotted to correspond to the primary antibody (anti-HA, 1: 500 dilution; anti-GFP, 1: 500 dilution, Clontech).

As a result of the above experiment, as shown in FIGS. 3 and 4, the interaction between NtTLP1 and CMV 1a protein is NtTLP1 mixed with GFP (green fluorescent protein), and CMV 1a has HA (hemagglutinin) epitope attached thereto. Mutual immunoprecipitation experiments using transgenic Arabidopsis protoplasts were identified.

As shown in Figure 3, the molecular weight band can be found at 50.0 kDa, which is the size estimated to be recombinant NtTLP1 :: GFP in Arabidopsis protoplast extracts transfected with NtTLP1 :: GFP and CMV 1a :: HA. . No significant interactions could be identified in lane 1 from Arabidopsis protoplast extracts transfected with GFP and CMV 1a :: HA. As shown in FIG. 4, the synthesis of the HA-tagged CMV la protein in the control experiment was confirmed by Western blot addition of polyclonal HA antibody. The results show that NtTLP1 and CMV 1a form a protein complex in plant cells.

Experimental Example 4 Location of NtTLP1 and Location of NtTLP1 and CMV 1a Protein Complexes

4-1. Locating NtTLP1

In vivo targeting experiments were performed using a green fluorescence microscope (GFP) to determine the location of NtTLP1 in vivo . NtTLP1 cDNAs with only stop codons were mixed with GFP at the N terminus of the transformed Arabidopsis protoplasts to observe the position of GFP protein and NtTLP1 :: GFP mixed protein using a fluorescence microscope.

As a result of the experiment, the fluorescence of the NtTLP1 :: GFP mixed protein was accumulated inside the cell as shown in FIG. 5, which shows that the protein was distributed in the cell substrate at high concentration (CH of FIG. 5). Is chlorophyll). This is similar to the results of the paper by Krispils et al., Who estimated that mature proteins could be retained inside the cytoplasm despite the presence of signal sequences (Chrispeels, MJ and Tague, BW, Protein sorting in the secretory system of plant cells., Int Rev. Cytol ., 125 , pp 1-45, 1991). The presence of secretory signal sequences suggests that NtTLP1 is sent extracellularly, and it is generally known that acidic TLPs are released into the extracellular space (Bednarek, SY, Wilkins, TA, Dombrowski, JE and Raikhel, NV, A carboxyl). -terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco., Plant Cell , 2 , pp 1145-1155, 1990). Computer analysis using the pSORT program showed that NtTLP1 was located in the ER membrane (endoplasmic reticulum, certainty = 0.55), cell substrate (certainty = 0.10) and extracellular space (certainty = 0.10).

4-2. Confirmation of NtTLP1 Secretion Potential into Extracellular Space

In order to confirm the possibility of NtTLP1 secretion into the extracellular space, the NtTLP1 :: GFP mixed protein obtained in Experimental Example 3-1 was inserted into onion epithelial cells by applying a method such as Itaya (Itaya, A., Hickman, H., Bao, Y., Nelson, R. and Ding, B., Cell-to-cell trafficking of Cucumber mosaic virus movement protein: green fluorescent protein fusion produced by biolistic gene bombardment in tobacco., Plant J. , 12 , pp 1223-1230, 1997). The location of GFP protein and NtTLP1 :: GFP mixed protein was observed using a fluorescence electron microscope.

As a result of the experiment, the gene inserted under the control of the CaMV 35S promoter was expressed at high levels in the epithelial cells of onion cells. After its expression, fluorescence of the NtTLP1 :: GFP mixed protein was first detected in the periphery between the cell membrane and the cell wall of the plant cell, and low levels were observed in the cell substrate (see FIG. 8). On the other hand, it was confirmed that the fluorescence of the GFP protein was accumulated throughout the cell (see FIG. 7). These results show that NtTLP1 was located intracellularly and some of them were secreted extracellularly or cell wall.

4-3. Confirmation of NtTLP1 and CMV 1 Co-location in Plant Cells

In order to confirm whether NtTLP1 and CMV 1a protein are co-located in plant cells, the entire CMV 1a cDNA, which contains only stop codons, is mixed at the N-terminus of RFP, and the position of CMV 1a :: RFP is determined using a fluorescent electron microscope. Confirmed by.

After expression, red fluorescent CMV 1a :: RFP was observed in aggregate form in the cytosol. In a previously published paper, CMV 1a protein is known to be located in tonoplasts and vascular membranes (Cillo, F., Roberts, IM and Palukaitis, P., In situ localization and tissue distribution of the replication -associated proteins of Cucumber mosaic virus in tobacco and cucumber., J. Virol ., 76 , pp 19654-19664, 2002).

Arabidopsis protoplasts transfected with NtTLP1 :: GFP and CMV 1a :: RFP complexes were observed by fluorescence microscopy 24 hours after transformation.

As a result of the experiment, as shown in Figure 6, the green and red signals of Arabidopsis protoplasts transfected with NtTLP1 :: GFP and CMV 1a :: RFP can be confirmed within 24 to 30 hours after transformation It was clearly seen that the NtTLP1 :: GFP and CMV 1a :: RFP proteins were combined, indicating that the two proteins were co-located in the cytoplasm.

Experimental Example 5. Confirmation of interaction pattern of CMV-encoded protein of three TLPs

TLPs have varying pH values from strong acidic to strong bases (range of 3.4 to 12), so acidic, neutral and basic TLPs are present. All three types have been identified in tobacco at the protein level (Koiwa, H., Sato, F. and Yamada, Y., Characterization of accumulation of tobacco PR-5 proteins by IEF-immunoblot analysis. Plant Cell Physiol ., 35 , pp 821-827, 1994). Depending on the different physicochemical properties of these three types of TLPs, their positions in their cells also differ. While TLPs in blood vessels (or other vesicles) are basic, in general extracellular TLPs are acidic. The biological significance of these trends is not yet known. The following experiments were conducted to determine whether these trends of TLPs are related to the interaction with the CMV 1a protein.

Osmotin (basic) and pTOL1 (neutral) were separated and mixed in the GAL4 activation site to observe the interaction with CMV 1a protein. In addition, in order to confirm the positional role of TLPs in the replication and movement of virus, whether the three TLPs can interact with CMV-encoded protein, RNA-dependent RNA polymerase 2a, CP and MP in Experimental Example 1 The same method was performed through the East to Hybrid analysis.

As a result of the experiment, as shown in Table 2, NtTLP1 interacted with CP, MP as well as CMV 1a protein, but did not interact with CMV 2a protein. In addition, interaction with NtTLPs, CMV 1a protein, and CP was confirmed by the East Three Hybrid analysis using the pBridge vector system, but it was confirmed that MP did not form a complex with NtTLP1 and CMV 1a in this system (see FIG. 9). Osmotin and pTOL1, however, only interact with CMV 1a and do not interact with any other protein. It was also confirmed whether NtTLP1 could also interact with TMV replicase or TMV-CP, but it was found that it did not interact with them. These results indicate that NtTLP1 specifically interacts with CMV viral proteins, which affects not only the proliferation of CMV but also the rate and extent of proliferation.

Interaction Between TLPs and CMV Viral Proteins in Yeast Cells CMV viral protein NtTLP1 pTOL1 Osmotin 1a + + + 2a - - - MP + - - CP + - -

+: As a result of the β-galactosidase activity blue colony

     -: white colony as a result of β-galactosidase activity

Experimental Example 6. Expression Pattern of TLPs for CMV Inoculation

Expression patterns of TLPs were observed by RT-PCR analysis in tobacco plants. Total RNA is described in Osbel et al. (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Seidman, JG, Smith, JA and Struhl, K., Short protocols in molecular biology, New York: Wiley , 1995) was isolated from tobacco leaves at 4 weeks. For RT-PCR analysis, cDNA was synthesized from DNase I treated with the whole RNAs obtained using MMLV-reverse transcriptase (Promega) and 6-hexamer random primers.

NtTLP1 (5'- ATGAACTTCCTCAAAAG-3 ', 5'-TCAAGGGCAGAAGACAAC-3'), pTOL1 (5'- ATGAGTCACTTGACAAC-3 ', 5'-CTACTTGGCCACTTCATG-3'), Osmotin (5'- ATGGGCAACTTGAGATC-3 ', 5' '-CTACTTAGCCACTTCATC-3'), PR-1 (5'- GCGAAAACCTAGCTTGGGGAAG-3 ', 5'-TATATAACGTGAAATGGACGC-3'), PR-2 (5'-CAATGCAGCAACTTACTTGAG-3 ', 5'- ACTTACGAGAGAAGCAGTAGC-3') 25 PCR was performed for 25 times under conditions of denaturation at 94 ° C. for 3 minutes; [1 minute at 94 ° C .; 45 seconds at 57 ° C .; 2 minutes at 72 ° C.]; .

In addition, in order to rule out the possibility of any wounding-induced TLP gene that occurs when rubbing the carborundum, the temporary immunization treatment was induced as a control.

As a result of the experiment, as shown in FIG. 10, no measurable accumulation of TLP gene was found in the temporarily vaccinated leaves. NtTLP1 transcription accumulated from 1 day after CMV inoculation and accumulated the most at 5 days. Similar patterns of gene expression could be observed in the case of osmotin and pTOL1, although there are some changes. In the case of osmotin, transcription was observed on day 1 of CMV vaccination (on day 1) and increased significantly up to 5 days later, whereas transcription of pTOL1 was observed on day 1 after CMV vaccination, although the decrease decreased from day 4 This dullness did not change gene expression levels until after 5 days. Expression of CMV viral genes was confirmed by observation of the conserved 3 ′ ends of CMV RNAs used as positive controls for CMV proliferation. In addition, gene expression patterns of PR-1 and PR-2, which are known to be induced during CMV infection, have been identified (Whitham et al., 2003). The level of 18S rRNA is estimated as an internal standard for cDNA quantity. Additionally, to determine whether the interaction between CMV 1a and PRs induced by CMV infection is a general property of PRs, the interaction between CMV 1a and two PRs (PR-1 and PR-2) was measured. PR-2 as well as 1 was not found to interact with the CMV 1a protein (data not shown).

These results confirm that all three types of different TLP genes are induced in response to CMV infection.

Experimental Example 7 Observation of Changes in Infection Levels of Transgenic Tobacco Plants Overexpressing NtTLP1 Against CMV Infection

To further observe the biological function of NtTLP1 in planta , sense and antisense-orientated NtTLP1 transgenic tobacco plants were produced and analyzed in the following manner.

The plasmids used for the transformation of tobacco consisted of the sense and antisense orientation of the entire NtTLP1 cDNA, and the binary vector pMBP2 (Han, SJ, Cho, HY, You JS, Nam YW, Park EK, Shin JS). , cloned into the polylinker site (polylinker site) of the YI Park, Park WM and Paek KH, gene silencing-mediated resistance in transgenic tobacco plants carrying potato virus Y coat protein gene., Mol. Cells, 9, pp376-383, 1999) It was done. The construct was inserted into the tobacco plant (tobacco cv Smasun NN) using Agrobacterium-mediated transformations described in Hoekema et al. (Hoekema, A., Hirsch, P., Hooykaas) , PJJ and Schilperoort, R., A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid., Nature , 303 , pp179-180, 1983).

7-1. RT-PCR Experiment

RNA was extracted from both sense and antisense transgenic tobacco plants and RNA was analyzed by the same method as RT-PCR performed in Experimental Example 5.

7-2. Western blot analysis

Total leaf protein in transgenic plants was measured in Western blot using polyclonal antibodies for NtTLP1.

Total soluble leaf protein was extracted from the leaf tissues of T1 transgenic tobacco plants using Lindbo et al. (Lindbo, JA and Dougherty, WG, Pathogen-derived resistance to potyvirus: Immune and resistant phenotypes in transgenic tobacco expressing altered forms of a potyvirus coat protein nucleotide sequence. Mol . Plant Microbe Interact., 5, pp144-153, 1992). The concentration of the protein was quantified by Bradford assay, separated on 12% polyacrylamide gels and transported to polyvinylidene difluoride membranes (Millipore) using Hoefer SemiPhor (Amersham Pharmacia Biotech). Antibody immunoassay blot analysis of proteins from membrane to tissue was performed using La's method (La, YJ, Han JH, Sim, GB, Kim, BD and Ahn, KK, Detection of cymbidum mosaic virus and odontoglossum ringspot virus). in orchid plants by tissue-blot immunoassay., J. Kor . Soc . Hort . Sci ., 40 , pp 481-484, 1999). The primary antibody against NtTLP1 was used at a concentration of 3 g / ml, and then treated with a secondary antibody (anti-goat-rat IgG conjugated with alkaline phosphatases, Sigma) at a concentration of 1 g / ml.

As a result of performing the experiment, Figure 11 shows the results of RT-PCR and Western blot analysis of RNA and protein expression levels in transgenic plants, where the level of endogeneous NtTLP1 gene expression was low in untransformed plants. In addition, the levels of low endogeneous NtTLP1 gene expression were reduced in antisense transgenic tobacco plants. In contrast, gene expression levels of NtTLP1 in sense transgenic tobacco plants increased significantly. Significant amounts of NtTLP1 protein were found in sense transgenic tobacco plants, whereas significant NtTLP1 protein measurement was not possible in antisense transgenic tobacco plants.

7-3. ELISA experiment

It was observed whether the NtTLP1 transgenic tobacco plants exhibited an altered response to the inoculation of the virus. In each independent line, 10 leaves of 6 transgenic tobacco T1 plants and 4 leaves of 10 non-transgenic controls were infected with CMV and the virus proliferated.

Initially, the accumulation pattern of CP (coat proteins) was tested. At various time points after infection, both the inoculated leaves of the infected plants and the upper leaves of the inoculated leaves were tested, and the dot ELISA test was performed for the accumulation of CMV-Kor CP.

Indirect ELISA tests (ELISA) were performed on CMV observations. All reagents were used at 150 μl / well except for the blocking step (200 μl / well) on microtiter plates. Leaf disks (8 mm diameter) were placed in a microcentrifuge tube and powdered with a grinder in 200 μl of extraction buffer (0.1 M Tris-HCl with 1% sodium sulfite, pH 7.4). Extracts diluted with threefold 0.15M carbonate buffer at pH 9.8 were incubated in wells at 37 ° C. for 3 hours. After three washes with 0.01 M Tris-HCl containing 0.85% sodium chloride and 0.05% Tween-20 (TBST) at pH 7.4, the plate was washed with 0.1 M Tris-HCl with 0.85% sodium chloride (TBS) at pH 7.4. Blocked with 1% BSA for 40 minutes. After incubation with the addition of the antigen, the plates were washed with TBST and incubated for 2 hours with the addition of 3 μg / ml of CMV antibody. The plate was washed again and incubated again with the addition of 1 μg / ml secondary antibody. After washing three times with TBST, a substrate solution containing p-nitrophenyl phosphate (1 mg / ml) was added to 0.2 M carbonate buffer at pH 9.8, and 30 minutes after addition at A ₄₀₅ nm with ELISA Reader (Bio-Rad). Measured.

As a result of the experiment, as shown in Figure 12, the accumulation of CMV-Kor CP in the inoculated leaves of the sense transgenic tobacco plants was 9-38% reduced compared to the non-transgenic plants. Similar results were seen in upper leaves vaccinated up to 12 days after infection (see FIG. 13). After 16 days of inoculation, NtTLP1-overexpressing transgenic plants showed a similar sensitivity to controls (see FIG. 13).

7-4. RNA blot analysis

Also, RNA blot analysis was performed for viral RNA accumulation in the uninoculated upper leaf tissue at 6 DPI 6 days post infection.

As a result of the above experiment, the degree of CMV RNA accumulation in sense transgenic tobacco plants was reduced by 4 -23% compared to that of the nontransformants as shown in FIG. In contrast, antisense transgenic tobacco plants were unaffected with respect to both CMV RNA accumulation or CP accumulation patterns. These results confirm that NtTLP1 overexpression inhibits CMV proliferation.

As described above, NtTLP1 (Nicotiana tabacum thaumatin-like protein 1) of the present invention as a host factor of the CMV 1a protein interacting with a protein belonging to the PR-5 group of the PR protein bunil from Nikko tiahna other bakeom, in NtTLP1 gene expression is directly the result of CMV attack in vitro as well as in planta , and transgenic plants overexpressing NtTLP1 gene have significantly reduced susceptibility to CMV infection, and NtTLP1 Since yeast interacts with CMV MP and CP, NtTLP1 inhibits the replication and migration of CMV virus and functions as an antiviral protein for CMV, which can be used as a CMV antiviral agent. In addition, by transgenic with pMBP2 recombinant vector containing the full-length cDNA of NtTLP1 and overexpressing NtTLP1, transgenic tobacco plants having resistance to the cucumber mosaic virus can be used in manufacturing to reduce damage to the cucumber mosaic virus.

<110> Korea University Industry and Academy Cooperation Foundation <120> An antiviral agent comprising NtTLP1 isolated from Nicotiana          tabacum to CMV infection <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 681 <212> DNA <213> Nicotiana tabacum <220> <221> CDS (222) (1) .. (678) <400> 1 atg aac ttc ctc aaa agc ttc ccc ttt ttt gcc ttc ctt tat ttt ggc 48 Met Asn Phe Leu Lys Ser Phe Pro Phe Phe Ala Phe Leu Tyr Phe Gly   1 5 10 15 caa tac ttt gta gct gtt act cat gct gcc act ttt gac att gtc aac 96 Gln Tyr Phe Val Ala Val Thr His Ala Ala Thr Phe Asp Ile Val Asn              20 25 30 aaa tgc acc tac aca gtc tgg gcc gcg gcc tct cca ggt gga ggc agg 144 Lys Cys Thr Tyr Thr Val Trp Ala Ala Ala Ser Pro Gly Gly Gly Arg          35 40 45 cgg ctc gac tca ggc caa tct tgg agc att aat gtg aac cca gga aca 192 Arg Leu Asp Ser Gly Gln Ser Trp Ser Ile Asn Val Asn Pro Gly Thr      50 55 60 gtc cag gct cgc att tgg ggt cga acc aat tgc aac ttc gat ggc agt 240 Val Gln Ala Arg Ile Trp Gly Arg Thr Asn Cys Asn Phe Asp Gly Ser  65 70 75 80 ggc cga ggt aat tgt gag act gga gac tgt aac ggg atg tta gag tgt 288 Gly Arg Gly Asn Cys Glu Thr Gly Asp Cys Asn Gly Met Leu Glu Cys                  85 90 95 caa ggc tat gga aaa gca cct aac act tta gct gaa ttt gca ctt aat 336 Gln Gly Tyr Gly Lys Ala Pro Asn Thr Leu Ala Glu Phe Ala Leu Asn             100 105 110 caa ccc aat cag gac ttt gtc gac atc tct ctt gtt gat gga ttt aac 384 Gln Pro Asn Gln Asp Phe Val Asp Ile Ser Leu Val Asp Gly Phe Asn         115 120 125 atc ccc atg gaa ttc agc ccg acc aat gga gga tgt cgt aat ctc aga 432 Ile Pro Met Glu Phe Ser Pro Thr Asn Gly Gly Cys Arg Asn Leu Arg     130 135 140 tgc aca gca cct att aac gaa caa tgc cca gca cag ttg aaa aca caa 480 Cys Thr Ala Pro Ile Asn Glu Gln Cys Pro Ala Gln Leu Lys Thr Gln 145 150 155 160 ggt gga tgt aac aac cca tgt act gtg ata aaa acc aat gaa tat tgt 528 Gly Gly Cys Asn Asn Pro Cys Thr Val Ile Lys Thr Asn Glu Tyr Cys                 165 170 175 tgt aca aat ggg cct gga tca tgt ggg cct act gat ttg tcg aga ttt 576 Cys Thr Asn Gly Pro Gly Ser Cys Gly Pro Thr Asp Leu Ser Arg Phe             180 185 190 ttt aag gaa aga tgc cct gat gct tat agt tat cca cag gat gat cca 624 Phe Lys Glu Arg Cys Pro Asp Ala Tyr Ser Tyr Pro Gln Asp Asp Pro         195 200 205 acc agt ttg ttt acg tgt cct tct ggt act aat tac agg gtt gtc ttc 672 Thr Ser Leu Phe Thr Cys Pro Ser Gly Thr Asn Tyr Arg Val Val Phe     210 215 220 tgc cct tg a 681 Cys pro 225 <210> 2 <211> 226 <212> PRT <213> Nicotiana tabacum <400> 2 Met Asn Phe Leu Lys Ser Phe Pro Phe Phe Ala Phe Leu Tyr Phe Gly   1 5 10 15 Gln Tyr Phe Val Ala Val Thr His Ala Ala Thr Phe Asp Ile Val Asn              20 25 30 Lys Cys Thr Tyr Thr Val Trp Ala Ala Ala Ser Pro Gly Gly Gly Arg          35 40 45 Arg Leu Asp Ser Gly Gln Ser Trp Ser Ile Asn Val Asn Pro Gly Thr      50 55 60 Val Gln Ala Arg Ile Trp Gly Arg Thr Asn Cys Asn Phe Asp Gly Ser  65 70 75 80 Gly Arg Gly Asn Cys Glu Thr Gly Asp Cys Asn Gly Met Leu Glu Cys                  85 90 95 Gln Gly Tyr Gly Lys Ala Pro Asn Thr Leu Ala Glu Phe Ala Leu Asn             100 105 110 Gln Pro Asn Gln Asp Phe Val Asp Ile Ser Leu Val Asp Gly Phe Asn         115 120 125 Ile Pro Met Glu Phe Ser Pro Thr Asn Gly Gly Cys Arg Asn Leu Arg     130 135 140 Cys Thr Ala Pro Ile Asn Glu Gln Cys Pro Ala Gln Leu Lys Thr Gln 145 150 155 160 Gly Gly Cys Asn Asn Pro Cys Thr Val Ile Lys Thr Asn Glu Tyr Cys                 165 170 175 Cys Thr Asn Gly Pro Gly Ser Cys Gly Pro Thr Asp Leu Ser Arg Phe             180 185 190 Phe Lys Glu Arg Cys Pro Asp Ala Tyr Ser Tyr Pro Gln Asp Asp Pro         195 200 205 Thr Ser Leu Phe Thr Cys Pro Ser Gly Thr Asn Tyr Arg Val Val Phe     210 215 220 Cys pro 225  

Claims (4)

Nicotiana tabacum Thaumatin-like protein 1 (NtTLP1) encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against Cucumber mosaic virus (CMV). According to claim 1, wherein the NtTLP1 protein, characterized in that belonging to the PR-5 group isolated from nicotiana tabacum. An antiviral agent containing NtTLP1 encoded by the gene sequence of SEQ ID NO: 1 having antiviral activity against cucumber mosaic virus as an active ingredient. A transgenic tobacco plant having resistance to a cucumber mosaic virus by transforming with a pMBP2 recombinant vector containing the gene sequence of SEQ ID NO: 1 and overexpressing NtTLP1.

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