KR101372363B1 - Method for Screening Anti-Viral Agents - Google Patents

Abstract

The present invention relates to a method for screening an antiviral agent, and more particularly, to a method for screening an antiviral agent comprising the following steps: (a) a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein Preparing a; (b) treating the sample of analysis to the virus-infected plant; And (c) measuring a fluorescence signal generated from the fluorescent protein in the virus-infected plant treated with the sample; The sample is determined to be an antiviral agent when it is determined that the fluorescence signal generated from the fluoroprotein is reduced compared to the untreated control sample. Compared with the prior art, the antiviral screening method of the present invention can rapidly assay multiple samples simultaneously.

Description

Method for Screening Anti-Viral Agents

The present invention relates to antiviral screening methods.

More than 1,200 plant viruses have been reported worldwide, many of them causing economic damage to crops. In particular, Korea suffered economic damage of 463 ha (Choi, 2001) and KRW 50 billion nationwide by CGMMV (Cucumber Green Mottle Mosaic Tobamovirus), which occurred in watermelon in 1998, and annually in peppers caused by viruses such as CMV (Cytomegalovirus). More than 30 billion won is incurred. In rice, RSV (Respiratory syncytial virus, Lee et al . (2008), which occurred at about 4,663 ha in Gyeonggi, Ganghwa, and 5,000 ha in 2007, in Buan, Jeonbuk, and Seocheon, South Chungcheong Province, caused huge damages in yield. . Since there is no direct control of such viruses, many researchers are trying to develop new plant virus control agents because of the difficulty in preventing the spread of damage. However, while the development of new plant virus control agents is difficult to develop materials, it also takes a lot of time and labor to commercialize the effects on the developed materials.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The inventors have sought to develop a rapid screening method for controlling pathogenic plant viruses. As a result, the present inventors completed the present invention by constructing a method for screening an antiviral agent using a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein.

Accordingly, it is an object of the present invention to provide a method for screening an antiviral agent.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention provides a method for screening an antiviral agent comprising the following steps:

(a) preparing a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein;

(b) treating the sample of analysis to the virus-infected plant; And

(c) measuring a fluorescence signal generated from the fluoroprotein in the virus-infected plant treated with the sample; The sample is determined to be an antiviral agent when it is determined that the fluorescence signal generated from the fluoroprotein is reduced compared to the untreated control sample.

The inventors have sought to develop a rapid screening method for controlling pathogenic plant viruses. As a result, the inventors have established a method for screening antiviral agents using virus-infected plants infected with plant viruses comprising nucleotide sequences encoding fluorescent proteins.

Hereinafter, the screening method of the present invention will be described in detail for each step.

Step (a): Preparation of Virus-Infected Plant

One of the main features of the present invention is the preparation of virus-infected plants infected with plant viruses comprising nucleotide sequences encoding fluorescent proteins.

As used herein, the term 'fluorescent protein' refers to Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Cyan Fluorescent Protein (CFP), Yellow Fluorescent Protein (YFP), Blue Fluorescent Protein (BFP), Enhanced Green Fluorescent Protein (EGFP). ), Enhanced Cyan Fluorescent Protein (ECFP), Enhanced Yellow Fluorescent Protein (EYFP), Enhanced Red Fluorescent Protein (ERFP), or Enhanced Blue Fluorescent Protein (EBFP).

According to a preferred embodiment of the present invention, the fluorescent protein is GFP (Green Fluorescent Protein).

Preferably, the plant virus that can be used in the screening method of the present invention is Tobacco mosaic virus (TMV), Pepper mottle virus (PepMoV), BBWV (3 strains BBWV-1, BBWV-2, BBWV-3; Broad bean wilt virus ), Bean common mosaic virus (BCMV), Butterbur mosaic virus (BMV), bean yellow mosaic virus (BYMV), Carnation mottle virus (CarMV), Chinese yam necrotic mosaic virus (ChYNMV), Carnation latent virus (CLV), CMV ( Cucumber mosaic virus), Chrysanthemum virus B (CVB), Cymbidium mosaic virus (CymMV), Daphne virus S (DVS), Garlic latent virus (GarLV), Garlic allexivirus (GarV), Kalanchoe latent virus (KLV), and LalacRSpot (Lilac ringspot) virus), LSV (Lily symptomless virus), LYSV (leek yellow stripe virus), NeLV (Nerine latent virus), NLV (Narcissus latent virus), OFV (Orchid fleck virus), ORSV (Odontoglossum ringspot virus), PeMoV (Peanut mottle) virus), PepMoV (Pepper mottle virus), PVM (Potato virus M), PVS (Potato virus S), SLV (Shallot latent virus) or TRV (Tobacco rattle virus), more preferred It is a TMV (Tobacco Mosaic Virus) or PepMoV (Pepper Mottle Virus).

A plant virus comprising a nucleotide sequence encoding the fluorescent protein of the present invention is infected with a plant to prepare a virus-infected plant.

As used herein, the term “plant” is understood as including all plant cells, plant tissues, plant organs (eg, leaves of plants), seeds of plants, and the like that can develop into mature plants.

Preferably, the plant is Nicotiana spp., Acineta spp., Aerides japonicum ), garlic ( Allium sativum ), Peanut ( Arachis hypogaea), Aran in Serra (Aranthera spp.), shrimps field (Calanthe discolor ), pepper ( Capsicum annuum ), Cattleya spp., Chenopodium amaranticolor ), chrysanthemum ( Chrysanthemum morifolium ), Crocus spp., Cymbidium spp., Daphne odora ), Dendrobium spp., Carnation ( Dianthus) caryophyllus ), hippo ( Dioscorea oposita cultivars ), Doritaenopsis spp., Epidendrum spp., Gladiolus hybridus ), Grammatophyllum spp., Kalanchoe blossfeldiana ), Lilium spp., Lycaste spp., Tomato ( Lycopersicon esculentum ), Narcissus spp., Nerine bowdenii cultivars , Odontoglossum spp., Petasites officinalis ), Phalenopsis spp., Potinara spp., Potato ( Solanum) tuberosum ), lilac ( Syringa vulgaris ), Burntweed ( Tetragonia) expansa ), Vanda spp., Vicia faba ), Eastern ( Vigna unguiculata ) or genus Zygopetalum spp., more preferably Nicotiana genus plants, most preferably Nicotiana benthamiana And Nicotiana tabacum It is a plant.

Preferably, the plant of step (b) is a leaf of a plant, more preferably a leaf disc of leaf.

According to a preferred embodiment of the present invention, the plant virus expressing a foreign fluorescent protein is a sequence in which the fluorescent protein-coding nucleotide sequence is inserted in-frame in the genome sequence of PepMoV (see FIG. 11) or the fluorescent protein in the genome sequence of TMV. -The coding nucleotide sequence is constructed using the sequence inserted in frame.

In the present invention, in the production of a virus-infected plant, a direct inoculation method of inoculating plants with a plant virus comprising the nucleotide sequence encoding the fluorescent protein, as well as a nucleotide sequence encoding the fluorescent protein to the plant to introduce the virus Indirect methods of inducing production and physical treatment (eg, grinding) to inoculate other plants.

In carrying out the indirect method, a method of introducing an exogenous nucleotide sequence into a plant is a method generally known in the art (Methods of Enzymology, Vol. 153, 1987, Wu and Grossman Edit, Academic Press; Is incorporated by reference). The exogenous polynucleotide can be inserted into a carrier such as a vector such as a plasmid or a virus to transform the plant, and Agrobacterium bacteria can be used as a medium (Chilton et al., 1977, Cell 11: 263: 271; Plants can be transformed by introducing foreign polynucleotides directly into plant cells (Lorz et al., 1985, Mol. Genet. 199: 178-182; the content of which is incorporated herein by reference). Inserted). For example, when a vector not containing a T-DNA region is used, electroporation, microparticle bombardment, and polyethylene glycol-mediated uptake may be used.

Step (b): Processing of Sample

According to the method of the present invention, a sample of analyte is treated to a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein.

As used herein, the term 'sample' refers to an unknown substance used in screening to test whether it affects the infectivity or survival of a plant virus.

The sample includes, but is not limited to, chemicals, antibodies, proteins, peptides, nucleotides, antisense-RNAs, small interference RNAs (siRNAs), and natural extracts. The sample analyzed by the screening method of the present invention is a single compound or a mixture of compounds (eg, a cell or tissue culture). Samples can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). Samples can be obtained by a variety of combinatorial library methods known in the art, for example biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution required By a synthetic library method, a “1-bead 1-compound” library method, and a synthetic library method using affinity chromatography screening. Methods of synthesizing molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed . Engl. 33,2059,1994; Carell et al., Angew . Chem . Int . Ed . Engl . 33, 2061; Gallop et al., J. Med. Chem . 37, 1233, 1994, and the like.

The method of treating a sample of the analyte of the present invention to a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein may be carried out using various methods known in the art. According to a preferred embodiment, a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein is fragmented into a section disk to contain a liquid medium containing a sample of analysis (eg, MS (Murashige-Skoog's)]. To process

One of the main features of the present invention is the fragmentation of virus-infected plants infected with plant viruses comprising nucleotide sequences encoding fluorescent proteins into fragment disks. When the leaves of the plant are sliced into slice discs, multiple samples can be screened quickly and simultaneously.

Step (c): Measurement of Fluorescence Signal

Finally, the fluorescence signal generated from a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding the treated fluorescent protein of the sample of analysis of the present invention is measured.

According to a preferred embodiment, the measurement of the fluorescent signal in step (c) of the present invention can be carried out using a variety of methods known in the art, most preferably using a fluorescence microscope.

A fluorescent signal generated from a virus-infected plant infected with a plant virus comprising a nucleotide sequence encoding a fluorescent protein treated with a sample of the analyte of the present invention is generated from the fluorescent protein, compared to a control without the sample. If it is determined that the fluorescence signal is reduced, the sample is determined to be an antiviral agent.

As used herein, the term 'decrease' refers to a fluorescence signal generated from a virus-infected plant that is infected with a plant virus that includes a nucleotide sequence that encodes a fluorescent protein to which the sample has been treated as compared to a control that has not been sampled (eg, conventional 10-100% reduction, preferably 30-100% reduction, more preferably 50-100% reduction, and most preferably 80-100% reduction). to be.

According to another aspect of the invention, the invention provides a method for screening an antiviral agent comprising the following steps:

(a) processing a sample of analyte on a plant;

(b) inoculating the sample treated plant with a plant virus transformed with a nucleotide sequence encoding a fluorescent protein and expressing the fluorescent protein; And

(c) measuring the fluorescent signal generated from the fluorescent protein in the plant; The sample is determined to be an antiviral agent when it is determined that the fluorescence signal generated from the fluoroprotein is reduced compared to the untreated control sample.

The method of the present invention is a method in which the steps of (a) and (b) of the screening method are reversed, and the contents common between the two are omitted in order to avoid excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an antiviral screening method.

(b) The antiviral screening method of the present invention can rapidly assay multiple samples simultaneously.

(c) The antiviral screening method of the present invention is more economical compared to the existing RT-PCR method.

1 shows N. tabacum cv. Inoculated with PepMoV-GFP. Show Samsun nn
Figure 2 is N. tabacum cv. Inoculated with PepMoV-GFP. Show control of Samsun nn.
Figure 3 shows the results confirming the antiviral activity against PepMoV-GFP of World Star Eco concentration of 0.2%, 0.4% and 1%.
4 shows the results of confirming antiviral activity against PepMoV-GFP of Good Morning at concentrations of 0.2%, 0.4% and 1%.
5 shows the results of confirming antiviral activity against PepMoV-GFP of KN1020 at concentrations of 0.5%, 1.0% and 2.0%.
Figure 5 shows the results confirmed by RT-PCR antiviral activity against TMV-GFP of World Star Eco, Good Morning and KN1020 at 0.2%, 0.4% and 1% concentration, respectively. FIG. 5A shows the results of RT-PCR using PepMoV specific primers, and FIG. 5B shows the results of RT-PCR using GFP specific primers. Columns M in FIGS. 5A and 5B are 1 kb markers, columns 1 to 3 are World Star Eco at 1.0%, 0.4% and 0.2% concentrations, and columns 4 to 6 are 1.0%, 0.4% and 0.2% concentrations. Good morning, and columns 7 to 9 are the results for KN1020 at 2.0%, 1.0% and 0.5% concentrations. Columns '-' and '+' indicate negative and positive controls.
Figure 6 shows the image of the antiviral screening method of sectioning leaves inoculated with TMV.
Figure 7 shows the results confirming the antiviral activity against TMV-GFP of World Star Eco concentration of 0.2%, 0.4% and 1.0%.
8 shows the results of confirming antiviral activity against TMV-GFP of Good Morning at concentrations of 0.2%, 0.4% and 1.0%.
9 shows the results of confirming antiviral activity against TMV-GFP of KN1020 at concentrations of 0.2%, 0.4% and 1%.
Figure 10 shows the results confirmed by RT-PCR antiviral activity against TMV-GFP of World Star Eco, Good Morning and KN1020 at concentrations of 0.5%, 1.0% and 2.0%, respectively.
Fig. 11 schematically shows a vector of PepMoV-GFP (pSP6PepMoV-Vb1 / GFP). NIb is Nuclear Inclusion b, CP is Coat Protein, P1 and P3 are Protein 1 and Protein 3, HC-Pro is Helper Component Protease, Cl is Cylindrical Inclusion, Vpg is 'Viral protein linked to the genome' , Pro is Protease.
12 shows a schematic of the vector of TMV-GFP. T7 is the T7 promoter, RdRP is the RNA-dependent RNA Polymerase, MP is the Movement Protein, and CP is the Coat Protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Antiviral agent

World Stae Eco (National Institute of Agricultural Research, Korea) and Good Morning (National Institute of Agricultural Research, Korea) and plant cell extracts, respectively, antiviral agents derived from Acinetobacter antiviralis and Pseudomonas oleovorans culture medium KN1020 (Gangwon-do Agricultural Research and Extension Services, Korea) was used.

The antiviral agent was diluted to distilled water or MS medium at an appropriate concentration. World Star Eco and Good Morning were tested at 1.0%, 4.0% and 0.2% concentrations, and KN1020 at 2.0%, 1.0% and 0.5% concentrations.

Plant viruses

To test antiviral activity, TMV (Tobacco Mosaic Virus, U1) and PepMoV (Pepper Mottle Virus) were used. TMV is the systemic host of TMV, Nicotiana tabacum cv. Maintained at Samsun nn.

To test antiviral activity, TMV-GFP (Shailaja et al., 1999) and PepMoV-GFP (SP6PepMoV-Vb1 / GFP (FIG. 11), labeled with TMG and PepMoV with Green Fluorscence Protein (GFP), Lee et al. , 2011]. Brief description of the preparation method of the TMV-GFP or PepMoV-GFP is as follows:

100 ng of TMV-GFP 1056 treated with KpnI restriction enzyme was added to T7 RNA polymerase (Promega, USA) and 100 ng of pSP6PepMoV-Vb1 / GFP treated with 4U or SacII restriction enzyme was added to SP6 RNA polymerase (Promega, USA). 10 μl of 4U and transcription optimization 5X buffer, 5 μl of 100 mM DTT, 2.5 μl rNTPs mix (rATP, rUTP and rCTP) 10 μl, 0.5 mM rGTP 10 μl, 2.5 mM RNA Cap analog (New England Biolabs, England) 5 μl After adding to 50 μl total with distilled water, and treated for 1 hour at 37 ℃ to prepare a transcript.

Nicotiana, two weeks old, prepared by transcript benthamiana is inoculated on carborundum-treated leaf surfaces (mechanical inoculation). 7-14 days after the inoculation, GFP was expressed by inoculating the plants to be prepared with leaf fragments used in the antiviral screening method using the juice of the leaves expressing the GFP with a UV lamp.

TMV-GFP consists of 5'NTR, replicaase and transfer proteins derived from TMV-U1. TMV-GFP was maintained in N. benthamiana , a systemic infection host.

All viruses were provided by the Plant Virus Gene Bank of Seoul Women's University.

Plant

Tobacco ( N. tobacum cv. Samaun NN) was used to test antiviral activity against TMV. N. benthamiana and N. tabacum cv. Samsun nn was used as a host for TMV-GFP and PepMoV-GFP, respectively. In each experiment, plants with similar growth stages were used and maintained in a greenhouse for 16 hours.

All plants were provided by Plant Virus GenBank (PVGB) of Seoul Women's University.

Antiviral Leaf Slice Screening System

To screen antiviral activity against TMV-GFP, N. benthamiana was inoculated with TMV-GFP and infected leaves were sectioned and placed on plates. After washing the surface of the leaves for 5 minutes with distilled water containing Tween 20, disinfect the surface of the leaves with 70% ethanol (including twin 20) for 30 seconds and after washing for 5 minutes with 2% hypochlorous acid (including twin 20) Washed with. A 0.5 ml tube was used to separate leaf sections approximately 0.5 cm in diameter and float in culture medium. All steps were performed in a clean-bench.

MS (Murashige-Skoog's) comprising vitamins (2.0 mg / l glycine, 100 mg / l myo-inositol, 0.5 mg / l nicotinic acid, 0.5 mg / l pyridoxine and 0.1 mg / l thiamine) and 20 g / l sucrose ) Liquid medium was used for leaf section culture (Murashige and Skoog, Duchefa, The Netherlands). The antiviral activity of the culture medium containing each antiviral agent (World Star Eco, Good Morning and KN1020) was analyzed. Antiviral agents were diluted to the appropriate concentration in MS liquid medium. World Star Eco and Good Morning were diluted to 1.0%, 0.4% and 0.2% concentrations and KN1020 to 2.0%, 1.0% and 0.5% concentrations. 24-well culture plates (SPL, South Korea) were used for the leaf section culture. Each well contains 1.5 ml MS medium containing the appropriate concentration of antiviral agent.

All leaf sections were incubated at room temperature for 7 days. Antiviral agents were examined daily by observing under fluorescence and visible light. The presence of virus in the leaf sections was confirmed by RT-PCR.

Antiviral agent Telegraph  Screening systems

To analyze antiviral activity against PepMoV-GFP, N. tabacum cv. Samsun nn was used. The leaves were treated with World Star Eco, Good Morning or KN1020 containing Tween 20 with a brush. As a control, distilled water containing Tween 20 was treated to the leaves. Antiviral agents were diluted in distilled water at appropriate concentrations. World Star Eco and Good Morning were tested at 1.0%, 0.4% and 0.2% concentrations, and KN1020 was tested at 2.0%, 1.0% and 0.5% concentrations.

After 1 hour, each leaf was inoculated with PepMoV-GFP. To prepare virus inoculum, systemic infected 0.01 g N. tabacum cv. Samsun nn leaves were ground in 2 ml inoculation buffer (0.01 M potassium phosphate buffer, pH 7.2). The virus was mobilized using N. tabacum cv. Using Carborundum (fisher Scientific, USA). Samsun nn leaves were inoculated. The plants were maintained in a greenhouse for 16 hours photoperiod and 25 ° C.

Botanical RNA  detach

Total RNA of the leaf sections was extracted. Leaf sections and leaf tissues were homogenized in 1.5 ml tubes of extraction solution (50 mM Tris-Cl, pH 8.0), 10 mM EDTA, 0.1% SDS and 0.1% 2-mercaptoethanol. The reaction mixture was extracted twice with phenol: chloroform: isoamyl alcohol (PCI, 25: 24: 1, v / v / v). Total RNA was precipitated by treatment with three volumes of complete ethanol and stored at −80 ° C. for 30 minutes. After centrifugation, the total RNA precipitated was washed with 70% ethanol and dried in a vacuum centrifuge (Tomy, USA). The result of RNA extraction was dissolved in RNase-free distilled water.

RT - PCR

RT-PCR was performed using RNA extracted from the leaf-section as a substrate. RT reaction was performed at 42 ° C. for 60 minutes at 3 μl total RNA, 50 mM KCl, 4 mM MgCl 2, 10 mM DTT, 2.5 mM each dNTP mixture, 0.5 μl 10 pMol reverse primer and 6.7 unit M-MuLV reverse transcriptase (Fermentas, USA ) Was performed with My Cycler ^™ Thermal Cycler (Bio-rad, USA) in 50 mM Tris-HCl buffer (pH 8.3).

1.7 in 30 μl containing synthetic cDNA, PCR buffer (10 mM Tris-HCl, pH 8.3), 1.5 mM MgCl ₂ and 50 mM KCl, 10 pMol primer pairs (forward and reverse primers) and 2.5 mM of each dNTP mixture CDNA was amplified using unit recombinant Taq DNA polymerase (Takara, Japan). cDNA was amplified 30 cycles using MyCycler ^™ Thermal Cycler (Bio-Rad, USA). Denaturation was performed at 94 ° C. for 30 seconds, primer annealing was performed at 55-58 ° C. (depending on the primer) for 30 seconds, and DNA extension was performed at 72 ° C. for 1 minute. 30 cycles were complete and extended at 72 ° C. for 10 minutes. RT-PCR products were identified through 1.0% agarose gel electrophoresis and Ethidium bromide (EtBr) gel staining.

Primers for identifying PePMoV-GFP and TMV-GFP are shown in Table 1. PePMoV-CP and turboGFP primers were used to detect PePMoV-GFP and TMVp50 primers were used for TMV-GFP detection.

Experiment result

PepMoV Antiviral activity against

The antiviral activity of antiviral agents (World Star Eco, Good Morning and KN1020) against PepMoV was tested. World Star Eco and Good Morning were treated at 1.0%, 0.4% and 0.2% concentrations, and KN1020 at 2.0%, 1.0% and 0.5% concentrations. N. tobacum cv. The leaves of Samsun nn were treated with antiviral agents and one hour later, inoculated with PepMoV-GFP.

Four days after inoculation, the leaves treated with 0.4% and 0.2% of World Star Eco showed low GFP expression compared to the control, and the mouth treated with 0.5% KN1020 showed no change compared to the control. In contrast, no other treatment group showed GFP expression (FIGS. 1-4).

At 8 days post inoculation, Good Morning treated with 0.4% and 0.2% World Star Eco and 1.0%, 0.4% and 0.2% concentrations showed lower GFP expression compared to the control. Leaves treated with 0.5% concentration of KN1020 showed no change compared to the control. In contrast, the leaves treated with 1.0% WorldStar Eco and 2.0% and 1.0% KN1020 did not show GFP expression (FIGS. 1-4).

At 12 and 16 days after inoculation, there was no difference from the control in the leaves treated with all concentrations of World Star Eco and Good Morning. However, leaves treated with KN1020 at 2.0% and 1.0% concentrations did not show GFP (FIGS. 1-4).

World Star Eco, Good Morning and KN1020 treated leaves were subjected to RT-PCR using primers specific for PepMoV and GFP. Leaves treated with KN1020 at 1.0% and 2.0% concentrations did not show bands by primers specific for PepMoV and GFP (FIG. 5).

TMV Antiviral activity against

Antiviral candidates (World Star Eco, Good Morning, and KN1020) were treated at different concentrations using a mass assay system of antiviral agents to observe GFP expression levels for one week. As a result, in the leaf sections treated with World Star Eco and Good Morning, GFP was observed up to 7 days irrespective of the concentration (Figs. 6 and 7), but in 1.0% and 0.4% KN1020, GFP expression decreased after one day of treatment, and 0.2%. In KN1020, GFP expression was decreased from two days after treatment, and thus GFP was not observed on day 7 (FIG. 7). To verify this result, RT-PCR analysis was carried out by separating total RNA of leaf fragments which observed GFP expression with UV lamps on the 7th day, and World Star Eco and Good Morning treated leaf fragments regardless of concentration. Infected virus was assayed (FIG. 10). On the other hand, RT-PCR analysis of the total RNA of KN1020-treated leaf sections showed that no infected virus was detected regardless of the treated concentration of KN1020 (FIGS. 9 and 10). These results showed the same results as the GFP expression observation results in the antiviral mass assay system, which could verify the reliability of the analysis. The antiviral mass assay system developed in this study can replace the RT-PCR assays used to test new antiviral agents, furthermore, has the economic advantages of time and cost.

references

Choi, GJ 2001. Occurrence of two tobamovirus diseases in cucurbits and control measures in korea. The Plant Pathology Journal . 17 (5): 243-248

Bartos, J., Kubin, S. and Setlik, I. 1960. Dry weight increase of leaf disks as measure of photosynthesis. Biologia Plantarum . 2 (3): 201-215

Lee, BC, Yoon, YN, Hong, SJ, Hong, YK, Kwak, DY, Lee, JH, Yae, US, Kang, HW and Hwang, HG 2008. Analysis on the occurrence of rice stripe virus. Research in plant Disease 14 (3): 210-213

Lee, MY, Song, YS and Ryu, KH 2011. Development of infectious transcripts from full-length and GFP-tagged cDNA clones of Pepper mottle virus and stable systemic expression of GFP in tobacco and pepper. Virus Research . 155 (2): 487-494

Shailaja, S., Gregory, PP, Dennis, JL, Joann, H., Jonathan, D., Laurence, KG and William, OD 1999.Heterologous Sequences Greatly Affect Foreign Gene Expression in Tobacco Mosaic Virus-Based Vectors, Viology 255: 312-323

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (8)

Screening method of antiviral agent comprising the following steps:
(a) preparing a virus-infected plant infected with Tobacco Mosaic Virus (TMV) or Peptper Mottle Virus (PepMoV) comprising a nucleotide sequence encoding a fluorescent protein;
(b) treating the sample of analysis to the virus-infected plant; And
(c) measuring a fluorescence signal generated from the fluoroprotein in the virus-infected plant treated with the sample; The sample is determined to be an antiviral agent when it is determined that the fluorescence signal generated from the fluoroprotein is reduced compared to the untreated control sample.
Screening method of antiviral agent comprising the following steps:
(a) processing a sample of analyte on a plant;
(b) inoculating the sample treated plant with a Tobacco Mosaic Virus (TMV) or Peptper Mottle Virus (PepMoV) transformed with a nucleotide sequence encoding a fluorescent protein and expressing the fluorescent protein; And
(c) measuring the fluorescent signal generated from the fluorescent protein in the plant; The sample is determined to be an antiviral agent when it is determined that the fluorescence signal generated from the fluoroprotein is reduced compared to the untreated control sample.
delete delete The method of claim 1 or claim 2, wherein the plant is a tobacco (Nicotiana spp.), Know Neta (Acineta spp.), I pungran (Aerides japonicum), garlic (Allium sativum), peanut (Arachis hypogaea), Aran sera ( Aranthera spp.), Calanthe discolor , Capsicum annuum , Cattleya spp., Chenopodium amaranticolor , Chrysanthemum morifolium , Crocus spp., Cymbidium spp. , Daphne odora , Dendrobium spp., Carnation ( Dianthus caryophyllus ), Hemp ( Dioscorea oposita cultivars ), Doritaenopsis spp., Epidendrum spp., Gladiolus hybrid ( Gladiolus hybrid ), Grammatophyllum spp., Kalanchoe blossfeldiana , Lilium spp., Lycaste spp., Tomato ( Lycopersicon esculentum ), Narcissus ( Narcissus spp.), Nerine bowdenii cultivars ), Odontoglossum spp., Butterbur ( Petasites officinalis), Palais higher system (Phalenopsis spp.), Fourteen Countries (Potinara spp.), Potato (Solanum tuberosum), lilac (Syringa vulgaris), tetragonia tetragonioides (Tetragonia expansa), Vanda (Vanda spp.), Broad bean (Vicia faba ), Eastern ( Vigna unguiculata ) or zygopetalum spp.
6. The method according to claim 5, wherein the plant is Nicotiana spp.
The method of claim 1, wherein the plant of step (b) is a leaf of a plant.
8. The method of claim 7, wherein the leaf of the plant is a slice disc of the leaf.

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