• Journal of the Korean Society of Tobacco Science
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• v.20 no.1
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• pp.50-56
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• 1998

The complementary DNA (cDNA) of potato virus Y- vein necrosis strain (PVY-VN) replicase gene (Nlb) was transformed into tobacco (Nicotiana tabacum cv. Burley 21) plants. Out of 25 putative transformants regenerated, 3 were resistant to PVY-VN, one highly resistant plant with no symptom until seed harvest time and the other two with mild chlorotic spot symptoms at late stages after infection. No symptom was observed in the highly resistant plant, while mild vein necrotic symptoms were developed on suckers of the moderately resistant plants after seed harvest time, In the first generation (T1) via self fertilization, resistance to susceptibility frequency in transgenic plants from the highly resistant transformant was about 3 : 1, while it was lowered much (about 1:2 and 1:19) in T1 of the moderately resistant transformants. In the second generation (T2) of the highly resistant plant, resistance frequencies were similar to T1, but resistance levels varied greatly and appeared to be decreased. Key words : potato virus Y, viral replicate gene, transgenic tobacco plants, resistance.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.144.1-144
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• 2003

Attenuated viruses can protect their hosts against challenge to their related viruses. Increasing evidence shows that mutations of the tobamoviral 126/183 kDa protein play a major role in the viral attenuation and contribute to the cross protection mechanism. In this study, four mutants of Pepper mild mottle virus (PMMoV) have been constructed by mutagenesis; two mutants, pTPpoly348 and pTPpoly762, were substituted in the middle of replicase gene, and the others, pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219, were deletion mutants made by deleting some parts of pseudoknot structures of the 3' noncoding region (NCR) of the virus. Progeny viruses generated from the four mutants were infectious on N. benthamiana plants with symptomless or mild mosaic symptom. Replication efficiency and viral product accumulations of four mutants were assessed by Northern and Western blot analyses on BY-2 protoplast cells. Accumulation of CP for the pTPL3D:: $\Delta$6207 and pTPL3D:: $\Delta$6219 were lower than that of other mutants and wild type virus. These data suggest that the 3'-NCR mutations contribute to the viral gene expression in host tissues, while mutants of replicase gene rather govern the symptom expression.

• Journal of the Korean Society of Tobacco Science
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• v.19 no.1
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• pp.11-17
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• 1997

A flue-cured tobacco variety (Nicotiana tabacum cv. Wisconsin) was used for Plant transformation with the complementary DNA (cDNA) of potato virus Y-necrosis strain (PVY-VN) replicase gone (Nb) which was synthesized through reverse-transcription Primed with oligo(dT) and Polymerization using RNase H-digested template. The cDNA was cloned into Plant expression vector Plasmid (PMBP2), and introduced into tobacco plants by co-culturing tobacco leaf disks with Agrobacterium tumefaciens LBA4404 containing the plasmid before Plant regeneration. Eight Plants, in which the inserted cDNA fragment was detected by Polymerase chain reaction (PCR), out of 70 putative transformants inserted with sense-oriented Mb cDNA showed no symptom at 3 weeks after inoculation, while the other 62 plants, and all plants with vector gone only and antisense-oriented NIb cDNA had susceptible vein-necrosis symptoms. However, only 2 of the 8 resistant plants were highly resistant, which remained symptomless up to 10 weeks after inoculation. Among the first progenies (T1) from self-fertilized seeds of the two resistant transgenic plants, less than 10 % of 71 plants appeared highly resistant (with no symptom), 70% moderately resistant (with mild symptoms on 1 - 2 leaves), and about 20% susceptible (with susceptible symptoms on 3 or more leaves) at 3 weeks after inoculation. These results suggest that the PVY resistance was inherited in the 71 generation. Key words : potato virus Y. viral replicase gene, transgenic tobacco Plants, resistance.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.115.2-116
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• 2003

Complete genomic nucleotide sequence of Daphe virus S (DVS), a member of the genus Carlavirus, causing leaf distortion and chlorotic spot disease symptoms in daphne plants, has been determined in this study. The genome of DVS contained six open reading fames coding for long viral replicase, triple gene block, 36 kDa viral coat protein (CP) and 12 kDa from the 5' to 3' ends, which is a typical genome structure of carlaviruses. Two Korean isolates of DVS isolates were 98.1％ and 93.6％ amino acid identical in the CP and 12kDa, respectively. The CP gene of DVS shares 25.2-55.2％ and 42.9-56.1％ similarities with that of 19 other carlaviruses at the amino acid and nucleotide levels, respectively. The 3'-proximal 12 kDa gene of DVS shares 20.2-57.8％ amino acid identities with that of 18 other members of the genus. The 3' noncoding region of DVS consists of 73 nucleotides with long excluding poly A tract, and shares 69.1-77.1％ identities to the known carlaviruses. In the phylogenetic analyses of the two proteins, DVS was closely related to Helenium virus S and Chrysanthemum virus B. This is the first complete sequence information for the DVS, and further confirms the classification of DVS as a distinct species of the genus Carlavirus.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.113.1-113
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• 2003

A potexvirus, Hosta virus X (HVX-Kr), causing mosaic and mottle symptoms was isolated from hosta plants (Hosta spp.), and its entire genome RNA sequence was determined. in Korea using cDNA library and RACE methods. The genome of HVX encodes five open reading frames coding for viral replicase, triple gene block (TGB), and viral coat protein (CP) from the 5'to 3' ends, which is a typical genome structure of potexviruses. The 3-terminal region of the virus includes the TGBI (26 kDa), TGB2 (13 kDa), TGB3 (8 kDa), and 23 kDa coat protein (CP) and the 3-nontranslated region (NTR). The CP gene of the type isolate of HVX (HVX-U) was amplified by RT-PCR and its nucleotide sequence was determined. The CPs of HVX-Kr and HVX-U had 100％ and 98.9％ identical amino acids and nucleotides, respectively. Most of the regions of the genome HVX had over 50％ nucleotide identical to other sequenced potexviruses. This is the first report of complete genome sequence information of HVX and molecular evidence supporting the virus as a distinct species of the genus Potexvirus.

• Journal of Microbiology and Biotechnology
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• v.29 no.3
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• pp.482-488
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• 2019

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. Replicase (Rep) proteins are considered essential for viral replication. Capsid (Cap) protein is the primary immunogenic protein that induces protective immunity. Little is known about comparison on the immunogenicity of PCV2 Rep and Cap fusion protein and Cap protein. In the present study, recombinant baculoviruses expressing the Rep-Cap fusion protein (Bac-Rep-Cap) and the Cap protein (Bac-Cap) of PCV2 were constructed and confirmed with western blot and indirect fluorescence assay. Immunogenicities of the two recombinant proteins were tested in mice. The titers of antibodies were determined with a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The $IFN-{\gamma}$ response of immunized mice was measured by ELISA. The mice immunized with the Bac-Rep-Cap and Bac-Cap successfully produced Cap-specific immunoreaction. The mice immunized with the Bac-Cap developed higher PCV2-specific neutralizing antibody titers than mice injected with the Bac-Rep-Cap. $IFN-{\gamma}$ in the Bac-Rep-Cap group was increased compared to those in the Bac-Cap group. Vaccination of mice with the Bac-Rep-Cap showed significantly decreased protective efficacy compared to the Bac-Cap. Our findings will indubitably not only lead to a better understanding of the immunogenicity of PCV2, but also improved vaccines.

• BMB Reports
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• v.47 no.6
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• pp.330-335
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• 2014

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3'-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected.

• Journal of Life Science
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• v.22 no.4
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• pp.552-558
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• 2012

Severe acute respiratory syndrome (SARS) is a severe respiratory infectious disease caused by a novel human coronavirus, SARS-CoV. The 3CL protease is a key enzyme in the proteolytic processing of replicase polyprotein precursors, pp1a and pp1ab, which mediate all the functions required for viral genomic replication and transcription. Therefore, this enzyme is a target for the development of chemotherapeutic agents against SARS. A large quantity of active SARS-3CL protease is required for development of anti-SARS agents. Here we have constructed overexpression vector for the production of the SARS-3CL protease. The gene encoding SARS-3CL protease was amplified using polymerase chain reaction and cloned into the pET29a expression vector, resulting in pET29a/SARS-3CLP. Recombinant SARS-3CL protease was successfully synthesized by the dialysis mode of the cell-free protein expression system, and purified by three-step fast protein liquid chromatography using HighQ and MonoP column chromatographies and Sephacryl S-300 gel filtration. In addition, the produced SARS-3CL protease was found to be an active mature form. This study provides efficient methods not only for the development of anti-SARS materials from natural sources, but also for the study of basic properties of the SARS-3CL protease.

• Proceedings of the Korean Society of Tobacco Science Conference
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• 1997.10a
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• pp.134-152
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• 1997

Plant viruses of tobacco including tobacco mosaic virus (TMV) and potato virus Y (PVY) cause severe economic losses in leaf-tobacco production. Cultural practices do not provide sufficient control against the viruses. Use of valuable resistant cultivars is most recommendable for the control of the viruses. However, conventional breeding programs are not always proper for the development of virus-resistant plants mostly owing to the frequent lack of genetic sources and introduction of their unwanted properties. Therefore, we tried to develop virus-resistant tobacco plants by transforming commercial tobacco cultivars, NC 82 and Burley 21, with coat protein (CP) or replicase (Nlb) genes of TMV and PVY necrosis strain (PVY-VN) with or without untranslated region (UTR) and with or without mutation. Each cDNA was cloned and inserted in plant expression vectors with 1 or 2 CaMV 35S promotors, and introduced into tobacco leaf tissues by Agrobacterium tumefaciens LBA 4404. Plants were regenerated in kanamycin-containing MS media. Regenerated plants were tested for resistance to TMV and PVY In these studies, we could obtain a TMV-resistant transgenic line transformed with TMV CP and 6 genetic lines with PVY-VN cDNAs out of 8 CP and replicase genes. In this presentation, resistance rates, verification of gene introduction in resistant plants, stability of resistance through generations, characteristics of viral multiplication and translocation in resistant plants, and resistance responses relative to inoculum potential and to various PVY strains will be shown. Yield and quality of leaf tobacco of a promising resistant tobacco line will be presented.