• 농업생명과학연구
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• 제52권6호
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• pp.27-36
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• 2018

김치가 세계 5대 건강식품으로 선정되어 세계인의 주목을 받게 되면서 갓의 수요도 점점 증가하고 있어 다양한 갓품종의 육성이 절실히 요구되는 실정이다. 그러나 아직 품종보급 체계가 미흡하여 자가 채종에 의한 자식약세현상, 순도저하, 상품의 불균일 등의 문제점이 발생하고 있다. 본 실험에서는 갓의 F1 품종 육성체제를 갖추기 위하여 유전자원으로 수집한 갓의 순계분리와 자식계통의 육성, 웅성불임인자의 도입을 통하여 고품질의 갓을 육성하기 위하여 각 조합을 공시하여 조합능력을 검정하고(인도자사이${\times}$고흥담양)조합과 (분리 MS 유기계-MS910${\times}$일본 적자색갓 8${\times}$강화갓 9) 분리계 조합이 우수하다고 판단되어 양친으로 선발하였다. PCR(Polymerase Chain Reaction)과 염기서열 분석을 통해 CMS type을 확인해 본 결과, MS계통 MSLS와 MSLC는 갓에서 보고된 orf263, orf220 및 orf288 CMS 유전자를 미토콘드리아 DNA에 포함하고 있는 것으로 나타났다. 최종적으로 생산력검정 및 농가 실증시험을 실시하였다.

• International Journal of Industrial Entomology and Biomaterials
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• 제15권2호
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• pp.115-122
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• 2007

As the genome of B.mori is available in GenBank and the EST database of B.mori is expanding, identification of novel genes of B.mori is conceivable by data-mining techniques. We used the in silico cloning method to get the vacuolar-type $H^+-ATP$ synthetase (V-ATPase) c subunit (16 kDa proteolipid subunit) gene of B.mori and analysed with bioinformatics tools. The result was confirmed by RT-PCR and sequencing. The V-ATPase c subunit cDNA contains a 468 bp ORF. The ORF encoded a 155-residue protein that showed extensive homology with V-ATPase c subunits from other 15 species and contained four membrane-spanning helices. Tissue expression pattern analysis revealed that V-ATPase c expressed strongly in Malpighian tubules, not in fat body. This gene has been registered in GenBank under the accession number EU082222.

• The Plant Pathology Journal
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• 제26권2호
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• pp.130-138
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• 2010

Orchid fleck virus (OFV) is an unassigned plant virus in the family Rhabdoviridae. OFV was isolated from Cymbidium sp. showing oval necrotic lesions on their leaves in Korea, and designated as OFV-NHHS1. The complete nucleotide sequence of the RNA1 (6,413 nt) (GenBank accession no. AB516442) and RNA2 (6,001 nt) (GenBank accession no. AB516441) was determined in this study. RNA1 and RNA2 contained five and one ORF respectively. RNA1 encodes nucleocapsid (N) of 49 kDa, ORF2 of 26 kDa, ORF3 of 38 kDa, ORF4 of 20 kDa and glycoprotein (G) of 61 kDa proteins, whereas RNA2 encodes a single polymerase of 212 kDa. OFV-NHHS1 shared extremely high similarity of 98.6-100% and 98.9-99.6% in nucleotidle and amino acid sequences with a Japanese isolate, OFV-so, respectively. However, the N, G and L of OFV-NHHS1 revealed 6.9-19.3%, 7.3-12.0%, and 13.4-26.6% identities to those of 29 Rhabdoviruses, respectively. To survey the infection rate of OFV in commercial orchids in Korea, 51 Cymbidium sp., 10 Phalaenopsis sp., 22 Oncidium sp. and 21 Dendrobium sp. plants that showed typical viral symptoms were collected. RT-PCR with specific primers for detection of Cymbidium mosaic virus (CymMV), ORSV and OFV showed high infection rate by ORSV alone and double infection by ORSV and CymMV. One of the orchids tested was infected with OFV. This is the first report of the complete nucleotide sequences of OFV isolated in Korea.

• International Journal of Industrial Entomology and Biomaterials
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• 제24권1호
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• pp.23-27
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• 2012

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus associated with Postweaning multisystemic wasting syndrome (PMWS), which is considered to be an important infectious swine viral disease. PCV2 capsid protein encoded by ORF2 is a structural protein and expected as the high immunogenicity protein. In this study, we generated recombinant baculovirus containing ORF2 of PCV2 and analyzed the optimal conditions for the production of capsid protein in insect cell. Production and status of recombinant capsid protein in insect cell were confirmed by SDS-PAGE and Western blot analysis using His tag antibody and anti-PCV2 serum. The yield of recombinant capsid protein was high like as shown visible on SDS-PAGE. Optimal multiplicity of infection (MOI) and infection time of recombinant virus were determined as 5 MOI and 4 days, respectively. ORF2 is known to have N-linked glycosylation site, but we couldn't detect the glycosylation of recombinant protein in insect cells.

• International Journal of Industrial Entomology and Biomaterials
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• 제30권2호
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• pp.50-57
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• 2015

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has type-specific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti-His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

• Journal of Microbiology and Biotechnology
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• 제15권4호
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• pp.873-879
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• 2005

Red sea bream iridovirus (RSIV) obtained from infected rock bream was propagated by Bluegill fry-2 (BF-2) cell culture. The virus titer was determined as $10^{5.5}\;TCID_{50}/ml$ on confluent BF-2 cell monolayers. The integrin binding site of ORF 055L of infectious spleen and kidney necrosis virus (ISKNV) was selected for the construction of a primer to obtain the RSIV ORF 055L gene. The genes were amplified using RSIV gene lyzate by PCR. The homologies of the ORF 055L sequence of RSIV with ISKNV and rock bream iridovirus (RBIV) were approximately $96\%$ and $100\%$, respectively. DNA vaccine was constructed by cloning the ORF 055L of RSN into pcDNA 3.1 (+), containing a cytomegalovirus (CMV) promoter. For antibody production, pcDNA-055 DNA vaccine was injected to BALB/c mice. The production of antibodies against pcDNA-055 DNA vaccine was confirmed by the Western blot analysis. The antibodies produced by the pcDNA-055 DNA vaccine showed efficacy to neutralize the RSIV in the neutralization test in BF-2 cell culture.

• Genomics & Informatics
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• 제19권4호
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• pp.47.1-47.12
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• 2021

Kaposi's sarcoma-associated herpesvirus (KSHV) is one of the few human oncogenic viruses, which causes a variety of malignancies, including Kaposi's sarcoma, multicentric Castleman disease, and primary effusion lymphoma, particularly in human immunodeficiency virus patients. The currently available treatment options cannot always prevent the invasion and dissemination of this virus. In recent times, siRNA-based therapeutics are gaining prominence over conventional medications as siRNA can be designed to target almost any gene of interest. The ORF57 is a crucial regulatory protein for lytic gene expression of KSHV. Disruption of this gene translation will inevitably inhibit the replication of the virus in the host cell. Therefore, the ORF57 of KSHV could be a potential target for designing siRNA-based therapeutics. Considering both sequence preferences and target site accessibility, several online tools (i-SCORE Designer, Sfold web server) had been utilized to predict the siRNA guide strand against the ORF57. Subsequently, off-target filtration (BLAST), conservancy test (fuzznuc), and thermodynamics analysis (RNAcofold, RNAalifold, and RNA Structure web server) were also performed to select the most suitable siRNA sequences. Finally, two siRNAs were identified that passed all of the filtration phases and fulfilled the thermodynamic criteria. We hope that the siRNAs predicted in this study would be helpful for the development of new effective therapeutics against KSHV.

• 식물병연구
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• 제12권3호
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• pp.213-220
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• 2006

2003년 청송지역 농가포장에서 심한 위축증상을 나타내는 콩 시료를 채집하였다. 이 시료를 RT-PCR로 진단한 결과 콩위축바이러스, Soybean dwarf virus(SbDV-L81)에 감염된 것으로 확인되었다. 본 연구는 우리나라에서 콩위축바이러스의 발생에 관한 최초 보고이다. 이 바이러스의 추가적인 특징을 알아보기 위하여 종 특이적 프라이머를 이용한 RT-PCR로 게놈의 부분 염기서열을 결정하였다. 결정된 염기서열은 병징 발현 유형과 매개충 특이성에 기초하여 이전에 조사된 SbDV 계통들과 비교분석하였다. ORF 2와 ORF 3 유전자사이 영역(intergenic region)과 ORF 2, ORF 3 및 ORF 4의 코팅영역에서 SbDV-L81은 황화계통(SbDV-YS and YP) 보다 위축계통(SbDV-DS and DP)과 유사한 특징을 보였다. 한편, ORF 5의 5'쪽 코딩영역은 완두수염진딧물(Acyrthosiphon pisum)이 매개충인 계통(SbDV-YP and DP)과 밀접한 관련이 있은 것으로 나타났다. SbDV-L81은 자연상태에서의 병정과 5가지 영역의 염기서열 비교결과에서 SbDV-DP 계통과 밀접한 관련이 있는 것으로 생각된다.

• Archives of Pharmacal Research
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• 제22권6호
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• pp.542-548
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• 1999

The UL47 gene (b 60390-b 60388) located in the unique long region of the human cytomegalovirus (HCMV) AD169 strain genome was analyzed RNA mapping. Northern blot analysis showed that the UL47 gene was expressed at late times after infection (72 h postinfection). The 9.7-kb transcript was expressed in the infected cells but not in phosphonoformate-treated cells at 72 hpi, indicating that the UL47 gene was only expressed at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer extension and RNase protection analysis were performed. Primer extension analysis revealed that the transcription initiation site of UL47 was located in 27 bp downstream (b 60323) of the TATA box motif. The sizes of UL47 ORF (approximately 2.9-kb) and UL48 ORF (approximately 6.7-kb) deduced from computer sequence analysis suggest that the expressed 9.7-kb transcript of UL47 uses the 3'-end polyadenylation signal of Ul48. The result of RNase protection determined that the 3'-end of UL47 RNA utilized the 3'-end polyadenylation signal of UL48, which is located in HCMV genome b 70082.

• Journal of Microbiology
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• 제38권4호
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• pp.230-238
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• 2000

Class III chitin synthases in filamentous fungi are important for hyphal growth and differentiation of several filamentous fungi. A genomic clone containing the full gene encoding Chs4, a class III chitin synthase in Penicillium chrysogenum, was cloned by PCR screening and colony hybridization from the genomic library. Nucleotide sequence analysis and transcript mapping of chs4 revealed an open reading frame (ORF) that consisted of 5 exons and 4 introns and encoded a putative protein of 915 amino acids. Nucleotide sequence analysis of the 5'flanking region of the ORF revealed a potential TATA box and several binding sites for transcription activators. The putative transcription initiation site at -716 position was identified by primer extension and the expression of the chs4 during the vegetative growth was confirmed by Northern blot analysis. Amino acid sequence analysis of the Chs4 revealed at least 5 transmembrane helices and several sites for past-transnational modifications. Comparison of the amino acid sequence of Chs4 with those of other fungi showed a close relationship between P chrysogenum and genus Aspergillus.