• Korean Journal of Agricultural Science
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• v.38 no.2
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• pp.249-255
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• 2011

This study was performed to investigate the characterization of infectious BLV env gene isolated form Korean Holstein Cattle and to determine its incoming origin. Gp51 region of BLV env gene known as having important role in immunological function was characterized using PCR-RFLP sequencing and phylogenetic analysis. BLV env gene was grouped into PCR-RFLP patterns with three restriction endonucleases including Pvu II, BamHI and Hae III, and we identified two new RFLP patterns from nucleotide sequences of each group. Phylogenetic analysis showed that 80% of the Korean Holstein was included in the USA and Japanese group. These results here can provide a valuable information about the character of the BLV env gene and research on infection route of BLV.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.7
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• pp.916-920
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• 2013

The triploid Pacific oyster, which is produced by mating tetraploid and diploid oysters, is favored by the aquaculture industry because of its better flavor and firmer texture, particularly during the summer. However, tetraploid oyster production is not feasible in all oysters; the development of tetraploid oysters is ongoing in some oyster species. Thus, a method for ploidy verification is necessary for this endeavor, in addition to ploidy verification in aquaculture farms and in the natural environment. In this study, a method for ploidy verification of triploid and diploid oysters was developed using multiplex polymerase chain reaction (PCR) panels containing primers for molecular microsatellite markers. Two microsatellite multiplex PCR panels consisting of three markers each were developed using previously developed microsatellite markers that were optimized for performance. Both panels were able to verify the ploidy levels of 30 triploid oysters with 100% accuracy, illustrating the utility of microsatellite markers as a tool for verifying the ploidy of individual oysters.

• BMB Reports
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• v.39 no.1
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• pp.22-25
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• 2006

The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.456-459
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• 1992

The genes of Halohacteria. gvpD and gvpE. take part in formation of gas vesicle. These mRNA cause a lot of experimental prohlems due to its eharacteristic instahility in the analysis of transcripts. This study allowed easy cloning and sequencing of RNA hy substituting a stable complementary DNA for the mRNA of the genes for an analysis. The weak 111 RNA was reverse transcribed to DNA using reverse transcriptase. and was amplified using PCR method. The transcripts confirmed in this ~,tudy have not heen round in the northern hybridization covering almost all ranges of ORF of the gene. gvpD.

• Food Science and Biotechnology
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• v.15 no.2
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• pp.232-237
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• 2006

Bacillus cereus group comprising B. cereus, B. thuringiensis, and B. mycoides was differentiated by polymerase chain reaction (PCR) and colony morphology. Prevalence of B. cereus group in rice and distribution of enterotoxin genes were determined as possible food poisoning agents. PCR using primers targeted for gyrB and cry genes could distinguish B. thuringiensis from B. cereus, and B. mycoides was differentiated by rhizoid morphological characteristics on nutrient agar. Among 136 rice and their processed products, prevalence of B. cereus group was 40%. B. cereus group consisted of 54 B. cereus, 11 B. thuringiensis, and 1 B. mycoides. Major isolates were B. cereus, with B. thuringiensis detected up to 10% among edible rice tested. Five enterotoxin genes, hbl, nhe, bceT, entFM, and cytK, were broadly distributed among B. cereus group, especially in B. cereus and B. thuringiensis. Prevalence of B. cereus group in rice and enterotoxin distribution suggest B. thuringiensis and B. cereus are toxigenic strain that should be controlled in rice and its products.

• Horticultural Science & Technology
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• v.32 no.4
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• pp.544-549
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• 2014

Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf and bulb samples showing characteristic symptoms of virus infection were collected in 2012, and 80 field samples were analyzed by reverse transcription polymerase chain reaction (RT-PCR). The infection frequencies were 79% for LMoV, 5% for LSV, and 3% for CMV. The LMoV coat protein gene was amplified and cloned into the pET21d(+) expression vector to develop serological diagnostic tools to detect LMoV. The resulting carboxy-terminal His-tagged coat proteins were expressed in Escherichia coli strain BL21 (DE3) by induction with IPTG. The recombinant proteins were purified using Ni-NTA agarose beads and used as an antigen to produce polyclonal antibodies in laying hens. The resulting egg yolk immunoglobulin (IgY) specifically recognized LMoV from infected plant tissues in immunoblotting assays and had comparable sensitivity to that of a mammalian antibody. In addition, method of immunocapture RT-PCR using this IgY was developed for sensitive, efficient, and rapid detection of LMoV. Based on these results, large-scale bulb tests and detection of LMoV in epidemiological studies can be performed routinely using this IgY. This is the first report of production of a polyclonal IgY against a plant virus and its use for diagnosis.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.182-190
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• 2003

A new phaC gene cluster encoding polyhydroxyalkanoate (PHA) synthase I PHA depolymerase, and PHA synthase II was cloned using the touchdown PCR method, from medium-chain length (mcl-) PHA-producing strain Pseudomonas putida KT2440. The molecular structure of the cloned phaCl gene was analyzed, and the phylogenic relationship was compared with other phaCl genes cloned from Pseudomonas species. The cloned phaCl gene was expressed in a recombinant E. coli to the similar level of PHA synthase in the parent strain P. putida KT2440, but no significant amount of mcl-PHA was accumulated. The isolated phaCl gene was re-introduced into the parent strain P. putida KT2440 to amplify the PHA synthase I activity, and the recombinant P. purida accumulated mcl-PHA more effectively, increasing from 26.6 to $43.5\%$. The monomer compositions of 3-hydroxylalkanoates in mcl-PHA were also modified significantly in the recombinant P. putida enforcing the cloned phaCl gene.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.334-339
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• 1999

More innovative molecular markers were investigated for rapid and consistent differentiation of Metarhizium anisopliae var. majus from M. anisopliae var. anisopliae. A total of 28 isolates were obtained from various countries and hosts: 13 isolates of M. anisopliae var. anisopliae, 12 isolates of M. anisopliae var. majus, and 3 isolates of M. anisopliae collected in Korea. This study involved restriction enzyme digestions of a PCR product amplified from nuclear internally transcribed spacer (ITS) and a portion of the 28S rDNA regions. Among 11 different restriction enzymes used in this study, MboⅠ digestion particularly produced a restriction pattern that had characteristics of M. anisopliae var. majus. This restriction pattern was consistent in all isolates of M. anisopliae var. majus regardless of their geographic origins and insect hosts. Mapping experiments revealed that MboⅠ sites of M. anisopliae var. majus are identical to those of M. anisopliae var. anisopliae with an exception for the presence of an additional site in the PCR product. Results from this study provide an additional method for identification and differentiation of isolates of these two varieties of M. anisopliae for use in the field and laboratory experiments.

• The Journal of Korean Society of Virology
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• v.29 no.1
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• pp.23-31
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• 1999

Hantavirus is a genus of the Bunyaviridae family causing two serious diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Puumala virus is a member of hantavirus originally found in Europe, and its natural reservoir is Clethrionomys glareolus. It is also associated with the human disease nephropathia epidemica, a milder form of HFRS. To identify the hantaviruses in bats, bats were collected from Jeong-Sun, Won-Joo, Chung-Ju and Hwa-Cheon area in Korea, and nested RT-PCR was performed with serotype specific primer from M segment. Interestingly, Puumala virus was detected in bats (Rhinolophus ferrum-equinum) only from Won-Joo. The 327 bp nested RT-PCR product, was sequenced. The sequence database search indicates that the sequence is homologous to the published sequence of Puumala viruses. The sequence similarities were ranged from 71% to 97%. The highest sequence similarity was 97% with Puumala virus Vranicam strain, and the lowest was 71% with Puumala virus K27 isolate. Puumala virus Vranicam strain was isolated from a bank vole (Clethrionomys glareolus) in Bosnia-Hercegovina. Puumala virus K27 was isolated from human in Russia. This analysis confirms that bats (Rhinolophus ferrum-equinum) in Korea are natural reservoir of Puumala virus.

• Horticultural Science & Technology
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• v.35 no.3
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• pp.323-332
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• 2017

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.