• 농업과학연구
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• 제42권2호
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• pp.99-103
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• 2015

Potato virus T (PVT) is a plant pathogen in the family Betaflexiviridae, group IV single-stranded positive sense RNA viruses. The major host of PVT is potato, and it has been reported in Ullucus tuberosus, Oxalis tuberosa and Tropaeolum tuberosum. This study aimed at developing reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR techniques for specific detection of PVT. Finally, Two RT-PCR primer sets were developed and verified. The RT-PCR products were amplified to 734 (PVT RT-PCR primer set 6) and 828 bp (PVT RT-PCR primer set 29) long to detect PVT. The nested PCR primer sets [PVT-N70/C20 ($734{\rightarrow}315bp$) and PVT-N75/C30 ($828{\rightarrow}529bp$)] were developed which are high sensitivity and verification for detection of PVT. Furthermore, a modified-positive control plasmid is use to verify contamination of laboratory in PVT detection. This study supported the diagnose PVT in potato or PVT related hosts.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 추계학술대회
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• pp.33-33
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• 2018

This study was conducted to examine the role of the transcription factors (TFs) (RsMYB1 and mPAP1+B-Peru) in the regulation of anthocyanin biosynthesis in the ornamental torenia cv. Kauai rose. In this study, we could produce several putative transgenic lines overexpressing the TFs via Agrobacterium-mediated transformation, and presence of the TFs in the randomly selected five transgenic lines was confirmed using polymerase chain reaction (PCR). According to results of reverse transcription-PCR analysis (RT-PCR), the expression of the TFs in all transgenic lines and of the anthocyanin structural genes (CHS, F3H, DFR, and ANS) in all transgenic lines and WT plants were distinctly detectable. However, transcript levels of the structural genes expressed in the transgenic lines overexpressing TFs were significantly higher than those expressed in WT plants. Therefore, it is suggested that anthocyanin content in flowers of the transgenic torenia would be significantly higher than that in flowers of WT plants. Moreover, these results indicate that the TFs (RsMYB1 and mPAP1+B-Peru) could be exploited as potential anthocyanin regulatory TFs to enhance anthocyanin content in the other horticultural plants.

• 대한약침학회지
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• 제15권2호
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• pp.20-23
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• 2012

Panax ginseng is a well-known herbal medicine in traditional Asian medicine, and wild ginseng is widely accepted to be more active than cultivated ginseng in chemoprevention. However, little has actually been reported on the difference between wild ginseng and cultivated ginseng. Thus, to identify and analyze those differences, we used suppressive subtraction hybridization (SSH) sequences with microarrays, realtime polymerase chain reaction (PCR), and reverse transcription PCRs (RT-PCRs). One of the clones isolated in this research was the chloroplast rpoC1 gene, a ${\beta}$subunit of RNA polymerase. Real-time RT-PCR results showed that the expression of the rpoC1 gene was significantly upregulated in wild ginseng as compared to cultivated ginseng, so, we conclude that the rpoC1 gene may be one of the important markers of wild ginseng.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.146.2-147
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• 2003

Two Korean isolates of Alfalfa mosaic virus (AHV-AZ, AMV-KR) were isolated from azuki bean and potato plants, respectively, and their pathologies were confirmed on some susceptible host plants including pepper, tobacco and red bean plants. Full length cDNAs to RNA1, RNA2 and RNA3 of the two Korean strains were amplified using the long-template reverse transcription (RT)-polymerase chain reaction (PCR) method. RT-PCR products covering entire regions for the three AMV genome RNAs were cloned. RNA transcripts were synthesized in vitro from each clones using T7 RNA polymerase and infectivity test was peformed in 9 reassortment sets of transcripts. All the combinations of reassorted transcripts were found to be infectious when inoculated onto Nicotiana benthamiana plants, and were not distinguishable to those of wild types. The full-length cDNA clones that were confirmed infectious were sequenced their nucleotide sequences. We will discuss sequence analysis of the two Korean isolates of AMV genomic RNA3 and compare reported foreign isolates of AMV.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.736-738
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• 2003

Ornithine decarboxylase (ODC) is the key enzyme in the synthetic pathway of polyamines. This enzyme is a homodimeric and a pyridoxal 5-phosphate (PLP) dependent enzyme. We have isolated, a cDNA clone encoding ODC from brain cDNA library constructed from flounder (Paralichthys olivaceus). The ODC cDNA contained a complete ORF consisting of 460 amino acids and one stop codon with comparison to nucleotide sequences of the flounder, zebrafish and rat ODC genes, the ODC genes were highly conserved. The transcription of ODC was analyzed with reverse transcription-polymerase chain reaction (RT-PCR) species in brain, kidney, liver, and embryo.

• Fisheries and Aquatic Sciences
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• 제4권1호
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• pp.25-31
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• 2001

4-Aminobutyrate aminotransferase plays an essential role in the 4-aminobutyric acid shunt, converting 4-aminobutyrate to succinic semi aldehyde. We isolated and sequenced' a fish cDNA fragment that encodes 4-aminobutyrate aminotransferase. A brain cDNA library from flounder (Paralichthys olivaceus) was constructed using the ZAP- III XR vector and screened for the fish 4-aminobutyrate aminotransferase gene using a probe derived from the conserved sequences of known mammalian 4-aminobutyrate aminotransferases. A partial cDNA for 4-aminobutyrate aminotransferase was cloned and found to be 700 bp in length corresponding to 66 amino acids. Nucleotide sequence of the clone was aligned with NCBI (National Center for Biotechnology Information) DNA sequence data base. The result showed high sequence identity with previously reported mammalian 4-aminobutyrate aminotransferases. The trans­criptional level of flounder 4-aminobutyrate aminotransferase was detected with the presence of mRNA at different flounder tissues by reverse transcription-polymerase chain reaction (RT-PCR). The expression of flounder 4-aminobutyrate aminotransferase was also tested and detected from the flounder tissues of the brain, liver, kidney and pancreas.

• Journal of Nutrition and Health
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• 제34권7호
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• pp.727-733
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• 2001

The present study was conducted to identify the mechanism about the regulation of appetite by examining the expression patterns of neuropeptide Y in the hypothalamus of mice either fasting mouse for 24 hours or with anorexia mutant mouse. In order to investigate the patterns of expression of neurpeptide Y, immunohistochemistry was employed for measurements at the tissue level, along with the molecular biological techniques of reverse transcription polymerase chain reaction(RT-PCR) and dot blotting. The results of this study are as follows. The level of expression of neruopeptide Y, a neuropeptide known to enhance appetite, was shown to be lowered in the arcuate nucleus(ARC), paraventricular nucleus(PVN), lateral hypothalamic area(LHA), and dorsomedial hypothalamic nucleus(DMN) in both the fasting and anorexia mutant groups when measured via immunohistochemistry, a tissue-level method. RT-PCR and dot blotting, the molecular biological methods employed in this study, revealed that the level of neuropeptide Y mRNA in the entire hypothalamus was similar in the control and fasting groups and lower in the anorexia mutant group. The results of the present study showed that while the levels of expression of the neuropeptide Y in the various hypothalamic regions studied did not exhibit regular increases or decreases when measured immunohistochemically. But the entire hypothalamus via molecular biological methods showed that the changes in these levels were more definite in the anorexia mutant group than in the fasting group.

• 원예과학기술지
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• 제32권4호
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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.

• 한국미생물·생명공학회지
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• 제49권3호
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• pp.440-448
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• 2021

Helicobacter pylori (H. pylori) establishes long-term infections associated with severe gastric diseases such as peptic ulceration and gastric cancer. Exposure to an antibacterial agent can help regulate the expression levels of its pathogenic genes. In this study, we analyzed the transcriptional changes in H. pylori genes induced by β-caryophyllene. We used next-generation sequencing (NGS) to analyze RNA expression changes, and reverse transcription-polymerase chain reaction (RT-PCR) was performed as required to verify the results. The NGS results showed that 30 out of 1,632 genes were expressed differentially by β-caryophyllene treatment. Eleven genes associated with DNA replication, virulence factors, and T4SS components were significantly downregulated. RT-PCR confirmed that treatment reduced the expression levels of 11 genes. RT-PCR showed the reduced expression of 11 genes (dnaE, dnaN, holB, gyrA, cagA, vacA, secA, flgE, virB2, virB4, and virB8) following β-caryophyllene treatment. These results suggest that β-caryophyllene can modulate various H. pylori pathogenic determinants and be a potential therapeutic agent for H. pylori infection.

• Asian Pacific Journal of Cancer Prevention
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• 제15권14호
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• pp.5815-5818
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• 2014

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously upregulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ${\beta}$-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.