• Journal of Plant Biotechnology
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• 제40권1호
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• pp.18-26
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• 2013

살충성 유전자 mcry1Ac1을 포함하고 있는 해충저항성 유전자변형(GM) 벼 Agb0101이 국내에서 개발되었다. 향후 Agb0101 벼의 환경방출에 따른 모니터링과 이력추적을 위해서는 신뢰성 있는 검출방법의 개발이 필요하다. 따라서, 본 연구에서 해충저항성 GM벼의 사후 안전관리를 위한 정성적 및 정량적 PCR 검정 방법을 개발하였다. 벼 녹말분지효소 유전자 RBE4를 PCR 분석의 내재유전자로 사용하였고, 이의 primer쌍 RBEgh-1/-2는 101bp의 PCR 증폭산물을 형성하였다. 정성 PCR 분석을 위해서 삽입된 T-DNA를 바탕으로 특이 primer를 제작하였고, 이벤트 특이적 검출 primer의 경우 Agb0101의 도입유전자 및 벼 염색체 DNA 사이의 5' 또는 3' 인접염기부위를 정확하게 특이적으로 PCR 증폭하였다. 반면, 대조구인 각종 작물, 국내 벼 품종 및 Agb0101과 동일 형질전환 벡터를 갖는 해충저항성 벼에서는 어떠한 PCR 증폭산물도 형성하지 않았다. 표준물질로써 내재유전자 및 이벤트 특이적 단편으로 제조된 pRBECrR을 이용한 real-time PCR 분석에 의해서 정량한계(LOQ)가 10 copies 농도의 범위인 것으로 확인되었고, 이의 유효성을 검증하기 위하여 상이한 농도의 Agb0101시료(10, 5, 3 및 1%)를 real-time PCR 분석하여 정량검정에 대한 표준편차 및 상대표준편차가 각각 0.06 ~ 0.40 및 3.80 ~ 7.01%의 낮은 범위에 포함되는 것을 확인할 수 있었다. 이들 결과로 본 연구에서 개발된 정성 및 정량 PCR 검정 방법이 해충저항성 GM벼 Agb0101의 모니터링 및 이력추적에 효과적으로 이용될 수 있을 것으로 본다.

• 대한화학회지
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• 제48권5호
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• pp.483-490
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• 2004

시판 중인 poly(vinylpyrrolidone) (PVP)와 hydroxy ethyl cellulose (HEC) 혼합물을 충진젤 기질로 이용하여 한국 재래산양에 감염된 orf virus (ORFV)를 빠른 시간에 검출하여 진단할 수 있는 새로운 효소중합연쇄반응 (polymerase chain reaction, PCR)-마이크칩젤 전기영동법 (microchip gel electrophoresis, MGE)을 개발하였다. Orf 바이러스 B2L 유전자에서 지표-DNA인 594-bp DNA를 PCR로 증폭시킨 뒤, MGE법을 이용하여 증폭된 DNA를 분석하였다. MGE법은 64 mm 총길이(유효길이 36 mm) ${\times}$90 ${\mu}$m 폭 ${\times}$20 ${\mu}$m 깊이의 유리로 제작된 마이크로칩을 사용하였다. 1.0% PVP ($M_r$ 360,000)와 1.0% HEC ($M_r$ 250,000)의 혼합 충진젤과 277.8 V/cm의 전기장에서 4분 안에 증폭된 594-bp DNA를 분석하였다. PVP와 HEC의 혼합된 충진젤을 사용시 DNA 단편의 길이에 영향이 없이 하나의 DNA 피크를 나타내며 향상된 분리도와 이동시간의 재현성을 보여주었다. 본 PCR-MGE법은 고전적인 슬랩젤 전기영동법에 비해 약 20배 이상의 빠른 검출시간과 정량분석이 가능한 효과적인 ORFV 유전자단편 검출법이었다.

• Journal of Animal Science and Technology
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• 제57권5호
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• pp.18.1-18.8
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• 2015

Background: Quantitative real time reverse transcription PCR (qRT-PCR) is one of the most important techniques for gene-expression analysis in molecular based studies. Selecting a proper internal control gene for normalizing data is a crucial step in gene expression analysis via this method. The expression levels of reference genes should be remained constant among cells in different tissues. However, it seems that the location of cells in different tissues might influence their expression. The purpose of this study was to determine whether the source of mesenchymal stem cells (MSCs) has any effect on expression level of three common reference genes (GAPDH, ${\beta}$-actin and ${\beta}2$-microglobulin) in equine marrow- and adipose-derived undifferentiated MSCs and consequently their reliability for comparative qRT-PCR. Materials and methods: Adipose tissue (AT) and bone marrow (BM) samples were harvested from 3 mares. MSCs were isolated and cultured until passage 3 (P3). Total RNA of P3 cells was extracted for cDNA synthesis. The generated cDNAs were analyzed by quantitative real-time PCR. The PCR reactions were ended with a melting curve analysis to verify the specificity of amplicon. Results: The expression levels of GAPDH were significantly different between AT- and BM-derived MSCs (p < 0.05). Differences in expression level of ${\beta}$-actin (P < 0.001) and B2M (P < 0.006.) between MSCs derived from AT and BM were substantially higher than GAPDH. In addition, the fold change in expression levels of GAPDH, ${\beta}$-actin and B2M in AT-derived MSCs compared to BM-derived MSCs were 2.38, 6.76 and 7.76, respectively. Conclusion: This study demonstrated that GAPDH and especially ${\beta}$-actin and B2M express in different levels in equine AT- and BM-derived MSCs. Thus they cannot be considered as reliable reference genes for comparative quantitative gene expression analysis in MSCs derived from equine bone marrow and adipose tissue.

• Journal of Microbiology and Biotechnology
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• 제14권3호
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• pp.620-627
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• 2004

On the foundation of a database of genome sequences and protein analyses, the ability to clone a gene based on a peptide analysis is becoming more feasible and effective for identifying a specific gene and its protein product of interest. As such, the current study conducted a protein analysis using 2-D PAGE followed by MALDI- TOF and ESI-MS to identify a highly expressed gene product of C. parasitica. A distinctive and highly expressed protein spot with a molecular size of 47.2 kDa was randomly selected and MALDI-TOF MS analysis was conducted. A homology search indicated that the protein appeared to be a fungal enolase (enol). Meanwhile, multiple alignments of fungal enolases revealed a conserved amino acid sequence, from which degenerated primers were designed. A screening of the genomic $\lambda$ library of C. parasitica, using the PCR amplicon as a probe, was conducted to obtain the full-length gene, while RT-PCR was performed for the cDNA. The E. coli-expressed eno 1 exhibited enolase enzymatic activity, indicating that the cloned gene encoded the C. parasitica enolase. Moreover, ESI-MS of two of the separated peptides resolved from the protein spot on 2-D PAGE revealed sequences identical to the deduced sequences, suggesting that the cloned gene indeed encoded the resolved protein spot. Northern blot analysis indicated a consistent accumulation of an eno1 transcript during the cultivation.

• 생명과학회지
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• 제27권5호
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• pp.524-529
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• 2017

본 연구에서는 zebrafish에 인간의 KCNE1 유전자가 삽입된 형광단백질 vector를 microinjection하고, 그 발현 여부를 확인하고자 하였다. 먼저 양 말단에 제한효소(EcoRΙ, BamHΙ) site를 넣어 제작한 primer들로 genomic DNA에서 KCNE1 유전자를 분리하였다. 그 결과는 약 402 bp 크기의 DNA band였고 이 PCR 산물을 형광단백질 vector인 pPB-CMVp-EF1-GreenPuro 속에 클로닝하여 pPB-CMVp-hKCNE1-EF1-GreenPuro plasmid를 제작하였다. 이렇게 준비된 형광 vector를 zebrafish 수정란에 microinjection하였고, 부화된 치어에서 RT-PCR과 DNA sequencing을 통해 GFP 및 hKCNE1의 발현을 최종 확인하였다. 본 연구는 향후 QT 연장증후군(LQTs)에 대한 동물 모델로써 신경자극 전도, 유전자 치료, 유용 유전자 클로닝을 위한 기술 개발에 응용될 수 있을 것으로 기대된다.

• Journal of Ginseng Research
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• 제41권1호
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• pp.31-35
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• 2017

Background: Korean ginseng (Panax ginseng) is a well-known medicinal plant of Oriental medicine that is still in practice today. Until now, a total of 11 Korean ginseng cultivars with unique features to Korean ginseng have been developed based on the pure-line-selection method. Among them, a new cultivar namely G-1 with different agricultural traits related to yield and content of ginsenosides, was developed in 2012. Methods: The aim of this study was to distinguish the new ginseng cultivar G-1 by identifying the unique single-nucleotide polymorphism (SNP) at its 45S ribosomal DNA and Panax quinquefolius region than other Korean ginseng cultivars using multiplex amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR). Results: A SNP at position of 45S ribosomal DNA region between G-1, P. quinquefolius, and the other Korean ginseng cultivars was identified. By designing modified allele-specific primers based on this site, we could specifically identified G-1 and P. quinquefolius via multiplex PCR. The unique primer for the SNP yielded an amplicon of size 449 bp in G-1 cultivar and P. quinquefolius. This study presents an effective method for the genetic identification of the G-1 cultivar and P. quinquefolius. Conclusion: The results from our study shows that this SNP-based approach to identify the G-1 cultivar will be a good way to distinguish accurately the G-1 cultivar and P. quinquefolius from other Korean ginseng cultivars using a SNP at 45S ribosomal DNA region.

• Journal of Veterinary Science
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• 제21권6호
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• pp.73.1-73.10
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• 2020

Background: Bovine papilloma is a neoplastic disease caused by bovine papillomaviruses (BPVs), which were recently divided into 5 genera and at least 24 genotypes. Objectives: The complete genome sequence of BPV type 15 (BPV Aks-02), a novel putative BPV type from skin samples from infected cows in Southern Xinjiang China, was determined by collecting warty lesions, followed by DNA extraction and amplicon sequencing. Methods: DNA was analyzed initially by polymerase chain reaction (PCR) using the degenerate primers FAP59 and FAP64. The complete genome sequences of the BPV Aks-02 were amplified by PCR using the amplification primers and sequencing primers. Sequence analysis and phylogenetic analysis were performed using bio-informatic software. Results: The nucleotide sequence of the L1 open reading frame (ORF) of BPV Aks-02 was 75% identity to the L1 ORF of BPV-9 reference strain from GenBank. The complete genome consisted of 7,189 base pairs (G + C content of 42.50%) that encoded 5 early (E8, E7, E1, E2, and E4) and 2 late (L1 and L2) genes. The E7 protein contained a consensus CX₂CX₂₉CX₂C zinc-binding domain and a LxCxE motif. Among the different members of this group, the percentages of the complete genome and ORFs (including 5 early and 2 late ORFs) sequence identity of BPV Aks-02 were closer to the genus Xipapillomavirus 1 of the Xipapillomavirus genus. Phylogenetic analysis and sequence similarities based on the L1 ORF of BPV Aks-02 revealed the same cluster. Conclusions: The results suggest that BPV type (BPV Aks-02) clustered with members of the Xipapillomavirus genus as BPV 15 and were closely related to Xipapillomavirus 1.

• The Plant Pathology Journal
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• 제25권1호
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• pp.47-53
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• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• Parasites, Hosts and Diseases
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• 제51권6호
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• pp.739-742
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• 2013

A 52-year-old woman presented with lower back pain, progressive symmetrical paraparesis with sensory impairment, and sphincter disturbance. Magnetic resonance imaging (MRI) of the whole spine revealed multiple intradural extramedullary serpiginous-mass lesions in the subarachnoid space continuously from the prepontine to the anterior part of the medulla oblongata levels, C7, T2-T8, and T12 vertebral levels distally until the end of the theca sac and filling-in the right S1 neural foramen. Sparganosis was diagnosed by demonstration of the sparganum in histopathological sections of surgically resected tissues and also by the presence of serum IgG antibodies by ELISA. DNA was extracted from unstained tissue sections, and a partial fragment of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using a primer set specific for Spirometra spp. cox1. After sequencing of the PCR-amplicon and alignment of the nucleotide sequence data, the causative agent was identified as the larva of Spirometra erinaceieuropaei.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.630-636
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• 2010

A survey of the endangered orchid Orchis militaris populations was carried out in north-eastern Italy. The occurrence of fungal root endophytes was investigated by light and electron microscopies and molecular techniques. Two main sites of presence were individuated in the Euganean Hills, differing as to the percentage of flowering individuals and of capsules completing maturity. Fluorescence microscopy revealed an intracellular cortical colonization by hyphal pelotons. Two ITS PCR products co-amplified. Sequencing revealed for the former an identity and a high similarity (99%) with a Tulasnellaceae (Basidiomycota) fungus found within tissues of the same host in independent studies in Hungary and Estonia, suggesting an interesting case of tight specificity throughout the Eurosiberian home range. The second amplicon had 99% similarity with Tetracladium species (Ascomycota) recently demonstrated as potential endophytes. TEM revealed two different hyphal structures. Double fungal colonization appears to occur in Orchis militaris and the possible requirement of a specific fungal partner throws light on the causes of this plant's rarity and threatened status.