• Journal of Plant Biotechnology
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• 제45권1호
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• pp.9-16
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• 2018

DNA 바코딩 기술은 다양한 약용식물 종들의 기원을 확인하기 위해 폭넓게 이용되고 있는 연구방법이다. 그러나, 시중에 판매되고 있는 유사 식물종을 재료로 사용한 상품이나 혼재되어 있는 상품에서 확인하고자 하는 약용식물을 선별 가능한 실질적인 기술의 개발은 아직 많이 미흡한 실정이다. 최근에는 보다 신속하고 정확도가 높은 기술을 개발하고자 DNA barcoding (Bar) 기술과 high-resolution melting (HRM) curve pattern 분석기술을 혼합한 Bar-HRM 분석기술을 이용한 연구가 진행 중에 있다. 본 리뷰논문에서는 국제적인 시장에서 다양한 기원의 약용식물 판별에 실질적으로 적용 가능한 Bar-HRM 기술의 최근의 발전 과정과 그 이용에 대해서 정리하였다. 다양한 연구들을 통해서 일부 성공적인 결과들이 보고되고 있지만, 제한된 DNA 바코드 및 단일염기다형성(Single Nucleotide Polymorphism, SNP) 등 아직 해결되어야 할 과제들이 많다. 특히, 핵 내 바코드로는 ribosomal DNA의 internal transcribed sequence (ITS)단편 이외에는 보고된 사례가 한건도 없었다. 또한, 약용식물을 끓는 물로 추출하여 가공한 약탕, 잼, 젤리, 쥬스 등의 제품은 DNA 단편이 분해되어 분리가 안 되는 경우에는 DNA바코딩 기술을 적용하기가 곤란한 것으로 알려져 있으나 비교적 짧은 DNA단편이 요구되는 Bar-HRM 분석기술을 이용하여 일부 성공한 보고도 있어 향후 그 응용사례가 증가할 것으로 전망된다.

• Asian-Australasian Journal of Animal Sciences
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• 제31권10호
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• 원예과학기술지
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• 제34권6호
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• pp.912-923
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• 2016

Watermelon production is often limited by powdery mildew in areas with a large daily temperature range. Development of resistant watermelon cultivars can protect against powdery mildew; however, little is known about the characteristics of its causal agents. Here, we identified the genus and race of a causal pathogen of powdery mildew in Ansung province of South Korea, and developed molecular markers for the generation of resistant watermelon cultivars. The causal pathogen was determined to be Podosphaera xanthii based on multiple sequence alignments of internal transcribed spacers (ITS) of rDNA. The physiological race was identified as 1W, and the Ansung isolate was named P. xanthii 1W-AN. Following inoculation with the identified P. xanthii 1W-AN, we found inheritance of the resistant gene fitting a single dominant Mendelian model in a segregated population ('SBA' ${\times}$ PI 254744). To develop molecular markers linked to fungus-resistant loci, random amplified polymorphic DNA (RAPD) was accomplished between DNA pooled from eight near-isogenic lines (NILs; $BC_4F_6$), originated from PI 254744 and susceptible 'SBB' watermelon. After sequencing bands from RAPD were identified in all eight NILs and PI254744, 42 sequence-characterized amplifiedregion (SCAR) markers were developed. Overall, 107 $F_2$ plants derived from $BC_4F_6$ NIL-1 ${\times}$ 'SBB' were tested, and one SCAR marker was selected. Sequence comparison between the SCAR marker and the reference watermelon genome identified three Nco I restriction enzyme sites harboring a single nucleotide polymorphism, and codominant cleavage-amplified polymorphic site markers were subsequently developed. A CAPS marker was converted to a high-resolution melt (HRM) marker, which can discriminate C/T SNP (254PMR-HRM3). The 254PMR-HRM3 marker was evaluated in 138 $F_{2:3}$ plants of a segregating population ('SBA' ${\times}$ PI254744) and was presumed to be 4.3 cM from the resistance locus. These results could ensure P. xanthii 1W-AN resistance in watermelon germplasm and aid watermelon cultivar development in marker-assist breeding programs.

• Molecules and Cells
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• 제40권2호
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• pp.100-108
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• 2017

Cathepsin F, which is encoded by CTSF, is a cysteine proteinase ubiquitously expressed in several tissues. In a previous study, novel transcripts of the CTSF gene were identified in the crab-eating monkey deriving from the integration of an Alu element-AluYRa1. The occurrence of AluYRa1-derived alternative transcripts and the mechanism of exonization events in the CTSF gene of human, rhesus monkey, and crabeating monkey were investigated using PCR and reverse transcription PCR on the genomic DNA and cDNA isolated from several tissues. Results demonstrated that AluYRa1 was only integrated into the genome of Macaca species and this lineage-specific integration led to exonization events by producing a conserved 3' splice site. Six transcript variants (V1-V6) were generated by alternative splicing (AS) events, including intron retention and alternative 5' splice sites in the 5' and 3' flanking regions of CTSF_AluYRa1. Among them, V3-V5 transcripts were ubiquitously expressed in all tissues of rhesus monkey and crab-eating monkey, whereas AluYRa1-exonized V1 was dominantly expressed in the testis of the crab-eating monkey, and V2 was only expressed in the testis of the two monkeys. These five transcript variants also had different amino acid sequences in the C-terminal region of CTSF, as compared to reference sequences. Thus, species-specific Alu-derived exonization by lineage-specific integration of Alu elements and AS events seems to have played an important role during primate evolution by producing transcript variants and gene diversification.

• Journal of Biomedical and Translational Research
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• 제19권4호
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• pp.116-124
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• 2018

Korean Native Pig (KNP) has a uniform black coat color, excellent meat quality, white colored fat, solid fat structure and good marbling. However, its growth performance is low, while the western origin Yorkshire pig has high growth performance. To take advantage of the unique performance of the two pig breeds, we raised crossbreeds (KNP ${\times}$ Yorkshire to make use of the heterotic effect. We then analyzed the liver transcriptome as it plays an important role in fat metabolism. We sampled at two stages: 10 weeks and at 26 weeks. The stages were chosen to correspond to the change in feeding system. A total of 16 pigs (8 from each stage) were sampled and RNA sequencing was performed. The reads were mapped to the reference genome and differential expression analysis was performed with edgeR package. A total of 324 genes were found to be significantly differentially expressed (${\left|log2FC\right|}$ > 1 & q < 0.01), out of which 180 genes were up-regulated and 144 genes were down-regulated. Principal Component Analysis (PCA) showed that the samples clustered according to stages. Functional annotation of significant DEGs (differentially expressed genes) showed that GO terms such as DNA replication, cell division, protein phosphorylation, regulation of signal transduction by p53 class mediator, ribosome, focal adhesion, DNA helicase activity, protein kinase activity etc. were enriched. KEGG pathway analysis showed that the DEGs functioned in cell cycle, Ras signaling pathway, p53 signaling pathway, MAPK signaling pathway etc. Twenty-nine transcripts were also part of the DEGs, these were predominantly Cys2His2-like fold group (C2H2) family of zinc fingers. A protein-protein interaction (PPI) network analysis showed that there were three highly interconnected clusters, suggesting an enrichment of genes with similar biological function. This study presents the first report of liver tissue specific gene regulation in a cross-bred Korean pig.

• 한국어병학회지
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• 제34권2호
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• pp.141-147
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• 2021

본 연구에서는 주요 새우 생산 국가에서 2017년 7월부터 2020년 11월 사이 생산되어 국내로 수입된 냉동 새우(29개 그룹)를 대상으로 흰반점바이러스(WSSV), covert mortality nodavirus (CMNV) 및 decapod iridescent virus 1 (DIV-1)의 검출여부를 조사하였다. 각 바이러스에 대한 nested PCR 결과, WSSV는 9개 그룹(9/29)에서 검출되었으며 CMNV와 DIV-1은 검출되지 않았다. Nested PCR에서 WSSV 양성으로 확인된 시료를 대상으로 WSSV genome variable loci로 알려진 VR 14/15 region에 대해 참조 서열들과 삽입/결손(insertion and deletion) 서열 비교 및 근연관계를 분석하였다. WSSV 양성 시료 중 1개 시료(20-CH-1 isolate, 2020년 10월 중국 생산)에서만 VR 14/15에 대한 PCR amplicon이 생성되었으며 염기서열 분석 결과, 20-CH-1 isolate는 2005년 인도에서 보고된 WSSV-IN-05-01과 99.84%의 상동성을 보였다. 이는 과거 알려진 바와 같이 새우의 교역을 통한 국가 간 WSSV가 확산되었음을 뒷받침해주는 결과이다.

• Journal of Microbiology and Biotechnology
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• 제34권7호
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• pp.1475-1483
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• 2024

Three Gram-stain-positive, aerobic, rod-shaped, and non-motile bacteria, labelled as W11^T, SW19^T, and YR1^T, were isolated from soil, and performed their polyphasic taxonomic investigation. The phylogenetic and 16S rRNA gene sequence analysis showed that strains W11^T, SW19^T, and YR1^T belonged to the genera Agromyces, Rathayibacter, and Nocardioides, respectively. Strain W11^T was closely affiliated with Agromyces cavernae SYSU K20354^T (98.1%), strain SW19^T showed the closest affiliation with Rathayibacter rubneri ZW T2_19^T (97.0%), and strain YR1^T was most closely related to Nocardioides marmorisolisilvae KIS18-7^T (98.0%). The genome sizes of strains W11^T, SW19^T, and YR1^T were 4,181,720 bp, 4,740,677 bp, and 4,228,226 bp, respectively, with DNA G+C contents of 70.5%, 64.2%, and 69.7%, respectively. Average nucleotide identity and digital DNA-DNA hybridization values of W11^T, SW19^T, and YR1^T with their respective reference species were <79.6% and <23.6%, respectively. The predominant cellular fatty acids detected in strain W11^T were anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0. In strain SW19^T, they were summed feature 9 (C_16:0 10-methyl and/or iso-C_17:1ω 9c), anteiso-C_17:0, and anteiso-C_15:0. Strain YR1^T exhibited C_18:1ω 9c, C_18:0 10-methyl, TBSA, and anteiso-C_15:0 as its major cellular fatty acids. Overall, the polyphasic taxonomic comparisons indicated that strains W11^T, SW19^T, and YR1^T represent novel species within the genera Agromyces, Rathayibacter, and Nocardioides, respectively. Accordingly, we propose the names Agromyces silvae sp. nov., with the type strain W11^T (=KCTC 49818^T =NBRC 115999^T), Rathayibacter soli sp. nov., with the type strain SW19^T (=KCTC 49860^T =NBRC 116108^T), and Nocardioides terrisoli sp. nov., with the type strain YR1^T (=KCTC 49863^T =NBRC 116165^T).

• 원예과학기술지
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• 제30권5호
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• pp.557-567
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• 2012

본 연구는 마커이용여교잡(marker-assisted backcross, MAB)을 통한 내병성 토마토 신품종육성에 필요한 기초 정보를 얻기 위해 수행되었다. TYLCV, 시들음역병, 청고병, 흰가루병에 내병성인 공여친 계통 10종과 이병성이지만 우수 원예형질을 지닌 회복친 계통 4종에 대해 병리검정과 TYLCV 내병성 연관 분자마커 분석을 수행하였다. MAB를 위한 회복친 유전자 선발(background selection)용 마커개발을 목표로 SOL Genomics Network에 공시된 토마토 유전자지도(reference map)로부터 전 게놈에 균등히 분포된 108개(염색체 당 평균 9개) SSR 마커를 분석하여, 총 303개의 다형성 마커를 기반으로 공여친, 회복친 계통 간 유연관계를 분석하였다. 그 결과, 유사도 값의 전체 범위는 0.33-0.80으로계통 간 가장 높은 유사도 값(0.80)을 나타낸 것은 청고병에 저항성인 '10BA333'와 '10BA424'이었고, 가장 낮은 유사도 값(0.33)을 나타낸 것은 시들음역병에 내병성인 야생종 L3708(Solanum pimpinelliforium L.)과 청고병에 저항성인 '10BA424'이었다. 유사도 값을 이용하여 UPGMA 분석한 결과, 유사도 0.58를 기준으로 나누었을 때 3개의 군(cluster)으로 분류되었는데, 대부분 동일한 내병성을 지닌 공여친계통 간 유전적 거리가 가까워 이들은 공통된 저항성 재료를 이용한 육성과정에서 파생된 계통일 것이라 판단되었다. 계통수(dendrogram)를 기준으로 유전적 거리가 지나치게 멀지 않으면서 비교적 다수의 회복친 유전자 선발용 SSR 마커의 확보가 가능한 여교배 조합(공여친 ${\times}$ 회복친)은 TYLCV 내병성의 경우 'TYR1' ${\times}$ 'RPL1', 청고병의 경우 '10BA333' 또는 '10BA424' ${\times}$ 'RPL2', 흰가루병의 경우 'KNU12' ${\times}$ 'AV107-4' 또는 'RPL2'로 판단되었다. 시들음역병의 경우 내병성 공여친인 'L3708'은 야생종으로서 모든 회복친 계통들과 유전적 거리가 매우 멀었으며, 적절한 조합은 유사도 값이 0.41이며 계통 간 45개의 다형성 SSR 마커가 선발된 'L3708' ${\times}$ 'AV107-4'로 판단되었다.

• 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.

• 생물정신의학
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• 제21권3호
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• pp.99-106
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• 2014

Objectives Previous studies suggest that the cannabinoid receptor 1 (CNR1) gene could be an important candidate gene for schizophrenia. According to linkage studies, this gene is located on chromosome 6q14-q15, which is known to harbor the schizophrenia susceptibility locus (locus 5, SCZ5, OMIM 803175). The pharmacological agent delta-9-tetrahydrocannabinol (${\Delta}$-9-THC) seems to elicit the symptoms of schizophrenia. The association between CNR1 polymorphisms and schizophrenia is actively being investigated, and some studies have linked the AAT-trinucleotide repeats in CNR1 to the onset of schizophrenia. In this study, we have investigated the association between the AAT-trinucleotide repeats in CNR1 and schizophrenia by studying schizophrenia patients and healthy individuals from Korea. Methods DNA was extracted from the blood samples of 394 control subjects and 337 patients diagnosed with schizophrenia (as per the Diagnostic and Statistical Manual of Mental Disorders, fourth edition criteria). After polymerase chain reaction amplification, a logistic regression analysis, with age and gender as the covariates, was performed to study the variations in the AAT-repeat polymorphisms between the two groups. Results In total, 8 types of trinucleotide repeats were identified, each containing 7, 8, 10, 11, 12, 13, 14, and 15 repeats, respectively. $(AAT)_{13}$ allele was most frequently observed, with a frequency of 33.6% and 31.6% in the patient and control groups, respectively. The frequency of the other repeat alleles in the patient group (in the decreasing order) was as follows : $(AAT)_{13}$ 33.6%, $(AAT)_{14}$ 21.6%, $(AAT)_{12}$ 18.5%, and $(AAT)_{7}$ 11.1%. The frequency of the repeat alleles in the control group (in the decreasing order) was as follows : $(AAT)_{13}$ 31.6%, $(AAT)_{14}$ 24.5%, $(AAT)_{12}$ 17.2%, and $(AAT)_{7}$ 11.6%. However, there were no significant differences in the AAT-repeat polymorphisms of the CNR1 gene between the patient group and the control group. Conclusions Although our study revealed no significant association of the AAT-repeat polymorphism of the CNR1 gene with schizophrenia, it will serve as a good reference for future studies designed to examine the cannabinoid hypothesis of schizophrenia.