• Title/Summary/Keyword: Cloning detection

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Probing Cell-Type Specific Gene Expression in the Ovarian Cells of Drosophila by P-Element Mediated Enhancer Detection (P-요소를 이용한 노랑초파리 난소에서의 세포특이적 유전자발현의 검출)

  • 계명찬;조경상;김경진;이정주
    • The Korean Journal of Zoology
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    • v.38 no.4
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    • pp.505-513
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    • 1995
  • P-lement mediated enhancer detector lines (EDla) were screened for reporter gene (1acZ expression In the ovary of Drosophila mejanogaster Cell-type spedfic 1acZ expression can be grouped Into three parts such as in the geimline, soma, and both. LacZ expression In germline cells was devided into 2 types; expression in nurse cells or in both of the nurse cells and oocote. In the stage-9 to stage-lO follicles, lacZ expression was observed either In the whole follicle cells around oocote or in the subpopulation of follicle cells in egg chamber. lacZ expression in the subset of follicle cells are showed in the centripetal follicle cells or the columnar follicle cells except centripetal follicle cells. Several lines showed anterior to postedor gradient pattern of lacZ expression in the follicle cells. Interestingly there were 3 lines in which lacZ was expressed In the polar cells and/or the horder cells of egg chamber. These lacZ expression patterns in the different ovarian cells of independent EDla reflect the cell type-spedflc expression of maternal genes nesr the P-element insertion, and might provide a basis for cloning of genes involved in oogenesis of Drosophila.

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Evaluation of Exogenous Promoters for Use in Brachiaria brizantha Transformation

  • Silveira Erica Duarte;Rodrigues Julio Carlyle Macedo;Cabral Glaucia Barbosa;Leite Juliana de Almeida;Costa Sidnei Souza;Carneiro Vera Tavares de Campos
    • Journal of Plant Biotechnology
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    • v.5 no.2
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    • pp.87-93
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    • 2003
  • Brachiaria (Poaceae) is the most important forage genus for cattle production in Brazil. The genetic breeding of this genus is limited by the incompatibility among species, differences in ploidy level and the natural cloning of plants by apomixis (Valle and Miles 1992). However, plant regeneration via tissue culture methods and genetic engineering provide an opportunity to introduce new characteristics in plants of this genus. We have developed methods for the 'genetic modification of Brachiaria brizantha cv. Marandu via biolistic transformation. A higher number of shoots was obtained with 4 mg/L 2.4-diclorophenoxyacetic acid and 0.2 mg/L benzylaminopurine in calli induction medium and 0.1 mg/L naphtaleneacetic acid and 4.0 mg/L kinetin in shoot regeneration medium. A selection curve for mannose was determined to use phospho mannose isomerase (PMI) gene of Escherichia coli as a selection marker. Calli formation was inhibited from 5 g/L mannose, even in the presence of sucrose while calli that were formed in the presence of mannose failed to develop embryos showing that PMI gene can be used for selection of transformants of this grass. Different promoters were tested to evaluate the efficiency based on the detection of the GUS gene expression (Jefferson et al. 1987). The monocot promoters, act1-D and ubi-1, resulted in higher expression levels than dicot promoters, ubi-3 and act-2, or the CaMV35S and CVMV promoters.

Development of Molecular Marker through Genome Realignment for Specific Detection of Xanthomonas campestris pv. campestris Race 5, a Pathogen of Black Rot Disease

  • Afrin, Khandker Shazia;Rahim, Md Abdur;Jung, Hee-Jeong;Park, Jong-In;Kim, Hoy-Taek;Nou, Ill-Sup
    • Journal of Microbiology and Biotechnology
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    • v.29 no.5
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    • pp.785-793
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    • 2019
  • Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is the most damaging disease in Brassica crops around the world. In this study, we developed a molecular marker specific to Xcc race 5. To do this, the available whole genome sequences of Xcc races/strains and Xc subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of Xcc races/strains, Xc subspecies and other plant-infecting bacterial strains (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to Xcc race 5. Moreover, they also amplified a 1,515-bp fragment for Xcc race 1 and an over 3,000-bp fragment for Xcc race 3. However, they did not amplify any fragments from the remaining Xcc races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown Xcc strains and ICMP8 was identified as race 5 among nine race-unknown Xcc strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify Xcc race 5 from Xcc races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an Xcc race 5-specific molecular marker.

Molecular Cloning of the 3'-Terminal Region of Garlic Potyviruses and Immunological Detection of Their Coat Proteins

  • Song, Sang-Ik;Song, Jong-Tae;Chang, Moo-Ung;Lee, Jong-Seob;Park, Yang-Do
    • The Plant Pathology Journal
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    • v.15 no.5
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    • pp.270-279
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    • 1999
  • cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

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Object Part Detection-based Manipulation with an Anthropomorphic Robot Hand Via Human Demonstration Augmented Deep Reinforcement Learning (행동 복제 강화학습 및 딥러닝 사물 부분 검출 기술에 기반한 사람형 로봇손의 사물 조작)

  • Oh, Ji Heon;Ryu, Ga Hyun;Park, Na Hyeon;Anazco, Edwin Valarezo;Lopez, Patricio Rivera;Won, Da Seul;Jeong, Jin Gyun;Chang, Yun Jung;Kim, Tae-Seong
    • Annual Conference of KIPS
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    • 2020.11a
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    • pp.854-857
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    • 2020
  • 최근 사람형(Anthropomorphic)로봇손의 사물조작 지능을 개발하기 위하여 행동복제(Behavior Cloning) Deep Reinforcement Learning(DRL) 연구가 진행중이다. 자유도(Degree of Freedom, DOF)가 높은 사람형 로봇손의 학습 문제점을 개선하기 위하여, 행동 복제를 통한 Human Demonstration Augmented(DA)강화 학습을 통하여 사람처럼 사물을 조작하는 지능을 학습시킬 수 있다. 그러나 사물 조작에 있어, 의미 있는 파지를 위해서는 사물의 특정 부위를 인식하고 파지하는 방법이 필수적이다. 본 연구에서는 딥러닝 YOLO기술을 적용하여 사물의 특정 부위를 인식하고, DA-DRL을 적용하여, 사물의 특정 부분을 파지하는 딥러닝 학습 기술을 제안하고, 2 종 사물(망치 및 칼)의 손잡이 부분을 인식하고 파지하여 검증한다. 본 연구에서 제안하는 학습방법은 사람과 상호작용하거나 도구를 용도에 맞게 사용해야하는 분야에서 유용할 것이다.

Genetic Insights into Domestication Loci Associated with Awn Development in Rice

  • Ngoc Ha Luong;Sangshetty G. Balkunde;Kyu-Chan Shim;Cheryl Adeva;Hyun-Sook Lee;Hyun-Jung Kim;Sang-Nag Ahn
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.33-33
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    • 2022
  • Rice (Oryza sativa L.) is a widely studied domesticated model plant. Seed awning is an unfavorable trait during rice harvesting and processing. Hence, awn was one of the target characters selected during domestication. However, the genetic mechanisms underlying awn development in rice are not well understood. In this study, we analyzed the genes for awn development using a mapping population derived from a cross between the Korean indica cultivar 'Milyang23' and NIL4/9 (derived from a cross between 'Hwaseong' and O. minuta). Two quantitative trait loci (QTLs), qAwn4 and qAwn9 were mapped on chromosome 4 and 9, respectively, increased awn length in an additive manner. Through comparative sequencing analyses parental lines, LABA1 was determined as the causal gene underlying qAwn4. qAwn9 was mapped to a 199-kb physical region between markers RM24663 and RM24679. Within this interval, 27 annotated genes were identified, and five genes, including a basic leucine zipper transcription factor 76 (OsbZIP76), were considered candidate genes for qAwn9 based on their functional annotations and sequence variations. Haplotype analysis using the candidate genes revealed tropical japonica specific sequence variants in the qAwn9 region, which partly explains the non-detection of qAwn9 in previous studies that used progenies from interspecific crosses. This provides further evidence that OsbZIP76 is possibly a causal gene for qAwn9. The O. minuta qAwn9 allele was identified as a major QTL associated with awn development in rice, providing an important molecular target for basic genetic research and domestication studies. Our results lay the foundation for further cloning of the awn gene underlying qAwn9.

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Development of Real-time Quantitative PCR Assay based on SYBR Green I and TaqMan Probe for Detection of Apple Viruses (사과 바이러스 검정을 위한 SYBR Green I 및 TaqMan probe 기반의 real-time PCR 검사법 개발)

  • Heo, Seong;Chung, Yong Suk
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.65 no.4
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    • pp.496-507
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    • 2020
  • Virus infections of apples result in lowered commercial qualities such as low sugar content, weakened tree vigor, and malformed fruits. An effective way to control viruses is to produce virus-free plants based on the development of an accurate and sensitive diagnostic method. In this study, real-time PCR assays based on SYBR Green I and TaqMan probes were developed for detecting ASGV, ASPV, and ApMV viruses. These methods can detect and quantify 103 to 1011 RNA copies/μL of each virus separately. Compared with methods with two different dyes, the SYBR Green I-based method was efficient for virus detection as well as for assay using the TaqMan probe. Field tests demonstrated that real-time PCR methods developed in this study were applicable to high-throughput diagnoses for virus research and plant quarantine.

Detection of a Microsporidium, Nosema ceranae, from Field Population of the Bumblebee, Bombus terrestris, via Quantitative Real-Time PCR (서양뒤영벌 야외개체군에서 Real-Time PCR을 이용한 Nosema ceranae의 검출)

  • Lee, Dae-Weon
    • Korean Journal of Microbiology
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    • v.49 no.3
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    • pp.270-274
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    • 2013
  • The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA (SSU rRNA) gene of N. ceranae was successfully amplified among examined genes and sequenced, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentration as low as $0.85ng/{\mu}l$ genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

Development of Direct Competitive Enzyme-Linked Immunosorbent Assay using Monoclonal Antibody (MAb) against Sulfamthazine (SMZ) and Establishment of Application Condition for Milk Sample (설파메타진에 단클론성 항체를 이용한 직접경쟁효소면역분석법의 개발과 우유 시료 적용 조건 확립)

  • Shim, Won-Bo;Mun, Chun-Sun;Kim, Jung-Sook;Choe, Ju-Mi;Kim, Ji-Hun;Park, Seon-Ja;Kang, Sung-Jo;Chung, Duck-Hwa
    • Korean Journal of Food Science and Technology
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    • v.38 no.2
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    • pp.176-182
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    • 2006
  • Sensitive and specific monoclonal antibody (MAb) was produced from hybridoma (1H11-5) obtained by fusion of myeloma cell (V653) and spleen cell isolated from mouse immunized sulfamthazine (SMZ)-HG-KLH. Direct competitive ELISA was developed for rapid detection of SMZ in milk samples using MAb against SMZ with optimized conditions between MAb and SMZ-HG-HRP conjugate, and applicable conditions for analysis of milk samples were established. Detection range of immunoassay was 0.1 to 100 ppb. Recoveries from spiked raw milk and processed milk samples averaged 82.1-120.7 and 82.1-97.1%, respectively.

Cloning and Characterization of a 5-Enolpyruvyl Shikimate 3-Phosphate Synthase (EPSPS) Gene from Korean Lawn Grass (Zoysia japonica) (들잔디 5-Enolpyruvyl Shikimate 3-Phosphate Synthase(EPSPS) 유전자 클로닝 및 특성)

  • Lee, Hye-Jung;Lee, Geung-Joo;Kim, Dong-Sub;Kim, Jin-Beak;Ku, Ja-Hyeong;Kang, Si-Yong
    • Horticultural Science & Technology
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    • v.28 no.4
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    • pp.648-655
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    • 2010
  • This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.