• 한국약용작물학회지
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• 제17권1호
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• pp.26-32
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• 2009

The region containing 18S rRNA gene, ITS 1 and part of the 5.8S rRNA gene of the Atractylodes japonica Koidz was amplified by PCR and the product cloned in a pBluescript SK II plasmid. DNA sequence of the cloned DNA was determined and submitted to the GenBank (accession number EU678363). Phylogenetic analysis of the ITS 1 DNA showed close similarity with the other plant species of the family Compositae. The extract of the plant materials of five different members of the family Compositae was analyzed by HPLC to detect atractylon. Extract of the A. japonica Koidz showed presence of significant amount of atractylon. However, noticeable amount of atractylon was not detected by the same analyses from the extracts of the other plants belonging to the family Compositae including Artemisia capillaris, Chrysantemum zawadskii, Eclipta prostrata or Taraxacum platycarpum.

• Mycobiology
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• 제37권3호
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• pp.240-242
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• 2009

An improved procedure for preparing PCR cloning vectors was developed. This procedure includes the incorporation of adapters to create XcmI restriction enzyme sites in pBluescript II SK(+) vectors, digestion with XcmI followed by further digestion of the small fragment produced by XcmI digestion with additional enzymes, and purification with PCR purification kits. Using this procedure, PCR cloning vectors with high ligation efficiencies and low blue or false-positive colonies were obtained.

• Journal of Ginseng Research
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• 제19권1호
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• pp.51-55
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• 1995

The DNA fragment containing ginseng ribulose-1,5-bisphosphate carboxytase/oxygenase large subunit(rbcL) gene was cloned from the ginseng chloroplast EcoRl library by colony lift hybridization with tobacco rbcL gene probe. From the screened clone, the DNA fragment containing ginseng rbcL gene was digested with several restriction enzyme and analyzed by Southern blot hybridization for the construction of restriction map. The ginseng rbcL gene fragment was subcloned in pBluescript II SK + vector and sequence analysis was performed. The nucleotide sequence of ginseng rbcL gene was compared with those of petunia, tobacco, alfalfa, rice and barley, which showed a homology of 93.1%, 95.2%, 90.5%, 85.5% and 84.3%, respectively.

• Journal of Microbiology
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• 제34권4호
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• pp.349-354
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• 1996

Pseudomonas sp. P20 is a natural isolate which is capable of degrading biphenyl and 4-chlorobiphenyl. From a clone of pCK1022 harboring pcbCD genes of Pseudomonas sp P20, a pcbD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA) hydrolase was subcloned in Escherichia coli XL-1-Blue by using pBluescript SK(+) vector. The 2.8-kb HindII fragment harboring the pcbD gene cloned in pCK 1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 carrying pCK0124 degraded HOPDA to benzoate and 2-hydroxypenta-2, 4-dienoate by HOPDA hydrolase encoded by pcbD gene as effectively as E coli CK 1022 HARBORING pcbCD genes.

• 농업생명과학연구
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• 제46권2호
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• pp.129-137
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• 2012

A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli $DH5{\alpha}$. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli $DH5{\alpha}$ exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about $50^{\circ}C$ for its enzymatic activity.

• 한국토양비료학회지
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• 제34권5호
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• pp.333-341
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• 2001

퇴비화 촉진 미생물인 Bacillus subtilis LYH201균주가 분비하는 섬유소 분해효소를 분자생물학적으로 연구한 결과는 다음과 같다. 섬유소를 분해하는 유전자는 유전자은행에 의해 구한 약 5,000개의 clone 중 CMC 배지 상에서 활성을 가지는 clone을 선발하여 bglC(pLYH7-39)로 명명하였다. 섬유소를 분해하는 bglC 유전자는 Pvu II, EcoRI, SspI의 제한효소 site를 가지고 있었으며, BglC는 Clostridium acetobutylicum GUN_CLOAB(P15704)와 57%의 identity와 71%의 homology를 나타내어 상동성이 가장 높았으며, CMC-SDS-PAGE 분석으로 56 kDa의 분자량을 나타냈고, 온도는 $50^{\circ}C$, pH는 7에서 활성이 가장 높았다.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.450-456
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• 2006

Pseudomonas fluorescens MC07 is a growth-promoting rhizobacterium that suppresses mycelial growth in fungi such as Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici. To determine the role of the bacterium's antifungal activity in disease suppression, we screened 2,500 colonies generated by Tn5lacZ insertions, and isolated a mutant 157 that had lost antifungal activity. The EcoRI fragment carrying Tn5lacZ was cloned into pBluescript II SK(+) and used as a probe to isolate wild-type clones from a genomic library of the parent strain, MC07. Two overlapping cosmid clones, pEH4 and pEH5, that had hybridized with the mutant clone were isolated. pEH4 conferred antifungal activity to the heterologous host P.fluorescens strain 1855.344, whereas pEH5 did not. Through transposon mutagenesis of pEH4 and complementation analyses, we delineated the 14.7-kb DNA region that is responsible for the biosynthesis of an antifungal compound. DNA sequence analysis of the region identified 11 possible open reading frames (ORF), ORF1 through ORF11. A BLAST search of each putative protein implied that the proteins may be involved in an antifungal activity similar to polyketides.

• 미생물학회지
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• 제36권1호
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• pp.46-51
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• 2000

장티푸스는 Salmonella typhi에 의해 유발되는 장감염성 질환으로 사람과 동물에 공통되는 질병이다. 본 연구에서는 국립보건원과 공동연구를 수행하여 한국형 장티푸스 유발균인 S. typhi KNIH100을 분리하였다. 분리된 S. typhi KNIH100의 염색체 DNA로부터 방향족 아미노산의 생합성에 관여하는 효소인 5-enolpyruvylshikimate-3-phosphate synthetase를 암호화하는 aroA 유전자를 포함하는 약 5.0 kb의 SalI절편을 pBluescriptII SK(+) vector와 aroA 돌연변이주인 E. coli CGSC2829를 이용하여 클로닝하였다. 그리고 이 클론을 pSAL80이라 명명하였다. 클로닝된 재조합 plasmid인 pSAL80에는 ATG 개시코돈과 TGA 종결코돈을 포함하는 1,284 염기로 구성된 aroA 유전자가 위치하고 있었으며, 다른 장내세균과 마찬가지로 serC와 하나의 오페론을 구성하고 있음을 밝혔다. 또한 S. typhi Ty2, S. typhimurium, 그리고 E. coli 등 다른 장내세균의 aroA 유전자와 상동성을 비교하여 본 결과 각각 99%, 95%, 77%의 상동성을 나타내었다.

• 원예과학기술지
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• 제17권6호
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• pp.770-772
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• 1999

새로 육성된 품종의 보호를 위한 분자마커를 대상 작물에 전이시키는 체계를 확립하고자 본 실험을 실시하였다. 식물에서는 전혀 존재하지 않는 mouse adenosine deaminase(ADA) gene으로부터 분자마커로 활용 가능한 크기의 DNA 단편을 획득하고 이를 pBI101에 삽입하여 chimeric gene을 만들었다. 분자마커를 포함하는 형질전환된 담배를 획득하기 위해 A. tumefaciens LBA4404를 이용하여 형질전환을 실시하였다. 담배 절편체에서 형질전환된 신초를 얻기 위해 BAP $1.5mg{\cdot}L^{-1}$, kanamycin $50mg{\cdot}L^{-1}$과 cefotaxim $200mg{\cdot}L^{-1}$이 혼용된 MS배지에서 선발하였으며 신초 발생후 kanamycin의 농도를 2배, 4배로 증가시켜 chimeric gene이 완전하게 전이되어 저항성을 가진 8개체를 얻었다. 항생제에 의해 선발된 8개체를 분자마커 primer로 PCR분석하여 분자마커가 식물체의 genome내로의 전이를 확인하였다.

• 미생물학회지
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• 제29권6호
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• pp.392-396
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• 1991

Pseudomonas sp. DJ77의 chromosomal DNA로부터 6.9kb XhoI 절편 상에 존재하는 phenanthrene 분해에 관련된 유전자군을 vector pBLUESCRIPT SK(+)에 클로닝하였다. 이렇게 얻은 재조합 plasmid인 pHENX7을 가지고 있는 JM101 균주는 3-methylcarechol을 노란색의 meta-cleavage 화합물로 전환할 수 있었다. 그러나 삽입된 절편의 방향이 반대가 되도록 제조한 pHENX7은 extradiol dioxygenase 활성을 나타내지 않기 때문에 전사방향을 알 수 있었다. pHENX7과 이의 듀도체들을 지니는 JH101균주에서 PhnC(24kDa), PhnD(31KDa), PhnE(34kDa), PhnF(KDa)의 4 polypeptide를 확인 할 수 있었고 개개의 유전자의 위치와 범위를 알 수 있었다. 유전자 순서는 phnC-phnD-phnE-phnF-phnG이었으며, phnC, phnD, phnE, phnF, phnG는 각기 glutathione S-transferase, meta-cleavage compound hydrolase extradiol dioxygenase, meta-cleavage compound dehydrogenase의 유전자이었다.