PCR 스크리닝 전략을 이용한 유용 희소방선균 선별 및 분자공정기법 구축

PCR 스크리닝 전략을 이용한 유용 희소방선균 선별 및 분자공정기법 구축
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Actinomycetes, gram positive soil bacteria, are valuable microorganisms which produce useful secondary metabolites including antibiotics, antiparasitic substances, anti-cancer drugs, and immunosuppressants. Although a major family of actinomycetes called streptomycetes has been intensively investigated, a potentially-valuable non-streptomycetes rare actinomycetes (NSRA) family is largely uncharacterized. In order to isolate NSRA strains among actinomycetes strain collection, a PCR-based genome screening was performed from 180 independently isolated antifungal actinomycetes strains (kindly provided by KRIBB). Using a streptomycetes-specific PCR screening strategy (1), we were able to screened out most of streptomyces species from the strain collection. The remaining several NSRA strains were then confirmed and classified based on 16S rRNA sequence information. Antimicrobial activity assay was performed to NSRA, one strain named N. MMBL010 shows both antifungal and antibacterial (gram positive, gram negative) activity. Additional PCR-based genome screening was performed to identify the stains whose genes might associate with production of valuable secondary metabolite. The presence of potentially-valuable genetic information such as typeⅠ PKS, type Ⅱ PKS and aminoglycoside phosphotransferase was screened from these NSRA strains followed by the establishment of genetic manipulation techniques (2). These results suggest that a PCR screening strategy for NSRA strains as well as NSRA genetic manipulation might lead to the isolation of potentially-valuable biosynthetic genes and compounds present in various rare actinomycetes.
Abstract Ⅰ Contents Ⅱ List of Tables IV Captions for Figures V 1. Introduction 1 2. Materials and Methods 2 2.1 Bacterial strains, isolation of chromosomal DNA 2 2.2 Streptomycetes screening PCR primer 2 2.3 Antimicrobial activity assay 2 2.4 Detection of TypeⅠ PKS, NRPS 3 2.5 Introduction of conjugation system 3 2.6 Construction of genomic library 4 3. Results and Discussion 5 3.1 Screening & isolation of Rare Actinomycetes 5 3.2 Identification of bioactive compounds produced by isolated strains 10 3.3 Detection of TypeⅠ PKS & NRPS which were related to antibiotics biosynthesis 12 3.4 Construction of genetic modification system based on conjugation 14 3.5 Construction of genomic library & Further study 16 4. Acknowledgements 17 5. References 18
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College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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