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Callyspongia elegans에서의 세균군집 분석 및 항균활성탐색

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Alternative Title
Bacterial community analysis and bacteria screening involving antimicrobial activity in Callyspongia elegans
Abstract
The aim of this study was to investigate the Bacterial community analysis and Screening of bacteria with antibacterial activities from Callyspongia elegans. In marine bacteria were isolated from the marine sponge C. elegans using marine agar. The resulting 112 isolated pure cultures were then used for further study. The characterization of the bacteria was done through microscopic and molecular analysis. Some of the isolated strains were determined to be novel bacterial species through taxonomic identification and classification .
A total of 112 culturable strains were isolated. They were characterized by determining morphological characteristics through, Gram' staining. The colony pigments of bacterial isolates were characterized as yellow, brown, ivory, and white. Thirty-seven strains were found to be Gram-positive and 75 strains were Gram-negative. Seventy-nine strains of all isolates were coccus-shaped, while 16 strains were rod-shaped.
The isolated strains were selected for phylogenetic investigation through a 16S rDNA gene sequence analysis. The bacterial genomic DNA was isolated and 16S rDNA regions were amplified using the primers 27F and 1522R. The sequences were then compared with the sequences in the NCBI Genebank database and a BLAST analysis was performed. On the basis of the results of the comparative analyses of 16S rDNA gene sequences, the 112 isolated bacteria were divided into 5 major groups: α-proteobacteria (39%), γ-proteobacteria (22%), Actinobacteria (14%), Fimicutes (9%), and Bacteroid-
etes (6%).
All of the isolated strains were tested for antibacterial activity using agar spot assays and amplifications of Polyketide synthase (PKS) genes. To detect the PKS genes, PCR was carried out using the genomic DNA of these strains as templates, as well as two oligonucleotides that bind specifically to the ketosynthase (KS) domains of PKS. PCR with the genomic DNA from all 16 strains yielded about 700 bp DNA fragments, which were the expected size for the PCR product. The results showed that KS , domains of PKS from 3 strains that had appeared to be novel actually had identities as Pseudovibrio sp. (90%) in the databases. Isolates demonstrating antibacterial activity represented 33% of the total. Twenty-eight strains also showed antibacterial activity against E. tarda.
It is strongly suggested that fifteen isolates are candidates for a new genus or species, based on the analyses of 16S rDNA gene sequences.
Author(s)
박소현
Issued Date
2012
Awarded Date
2012. 8
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000006000
Alternative Author(s)
Park, So-Hyun
Affiliation
제주대학교
Department
대학원 해양생명과학과
Advisor
허문수
Table Of Contents
목 차 ⅰ

List of Tables ⅲ
List of Figures ⅳ
Abstract ⅴ

Ⅰ. 서 론 1

Ⅱ. 재료 및 방법 4

2.1. 시료채집 4

2.2. 미생물의 분리 및 배양 5
2.2.1. 미생물의 분리·배양 및 보관 5

2.3. 표현형질에 따른 특징 8
2.3.1. 배양상 특징 8
2.3.2. 형태학적 특징 8

2.4. 분자생물학적 방법 9
2.4.1. Genomic DNA 분리 9
2.4.2. 16S rDNA 유전자 증폭 10
2.4.3. 유전자 cloning 및 형질전환 11
2.4.4. 염기서열 분석 및 계통학적 분석 12

2.5. 항균물질 탐색 13
2.5.1. 항균활성 유전자 탐색 13
2.5.2. 분리 균주의 항균활성 측정 14

Ⅲ. 결 과 16

3.1. C. elegans 미생물의 순수분리 16
3.1.1. 세균수 측정 16

3.2. 표현형질에 따른 특징 17
3.2.1. 분리균주의 배양상 및 형태학적 특성 17

3.3. 16S rDNA 염기서열의 계통학적 분석 23
3.3.1. C. elegans 내 세균군집의 계통학적 특성 23
3.3.2. C. elegans bacteria 군집분석 33

3.4. 항균물질 탐색 36
3.4.1. 항균활성유전자 탐색 36
3.4.2. 분리균주의 항균활성 측정 38


Ⅳ. 요 약 40


Ⅴ. 참고 문헌 42


Ⅵ. 감사의 글 49
Degree
Master
Publisher
제주대학교 대학원
Citation
박소현. (2012). Callyspongia elegans에서의 세균군집 분석 및 항균활성탐색
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General Graduate School > Marine Life Sciences
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