- 주요 양식장 물고기로부터 다양한 어병 관련 세균의 분리 확보 - 분리한 어병 세균으로부터 신호전달 물질 탐색 및 병원성 phenotype과의 관련성 조사 - 본 연구팀이 확보하고 있는 bioassay system을 이용하여 미생물 배양액 및 식물 추출액으로부터 어병 관련 신호 전달 억제제 탐색 및 발굴 - 분리한 어병 세균을 대상으로한 신호전달 억제제의 분리, 정제 및 특성화 - 분리한 어병 세균으로부터 신호전달 관련 유전자 분리 및 신호물질 분해 효소 발현 - 억제제 및 신호전달 관련 유전자를 활용한
- 주요 양식장 물고기로부터 다양한 어병 관련 세균의 분리 확보 - 분리한 어병 세균으로부터 신호전달 물질 탐색 및 병원성 phenotype과의 관련성 조사 - 본 연구팀이 확보하고 있는 bioassay system을 이용하여 미생물 배양액 및 식물 추출액으로부터 어병 관련 신호 전달 억제제 탐색 및 발굴 - 분리한 어병 세균을 대상으로한 신호전달 억제제의 분리, 정제 및 특성화 - 분리한 어병 세균으로부터 신호전달 관련 유전자 분리 및 신호물질 분해 효소 발현 - 억제제 및 신호전달 관련 유전자를 활용한 어류 병원성 제어 연구 - 신호물질 분해 효소를 포함한 억제제를 이용해 양식장 어병 제어의 현장 적용시험 및 병원성 제어 효능 시험 - 신호물질 억제제 생산 균주 및 식물체로부터 antagonist의 대량 생산 - 양식장에서의 처리를 위한 분리 신호억제 물질의 제제화 기술 개발 및 상품화
Abstract▼
The purpose of this study is to develope biocontrol agents, based on blocking of quorum sensing, against pathogens in fish culture. Quorum-sensing is a cell-density dependent signaling mechanism that controls diverse biological functions, including virulence factors via N-acyl-homoserine lactone (AH
The purpose of this study is to develope biocontrol agents, based on blocking of quorum sensing, against pathogens in fish culture. Quorum-sensing is a cell-density dependent signaling mechanism that controls diverse biological functions, including virulence factors via N-acyl-homoserine lactone (AHL) signal molecules in gram-negative bacteria. Since AHL-mediated quorum sensing mechanisms were widespread and highly conserved in many pathogenic bacteria, it has been postulated that blocking of quorum sensing could be a target for control of pathogens. In this study, AHL competitor and AHL-degrading enzyme among several strategies were used to block quorum sensing. Study were undertaken to investigate the profile of AHLs produced by Vibrio spp., Edwardsiella spp. and Aeromonas hydrophila isolated from diseased fish (Paralichthys olivaceus). All tested 24 fish pathogens were found to produce quorum-sensing signal molecules, N-acyl homoserine lactones (AHLs). TLC analysis of AHLs showed that various AHLs (OHHL, OHL, DHL, and OdDHL) were produced, and N-3-oxo hexanoyl homoserine lactone (OHHL) was synthesized as a major signal molecule in all the strains. Edwardsiella spp. were found to produce hexanoyl homoserine lactone (HHL) dominantly besides OHHL.Several strains produced virulence factors including protease, lipase, siderophore and hemolysin activity. AHL synthase genes were cloned from the 6 Vibrio spp, showed high similarity (87-97%) to vanI of Vibrio anguillarum. The edI gene that encodes AHL synthase from Edwardsiella sp. E7 was cloned. And the lipked edR gene encoding a transcriptional regulator gene was identified using Southern hybridization and primer walking. This is a first report that Edwardsiella strains have quorum-sensing mechanisms. Comparison of EdI with Erwinia carotovoa CarI and Enterobacter agglomerans EagI showed identities of 53% and 50% in the deduced amino acid sequences, respectively. EdR had 59% homology with Erwinia chrysanthemi EchR. The edIR homologue genes were also found in other two Edwardsiella strains. These results indicate that quorum-sensing mechanisms were also conserved in many fish pathogens. To investigate that the virulences of fish pathogens were regulated by quorum-sensing, the aiiA gene, encoding AHL-degrading enzyme of Bacillus thuringiensis, was expressed in Vibrio sp. PT2. As a result, the expression of AiiA reduced AHL production and protease activity of Vibrio sp. PT2, suggesting that virulence factor such like the protease activity was regulated by quorum-sensing. For the attenuation of AHL-regulated virulence, AHL antagonists have been screened from the plant extracts and the supernatants of Streptomyces spp.. Antagonist activity was. detected from 65 plant extracts. The antagonists of Buxus. mibriophylla var. koreana extract, showing the highest antagonist activity, were purified, and the structures of compounds were determined by NMR analysis. As a result, theses compounds were identified as octadecane-l-ol, scopoletin, betuline, scopolin, and rutin containing ring structure.The antagonist purified from Streptomyces G406 showed the structure similar to genistein through ESI-Mass analysis. Unfortunately, the antagonist activity was decreased according to the progress of purification step. As a result, all the purified antagonists showed relatively lower activity than the compound mixtures before purification.
목차 Contents
제 1 장 서론...16
제 2 장 국내외 기술개발 현황...19
제 1 절 세계적 기술현황 및 전망...19
제 2 절 국내 기술현황 및 전망...19
제 3 장 연구수행 방법 및 내용...20
제 1 절 어병 관련 세균의 분리 및 이들의 신호물질과 병원성 phenotype 확인...20
1. 양식장 물고기로부터 다양한 어병 관련세균의 분리...20
2. 분리된 어병 세균으로부터 신호물질 (AHL) 탐색...20
3. 어병 세균의 병원성 phenotype 조사...21
가. Protease 활성 조사...21
나. Lipase 활성 조사...21
다. Hemolytic 활성 조사...21
라. Siderophore 활성 조사...21
4. 어병 세균 및 방선균의 동정...22
5. 어병 세균의 신호전달 관련 유전자 분리 및 신호물질 분해 효소 발현...22
가. DNA 조작...22
나. 신호물질 합성 유전자 (AHL synthase)와 regulator 유전자 분리...22
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