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해면에서 분리된 Bacillus Megaterium의 저작자표시-비영리-변경금지 2.0 대한민국 이용자는 아래의 조건을 따르는 경우에 한하여 자유롭게 l 이 저작물을 복제, 배포, 전송, 전시, 공연 및 방송할 수 있습니다. 다음과 같은 조건을 따라야 합니다: 저작자표시. 귀하는 원저작자를 표시하여야 합니다. 비영리. 귀하는 이 저작물을 영리 목적으로 이용할 수 없습니다. 변경금지. 귀하는 이 저작물을 개작, 변형 또는 가공할 수 없습니다. l 귀하는, 이 저작물의 재이용이나 배포의 경우, 이 저작물에 적용된 이용허락조건 을 명확하게 나타내어야 합니다. l 저작권자로부터 별도의 허가를 받으면 이러한 조건들은 적용되지 않습니다. 저작권법에 따른 이용자의 권리는 위의 내용에 의하여 영향을 받지 않습니다. 이것은 이용허락규약(Legal Code)을 이해하기 쉽게 요약한 것입니다. Disclaimer Probiotics Properties of Bacillus megaterium Isolated from Marine Sponges and Effect of Enhance Immune Response in Olive flounder Paralichthys olivaceus SoHyun Park (Supervised by professor MoonSoo Heo) A thesis submitted in partial fulfillment of the requirement for the degree of Doctor of Science Department of Marine Life Science GRADUATE SCHOOL JEJU NATIONAL UNIVERSITY 목 차 목 차 ····································································································································· Ⅰ Abstract ······························································································································· Ⅴ List of Tables ···················································································································· Ⅸ List of Figures ·················································································································· Ⅺ 제 1장. 제주 해면에서 분리된 세균의 세균군집 구조 분석 및 신종후보균주의 분류학적 연구 및 동정 1.1. 서론 ································································································································ 1 1.2. 재료 및 방법 1.2.1. 시료채집 ················································································································· 3 1.2.2. 해양세균 분리 및 배양 ······················································································· 3 1.2.3. 16S rRNA 염기서열 분석 및 계통학적 분석 ················································· 5 1.2.4. 해면에서 분리된 신종후보균주의 다상학적 분석 1.2.4.1. 16S rRNA 염기서열 분석 및 계통학적 분석 ········································· 8 1.2.4.2. 형태학적 분석 ································································································ 9 1.2.4.3. 생리학적 및 생화학적 분석 ······································································ 11 1.2.4.4. 화학분류학적 분석 ······················································································ 13 1.2.4.5. Genomic 분석 ···························································································· 16 1.3. 결과 및 고찰 1.3.1. 해면 내 분리세균의 16S RNA 염기서열 및 계통학적 분석 1.3.1.1. 보라예쁜이해면 ···························································································· 17 1.3.1.2. 굵은나선별해면 ···························································································· 24 1.3.1.3. 호박해면 ······································································································· 30 - I - 1.3.1.4. 주황해변해면 ······························································································· 36 1.3.2. 주요 세균군집구조 분석 ··················································································· 42 1.3.3. 해면에서 분리된 신종후보균주의 다상학적 분석 1.3.3.1. 계통학적 분석 ····························································································· 44 1.3.3.2. 형태학적, 생리학적 및 생화학적 특성 ··················································· 46 1.3.3.3. pufLM gene ······························································································· 48 1.3.3.4. 화학분류학적 특성 ······················································································ 49 1.3.3.5. 균주기탁 ······································································································· 53 1.3.3.6. GenBank Accession Number ······························································· 53 1.3.4. Description of Labrenzia callyspongiae sp. nov., ······························ 54 제 2장. 해면에서 분리된 세균의 항균활성 탐색 및 Probiotics 특성 연구 2.1. 서론 ······························································································································ 57 2.2. 재료 및 방법 2.2.1. 분리된 세균의 어류질병에 대한 항균활성 탐색 ·········································· 58 2.2.2. 용혈성 및 유해효소 생성 ················································································· 60 2.2.3. pH, NaCl, 온도 ································································································· 61 2.2.4. 고분자 유기물질 분해능 ··················································································· 62 2.2.5. 인공위액과 인공담즙에서의 생존력 ································································ 63 2.2.6. API 50CHB 시험 ······························································································· 64 2.2.7. 항생제 감수성 시험 ··························································································· 65 2.2.8. 배양액 물질분석 ································································································· 66 2.3. 결과 및 고찰 2.3.1. 어류질병세균에 대한 항균활성 탐색 ······························································ 67 2.3.2. 용혈성과 유해효소 생성여부 확인 ·································································· 69 2.3.3. 고분자 유기물질 분해능 ··················································································· 70 2.3.4. pH, NaCl, 온도 ································································································· 72 - II - 2.3.5. 인공위액과 담즙내성 ························································································· 74 2.3.6. Probiotics 균주 선정 ······················································································· 74 2.3.7. API 50CHB 시험 ······························································································· 75 2.3.8. 항생제 감수성 시험 ··························································································· 76 2.3.9. 배양액 물질분석 ································································································· 77 2.3.10. 최종 선별균주 동정 및 계통수 작성 ···························································· 78 제 3장. Bacillus megaterium PPR15T 첨가사료를 급이시 넙치의 비특이적 면역반응과 질병저항성에 미치는 영향 3.1. 서론 ······························································································································ 80 3.2. 재료 및 실험방법 3.2.1. 실험사료 제작 ····································································································· 82 3.2.2. 어류 준비 및 사육조건 ····················································································· 82 3.2.3. 성장도 조사 및 혈액학적 분석 ········································································ 83 3.2.4. 식세포 활성 ········································································································· 84 3.2.5. Lysozyme 활성 ·································································································· 85 3.2.6. Myeloperoxidase 활성 ···················································································· 86 3.2.7. Superoxide dismutase (SOD) activity ······················································ 87 3.2.8. RNA 추출 ············································································································ 88 3.2.9. cDNA 합성 ·········································································································· 90 3.2.10. qRT-PCR ·········································································································· 91 3.2.11. 인위감염에 대한 질병 저항성 ······································································· 92 3.2.12. 유의학적 통계분석 ··························································································· 92 3.3. 결과 및 고찰 3.3.1. 성장도 조사 ········································································································· 93 3.3.2. 혈액학적 분석 ····································································································· 94 3.3.3. 식세포 활성 ········································································································· 97 - III - 3.3.4. Lysozyme 및 Myeloperoxidase 활성 ·························································· 98 3.3.5. Superoxide dismutase (SOD) 활성 ·························································· 100 3.3.6. qRT-PCR을 통한 면역관련 유전자 발현 ···················································· 101 3.3.7. 인위감염에 대한 질병 저항성 ······································································· 104 국문요약 ····························································································································· 105 참고문헌 ····························································································································· 108 감사의 글 ··························································································································· 125 - IV - Abstract The ocean is a vast ecosystem that is home to more than 80% of living organisms, and is one of the most valuable biological resources. Marine organisms found in the ocean are indigenous to special environments compared to those living on land, in terms of salinity, temperature, and chlorophyll, and thus have the ability to produce unique substances and can produce various biologically active substances. Of all the marine life forms, sea sponges are important resources for studying new materials. The emergence of antibiotic resistant bacteria strains has led to an increase in the research for new substances, and the number of bioactive substances that are isolated from marine sponges is also on the rise. These bioactive substances have been reported to be produced by commensal bacteria existing in the sponges. Bacteria
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