북방전복(Haliotis discus hannai)의 allatotropin 전구체와 allatotropin 수용체의 동정 및 allatotropin에 의한 심장 박동에 미치는 영향
Identification of allatotropin precursor and allatotropin receptor, and effect of allatotropin on heart rate in Pacific abalone (Haliotis discus hannai)
- 주제(키워드) 도움말 neuropeptide , AT
- 발행기관 강릉원주대학교 일반대학원
- 지도교수 도움말 손영창
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 해양분자생명과학과
- 세부분야 해당없음
- 실제URI http://www.dcollection.net/handler/kangnung/000000011742
- UCI I804:42001-000000011742
- 본문언어 한국어
초록/요약 도움말
1989년 신경펩타이드 알라토트로핀(allatotropin, AT)이 나방의 한 종인 박각시 나방 Manduca sexta의 중추신경계에서 최초로 동정된 이후, AT 펩타 이드는 장 수축, 심박수, 이온 수송, 일주기 리듬 및 소화효소 분비를 조절 한다고 알려져 있다. 본 연구는 해양 연체동물의 AT 신호전달분자를 동정 하고 기능을 탐색하기 위하여, 북방전복(Haliotis discus hannai, Hdh)의 AT 전 구체의 엑손-인트론 구조를 조사하고, AT 수용체(ATR)의 cDNA를 cloning하 여 발현벡터에 삽입한 후 포유동물 유래의 세포에 형질도입하였다. Hdh-AT 및 Hdh-AT의 2번째 L형 페닐알라닌(Phe) 잔기를 D형으로 이성질체화 한 Hdh-D2-AT를 합성하여 Hdh-ATR 발현 세포에 처리하고 신호전달 반응을 리포터시스템을 이용하여 조사하였다. Hdh-ATR은 404개의 아미노산으로 구성되어 있으며, intracellular loop 2 (ICL2)에 보존된 E/DRW 모티프, 로돕신계열 G-단백질 연계 수용체(GPCR) 의 특징인 transmembrane domain 6 (TMD6)에 잘 보존된 트립토판(Trp) 잔기 는 다양한 무척추동물의 ATR 상동체와 동일하게 티로신(Tyr) 잔기로 치환 되어 있었다. Hdh-AT 전구체는 14개 아미노산으로 구성된 한 개의 성숙형 펩타이드가 포함된 전체 107개 아미노산으로 암호화되어 있었으며, Hdh-AT 전구체 및 Hdh-ATR의 조직별 발현율을 조사하기 위해 실시간 정량PCR을 실시한 결과, Hdh-ATR 및 Hdh-AT 전구체 mRNA 발현은 말초조직에 비해1 신경절에서 높게 발현되었다. 리포터벡터와 Hdh-ATR 발현벡터를 형질도입 한 포유동물 유래의 세포에 Hdh-AT 및 Hdh-D2-AT를 처리하였을 때, Ca2+ 반응형 리포터 활성은 Hdh-AT 펩티드 농도의존적으로 증가하였고, ERK1/2 인산화를 유도하였다. Hdh-AT와 Hdh-D2-AT를 북방전복의 혈동에 주사한 결과 농도의존적으로 심박수가 감소하였으며, 고농도 Hdh-D2-AT에 의해 폐사가 유발되었다. 본 연구의 결과를 요약하면, Hdh-AT 신호전달 시스템은 신경계에 주로 발현되며, Gαq/PKC 활성화 경로와 상관관계가 있고, Hdh-D2- AT는 심박수를 저하시키는 생물학적 활성을 보였다.
more목차 도움말
Ⅰ. 서론 ·······························································································1
Ⅱ. 재료 및 방법 ·················································································4
1. 염기서열 및 아미노산 서열 탐색과 분자 계통수 분석···············4
2. 북방전복의 AT 수용체 cloning···················································4
3. 펩타이드 합성···········································································5
4. 실시간 정량 PCR·······································································6
5. Luciferase reporter assay 분석························································6
6. Ca2+ mobilization assay ·································································7
7. ERK 1/2 인산화··········································································8
8. 북방전복의 AT 수용체 3D 모델링 ·············································8
9. 북방전복의 ATs에 의한 전복의 심박수 조사·········································9
10. 북방전복의 ATs에 의한 전복의 폐사율 조사······························9
11. 북방전복의 Hdh-D2-AT에 의한 굴 (Crassostrea gigas)의 심박수 조사
················································································································································9
12. 통계분석····················································································10
Ⅲ. 결과 ······························································································11
1. 북방전복의 AT 전구체 서열 분석 ·············································11
2. 북방전복의 AT 수용체 서열 분석 ·············································12
3. 북방전복의 AT 수용체 및 전구체 조직별 mRNA 발현 분석······16
4. 북방전복의 luciferase reporter assay 분석······································16
5. 북방전복의 AT Ca2+ mobilization assay 분석 ·································21
6. 북방전복의 AT ERK 1/2 인산화 분석··········································22
7. 북방전복의 AT 수용체 3D 모델링 분석·····································23
8. 북방전복의 ATs에 의한 전복의 심박수 ····································25
9. 북방전복의 ATs에 의한 전복의 폐사율 ·················································25
10. 북방전복의 Hdh-D2-AT에 의한 굴의 심박수·······························26
Ⅳ 고찰 ·······························································································27
참고문헌··········································································30
Abstract············································································33
