淡水长臂大虾异型蛋白激酶C之cDNA分子选殖与特性分析及其与脱壳.DOC

淡水長臂大蝦異型蛋白激酶C之cDNA分子選殖與特性分析及其與脫殼週期及外來物質注射之關聯性 Molecular cloning and characterisation of atypical protein kinase C cDNA from the freshwater giant prawn, Macrobrachium rosenbergii in relation with moult stages and foreign materials injection 研究生:葉怡均 Yi-Chun Yeh 指導教授: 鄭文騰 Winton Cheng 邱謝聰 Shieh-Tsung Chiu 【摘要】 本研究目的在於選殖淡水長臂大蝦（Macrobrachium rosenbergii）異型蛋白激酶C（atypical protein kinase C, aPKC）之序列，並探討內、外因子對淡水長臂大蝦血球aPKC mRNA表現之影響。利用退化性寡核苷酸引子進行反轉錄聚合酶鏈鎖反應，選殖淡水長臂大蝦aPKC cDNA序列，其部份序列為1403 bp，可轉譯467個胺基酸，序列中有發現2個功能區，分別為C1富含半胱胺酸鋅指功能區（Cys-rich zinc-finger damain）以及羧基端之激酶功能區（kinase domain）。經由反轉錄聚合酶鏈鎖反應分析發現aPKC mRNA轉錄在各組織均有表現。以即時聚合酶鏈鎖反應分析，探討內、外因子對淡水長臂大蝦血球aPKC mRNA表現之影響，結果顯示：在不同脫殼階段中aPKC基因表現量C期明顯高於其他脫殼階段（A、B、D0/1及D2/3）。注射乳酸鏈球菌經6小時及注射肽聚醣經12小時，血球aPKC之表現量顯著增加；注射脂多醣及溶藻弧菌，分別在12及3小時，血球aPKC之表現量顯著降低；注射干擾素誘導物poly：IC及白斑病毒經12小時，血球aPKC之表現量顯著降低。結論，細菌性病原及其成份可誘導aPKC之表現，但非病原及其成份會降低aPKC之表現。 關鍵字：淡水長臂大蝦、異型蛋白激酶C、即時聚合酶鏈鎖反應、脫殼週期、外來物質注射 【Abstract】 This research is aimed to resolve the molecular characteristics of atypical protein kinase C (aPKC) in freshwater giant prawn, Macrobrachium rosenbergii, via aPKC cDNA cloning and expressional profiling. The extrinsic and intrinsic factors that affect expression of aPKC were also addressed. The aPKC cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) using degenerated primers. The partial cloned cDNA of aPKC consists of 1403 bp and encoded 467 amino acids. The predicted protein consists of a cys-rich zinc-finger domain and a kinase domain observed in the partial sequence. Transcription of aPKC by RT-PCR was detected in all tissues examined. To evaluate the extrinsic and intrinsic factors that affect expressions of aPKC gene, the results analyzed by quantitative real-time PCR were as follows: In different moult stages, aPKC gene expression at C stage were significantly higher than the other moult stages (A, B, D0/1, D2/3). The aPKC transcripts expressed in haemocytes was significantly increased at 6 h after Lactococcus garvieae