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標題: 魚類適應鹽度生理機制之新觀點-魚類適應鹽度過程離子運輸蛋白的調控FXYD蛋白的角色
Role of Fxyd Proteins in Regulation of Ion Transporters during Acclimation to Salinity
作者: 李宗翰
關鍵字: 漁業類, 生物科學類;基礎研究
本整合型計畫擬以分子, 細胞, 生理學等多種研究方法, 進行整合性探討, 以暸解魚類在環境鹽度變化時, 上游的內分泌(CRH與cortisol)啟動魚鰓的肝醣代謝, 提供額外的能量供應下游的FXYD蛋白質進行調控Na+/K+-ATPase相關之離子運輸機制之運作. 本子計畫即負責探討魚類適應鹽度機制下游的FXYD蛋白質對Na+/K+-ATPase的調控. 吳郭魚在環境鹽度升高時, 鰓的Na+/K+-ATPase活性也隨之增加, 但是調控Na+/K+-ATPase表現的機制至今仍不清楚. 近年來在哺乳動物發現的FXYD蛋白質有至少七個成員, 共同特徵是有ㄧ個穿膜domain及位於膜外的ㄧ個FxYD motif, 多數成員都具有調節Na+/K+-ATPase表現的功能. 本子計畫的目的是(1)提供FXYD蛋白質及其mRNA存在於硬骨魚類的證據; (2)闡明FXYD蛋白質與Na+/K+-ATPase在魚類?組織上的交互作用; (3)呈現魚類FXYD蛋白質調控Na+/K+-ATPase的功能. 本子計畫擬分三年進行: 第一年, 利用黃鵬鵬教授(子計畫1主持人)所提供的吳郭魚cDNA library選殖廣鹽性魚類可能存在的FXYD蛋白質, 測定其組織分佈, 並且製作抗體, 供後續實驗使用; 第二年, 探討吳郭魚鰓在環境鹽度變化時, Na+/K+-ATPase以及FXYD蛋白質的表現變化, 並且藉由免疫螢光染色與共同免疫沉降法證明Na+/K+-ATPase與FXYD蛋白質在鰓上有交互作用; 第三年, 與黃鵬鵬教授合作用非洲爪蟾的卵作為表現系統, 經由人工方式將Na+/K+-ATPase與FXYD蛋白質分別表現在爪蟾卵上, 再經由分析Na+/K+-ATPase活性比較FXYD蛋白質對Na+/K+-ATPase的調控機制. 本子計畫的研究成果配合其他兩個子計畫的結果, 將可提供魚類適應鹽度機制之創新之觀點.

The present project use molecular, cellular, and physiological approaches to study the axis of salinity adaptation from trigger of the glycogen metabolism (energy) in fish gills by up-stream endocrine (CRH and cortisol) to provide energy for the operation of the down-stream FXYD-mediated Na+/K+-ATPase regulation. This sub-project (sub-project 3) will focus on the down-stream FXYD regulatory mechanisms upon salinity challenge.The Na+/K+-ATPase activity in gills of tilapia elevated with increasing salinities, while little is known about the regulatory mechanisms of Na+/K+-ATPase expression. Recently found mammalian FXYD proteins were consisted of at least 7 members and characterized by a transmembrane domain and an extra-membrane FxYD motif. Most members function as Na+/K+-ATPase regulators.The specific aims of this sub-project are to (1) provide the evidence that FXYD protein (and mRNA) exhibit in teleostean fish; (2) elucidate the interaction of FXYD proteins and Na+/K+-ATPase; (3) reveal the regulatory functions of FXYD proteins. This three-year sub-project will focus on (1) the 1st year- cloning the FXYD proteins from the tilapia cDNA library provided by Prof. P.P. Hwang (sub-project 1) and production of antisera against selected FXYD proteins; (2) the 2nd year- (i) time-course changes in transcripts and relative protein abundance of FXYD proteins and Na+/K+-ATPase in gills of tilapia upon salinity challenge; (ii) immunolocalization and co-immunoprecipitation of FXYD proteins and Na+/K+-ATPase in tilapia gills; (3) the 3rd year- using Xenopus oocytes to carry out functional interaction between FXYD proteins and Na+/K+-ATPase. The results of this sub-project together with data of the other two sub-projects will provide new insights through the energy-axis to the mechanisms of salinity adaptation.
其他識別: NSC96-2313-B005-010-MY3
Appears in Collections:生命科學系所

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