Na，K-ATPase α次單元在?郭魚滲透壓調節中的角色：mRNA和蛋白質的表現

Abstract

利用快速放大cDNA基因（rapid amplification of cDNA ends, RACE）的方式，選殖到吳郭魚Na, K-ATPase α1和α3 兩種次單元殖株TG33和TH3。其中TG33之全長cDNA共3390bp，可轉譯出1023個胺基酸；TH3之全長cDNA共3581bP，可轉譯出1010個胺基酸。與其他動物的同型次單元相比較，分別有高達91％和88％的相同度，而α1輿α3間則只有80％的相同度，隨後經由離體轉錄及轉譯的方式證實α1和α3為100kDa的蛋白質。 比較TG33和TH3之cDNA序列，選擇具明顯差異的3’-Untranslated region設計探針P1和P3。利用北方和南方雜合試驗證實，P1和P3兩種探針彼此沒有交互作用（cross reaction）產生。由北方墨點法的結果顯示，吳郭魚的TG33主要以腎臟和腸組織表現量較高；TH3則是在腦部和心臟的含量較豐富。另外，TG33在吳郭魚鰓部的表現有隨著鹽度提高而增加的趨勢，海水魚鰓部的表現約是淡水魚的10倍，表示Na, K-ATPase α1次單元與吳郭魚滲透壓調節有關。 進一步參考α1和α3之胺基酸序列，選擇可供區分兩者之序列合成oligopeptides，並以之為抗原注射兔子以產生抗體Tal-2。而經由西方墨點法確認，Tal-2對於Na, K-ATPase α1次單元具有專一性，隨後便利用此抗體調查α1在吳郭魚各組織的表現量，發現腎臟和腸組織的含量較高。這樣的結果與北方墨點法的資料相吻合，不過，其他組織也有相當量的表現。此外，鹽度的變化會影響α1蛋白的表現，如海水馴化之吳郭魚鰓部α1的蛋白質表現量約是淡水馴化吳郭魚的15倍。總之，吳郭魚在面對不同鹽度環境時，可經由調節鰓部Na, K-ATPase α1次單元的mRNA和蛋白質的表現而維持體內滲透壓的平衡。

Using rapid amplification of cDNA ends (RACE), full length cDNAs of Na, K-ATPase α1 and α3 subunits of tilapia (Oreochromis mossambicus) were cloned and sequenced. Clone TG33 is 3390bp in length and encodes a polypeptide of 1023 amino acids, while clone TH3 is 3581bp in length and encodes a polypeptide of 1010 amino acids. Clones TG33 and TH3 showed 91% and 88% identity at amino acid level with previously described animal Na, K-ATPase α1 and α3 subunits, respectively. Northern blot analysis showed that α1 subunit is predominantly expressed in kidney and intestine and α3 subunits is expressed in brain and heart. Results of immunoblotting and RT-PCR indicate that Na, K-ATPase α1 subunit is ubiquitously expressed. The amount of α1 subunit mRNA and protein in gill tissue increased with the level of environmental salinity. Our results indicate that the salinity-dependent stimulation of mRNA expression of gill Na, K-ATPase α1 subunit is associated with corresponding stimulation at the protein level. This provides direct evidence of enhanced transcription and translation of Na, K-ATPase α1 subunit gene upon salinity challenge.