Abstract
金屬硫蛋白(metallothionein) 是一種普遍存在於生物體內的低分子量蛋 白質, 與金屬離子具有高度的結合能力. 所以被認為與生物體內重金屬的 解毒作用有關, 或參與了細胞內微量金屬如銅, 鋅等的代謝作用. 哺乳類 動物中含有多種型式的金屬硫蛋白, 根據陰離子交換樹脂的分析特性主要 可分為兩大類, MT-I及MT-II.可是, 在一些報告中指出, 鳥類.爬蟲類及 兩棲類似乎只表現一種或兩種金屬硫蛋白.由以前的實驗已知,臺灣常見的 兩種水禽類--番鴨及菜鴨--只表現一種金屬硫蛋白, 且其氨基酸序列與雞 的完全相同. 由於雞和鴨是完全不同屬的鳥類, 因此比較二者的金屬硫蛋 白基因結構將有助於瞭解其在鳥類中的的演變情形. 在此,我們利用番鴨 為實驗材料,以鎘誘導金屬硫蛋白的表現後, 從肝臟抽取出核糖核酸再經 由逆轉錄酵素的作用逆轉錄成互補去氧核糖核酸(cDNA), 送入寄主中篩 選. 在找了二千多個轉形株後, 仍無法找到番鴨的金屬硫蛋白互補去氧核 糖核酸, 因此改以聚合酵素連鎖反應從合成的互補去氧核糖核酸中選殖番 鴨金屬硫蛋白的轉譯區域, 經過核酸序列後顯示此轉譯區域只有六個核酸 位置與雞的完全不同, 其餘序列則完全相同. 此外, 我們也探討了銅, 鋅,鎘,等金屬對番鴨金屬硫蛋白的表現.而在胚胎發育過程中, 金屬硫蛋 白在肝臟中的表現, 其在小鴨孵出前兩天會達到最高點而在孵出當天又回 復到基本表現量.這種在胚胎過程中表現的變化顯示金屬硫蛋白在發育過 程中扮演某種角色. Metallothionein (MTs)are small molecular weight, cysteine-rich proteins which bind heavy metal ions with high affinity. The expression of MT is induced in response to the challenfe of metal ions. It is suggested that MT may play roiles in detoxification of heavy metals and metabolism of essential metals such as zinc and copper ions. Two major isoforms (MT-I and MT-II) have been identified in mammals. However, only one predominant form of MT protein was found in birds, reptiles and amphibians. For domesticated fowls, a complete homology in protein sequence between MT isolated from chick and two genera ducks-- the Mascovy duck and the Tsai duck-- has been shown. In order to investigate the gene organization of MT in the aves, cDNA was synthesized from hepatic mRNA of Mascovy (Cairina muschata) to clone MT gene. However, the attempt was not succeded. A 0.2 kb DNA product was obtained by reverse-PCR using a pairs of primer corresponding to the coding sequence of chicken MT gene. After cloning and sequencing, the Mascovy MT coding region was revealed. The DNA sequence of Mascovy MT differs form that of chicken in six nucleotides. We investigated the expression of MT mRNA induction in Mascovy by zinc, cadmium and copper treatments. In addition, hepatic MT expression during development was also studied. It was found that Mascovy MT mRNA reaches the highest level on two days before hatching, which is quite different from what was found in mammals.