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標題: 應用金黃色葡萄球菌鎘離子運輸蛋白CadA於斑馬魚重金屬抗性之研究
Application of the Staphylococcus aureus cadmium-transporter CadA in zebrafish(Danio rerio) for heavy metal resistance
作者: 李郁蕙
Li, Yu-Hui
關鍵字: cadmium

heavy metal resistance
出版社: 生命科學系所
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摘要: 鎘用於電鍍工業、礦業、冶鍊、電池、廢五金、染料、焊料,以及用於製造塑膠之穩定劑。離子形式的鎘對生物有很大的威脅,可經由食物鏈之關係進而危害到人類的健康,日本就曾經出現鎘中毒引起的「痛痛病」(Itai-itai disease),因此鎘污染所造成的生態環境破壞以及對人類構成的危險引起國際上的重視。鎘對魚類的影響,包括造成不正常顫抖、激烈泳動、破壞表皮、滲透壓之不平衡、影響鰓之結構與功能、生長遲緩、畸形、電解質失調等。為了解除鎘的毒性,生物體自低等的細菌至高等的哺乳動物都發展出一些獨特的解毒機制。其中,金黃色葡萄球菌(Staphylococcus aureus)之質體pI258上基因cadA,其產物CadA屬於P-type ATPase,為一種需要水解ATP以提供能量的鎘離子排出系統。本研究的主要目的是將pI258上cadA基因,以金屬硫蛋白MT(metallothionein)啟動子構築於帶有綠螢光蛋白(green fluorescent protein, GFP)報告基因的載體pIRES-hrGFP-1a上,並利用顯微注射法(microinjection)轉殖至斑馬魚(Danio rerio),期可以藉P-type ATPase將鎘排出細胞外而不累積在細胞內的方式,探討cadA基因轉殖斑馬魚對鎘的抗性是否能增加及減少累積量,解決鎘等重金屬累積在魚類食品的問題。本研究利用聚合酶連鎖反應及反轉錄聚合酶連鎖反應篩選表現cadA基因的轉殖斑馬魚,並且繼續飼養孵育。在進行受精後72小時孵化完成之野生種斑馬魚稚魚之鎘、鋅、鉛重金屬96小時抗性測試中發現,野生種斑馬魚稚魚在鎘溶液5 μM、鋅溶液76.5 μM ,或鉛溶液12.0 μM 時,分別能夠約有50 %存活率。而另在鎘溶液5、10 μM及鋅溶液51.0、61.2 μM中進行抗性測試的結果顯示,短暫性(transient)表現cadA基因的轉殖斑馬魚在鎘及鋅抗性表現上確實比野生種高。
Cadmium(Cd) is a non-essential metal for the organisms, widely used in industrialized countries. The Itai-itai disease caused by cadmium contamination has made a serious impact on human health for its bioaccumulation through the food chain. Cadmium represents a heavy metal pollutant in Taiwan. Under the Cd2+ exposure, fresh water fish experiences growth inhibition and even death. Homeostatic mechanisms are required for all organisms to minimize the toxicity of cadmium. The motivation of this study is to clone and to transfer the cadA gene originated from bacterial plasmid, Staphylococcus aureus plasmid pI258, into Danio rerio. Experiments were conducted to examine whether it would confer resistance to cadmium, lead(Pb), zinc(Zn) as well as decrease the heavy metal content of the transgenic zebrafish. The stable transgenic germ-lines were confirmed both in DNA, and RNA level. Furthermore, fish embryos during developing or larvae in general showed the highest susceptibility in the fish life cycle. Posthatch larvae were continuously exposed to 5 μM Cd, 76.5 μM Zn, or 12.0 μM Pb for 96 hours beginning at the 72nd hour after fertilization, and resulting in a larval survival of near 50%.On the other hand, transient zebrafish expressing cadA showed that CadA was functionally active and that the zebrafish had enhanced resistance to Cd(II), and Zn(II). These results suggest that transgenic zebrafish expressing cadA can enhance metal resistance probably due to the ability to reduce the heavy metal content in fish.
其他識別: U0005-0307200600152900
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