吳郭魚仔稚魚金屬硫蛋白的表現與其生理角色

Expression and function of metallothionein in developing larvae of tilapia (Oreochromis mossambicus)

吳淑美(Su-Mei Wu)

指導教授 ： 黃基礎博士 ； 黃鵬鵬博士

國立臺灣師範大學/理學院/生物學系/博士(2000年)

摘要

中文摘要
本論文以吳郭魚仔魚(Oreochromis mossambicus)為材料，探討金屬硫蛋白(MT)之生理功能。首先進行MT純化以暸解其生化特性並建立定量方法，以探討MT在仔魚發育與重金屬解毒機制的角色。重要結果如下：
(1)、純化吳郭魚之MT，其分離係數是 2.07，因此推論MT在吳郭魚體內是以聚元體方式存在，而且因為需應用較高濃度之Tris-HCl與Acetonitrile，才能將MT分離出陰離子管柱與高壓液相層析管柱，因此推論吳郭魚之MT是一個極性高的蛋白質。
(2)、利用合成之多月太類(peptide)製作抗體，確立抗體之專一性，並且應用酵素免疫連結反應來定量MT蛋白質。
(3)、仔魚自孵化後到發育第七天，其MT之表現呈現戲劇化的變化，剛孵化(H0)及第一天(H1)顯著的高於其他時間，此結果與發育階段鋅及銅的含量變化趨勢相近，因此推論發育中之MT可能與鋅及銅之調節有關。
(4)、將H0與H3之仔魚以35 μg/L鎘處理24小時，H0顯著誘導MT，但H3則無誘導效果，比較H0和H3之鈣含量變化，結果經鎘處理後的H0仔魚，其鈣含量無顯著變化，而H3則顯著下降，因此推論H3仔魚之 MT的表現受到抑制是因為其生理狀況已不正常之緣故。
(5)、進一步以膠體管柱層析分析比較，H0 之蛋白質與鎘含量分布高峰一致，而H3之蛋白質與鎘含量分布高峰則不一致，顯示H3蛋白質與鎘之結合已有不正常之現象。
(6)、重金屬鎘、銅和鋅可誘導吳郭魚仔魚MT表現，而且呈現劑量與時間序列之效應。
(7)、剛孵化仔魚利用低劑量之鋅、鎘及銅前處理再轉移到高鎘，可顯著的增加仔魚的存活力。本論文從MT，Cd2+，Ca2+，Na+含量變化去比較，發現其原因是因為低劑量重金屬前處理可以誘導MT的產生；增加陽離子吸收或減少鎘累積之緣故。
綜合以上之結果，本論文認為吳郭魚仔魚MT之生理功能可能是參與發育過程鋅及銅離子之調節，以及與重金屬之解毒作用有關。

關鍵字

吳郭魚 ； 仔稚魚 ； 金屬硫蛋白

並列摘要

Abstract
The purpose of the present thesis was to study the physiological functions of metallothionein (MT) in developing larvae of tilapia (Oreochromis mossambicus). Firstly, MT was purified and characterized, and the quantitative method for MT was also established. The roles of MT in larval devlopment and metal detoxification were studied. The results were surrmanized as follows:
(1) Purification and characterization revealed that tilapia MT, presented as a polymer protein, and is more electronegative than the MTs from other animals.
(2) Synthetic peptides were designed to raise an antibody specifically against tilapia MT, and an ELISA system using the antibody was established to measure the MT contents in tilapia and some other species.
(3) MT contents appeared a dramatic change in development of tilapia. Amount of whole-body MT in tilapia larvae increased to a peak at 1 d after hatching (H1), decreased rapidly thereafter. The change pattern in MT amount was similar to that in Zn and Cu contents during larval development, suggesting that MT is associated with the Zn and Cu regulation in developing larvae.
(4) H0 larvae, which were treated with 35 μg /L Cd2+ for 24 h, showed significant increases in the MT amount (1.7-fold) and accumulated Cd2+ (6.5-fold), but no significant change in Ca2+ content. The H3 larvae with the same treatment revealed about a 10-fold increase in the accumulated Cd2+, and a 10% decrease in Ca2+ content, but no any change in MT. 35 μg /L Cd2+ caused to experience hypocalcemia, an abnormal physiological condition, in which H3 larvae could not synthesize sufficient MT and thus showed greater mortality.
(5) Results of gel filtration chromatography showed that peaks of protein and Cd were concurrent in H0 while those in H3 were inconsistent, suggestion the occurrence of abnormal binding of Cd to the protein in H3.
(6) Waterborne Cd, Cu and Zn could induce MT in new-hatched larvae with dose-dependent and time-dependent patterns.
(7) Pretreatment with low-dose Cu, Zn and Cu were found to enhance the survival of larvae upon the subsequent exposure to a high-dose Cd. Stimulation of MT and/or activation of ionic uptake may achieve this enhancement.
Based on the above results, We concluded that MT might play some roles in the development and metal-detoxification of tilapia larvae.

並列關鍵字

tilapia ； larvae ； metallothionein