本研究主要目的在於，過度表現ADH3p對啤酒酵母(Saccharomyces cerevisiae)粒線體形態上的影響，我們先建構了含有正常ADH3序列的質體pYES2-ADH3跟點突變過的ADH3序列的質體pYES2-ADH3G211A ，將這兩個質體和不含ADH3序列的質體pYES2分別transform到啤酒酵母菌內，再使用半乳糖(galactose)誘導使ADH3p跟ADH3pG211A過度表現，將誘導後的菌體打破，取出所含的蛋白質用SDS-PAGE觀察誘導的狀況跟測定比活性，確定ADH3p和ADH3pG211A有被誘導出來，且ADH3pG211A的點突變有效之後，使用MitoTracker Green FM染色後，在螢光顯微鏡下觀察粒線體形態上的變化，我們發現當ADH3過度表現的時候，網狀的粒線體比例會下降，而分散呈點狀的粒線體比例上升；此外我們也使用FACS (Fluorescence-activated cell sorting)對染色之後的酵母菌所發出的螢光強度進行測量，結果發現有過度表現ADH3p的實驗組，螢光強度上升。

The main purpose of this research is to study the effect of over-expression of ADH3p on mitochondrial morphology in Saccharomyces cerevisiae. We constructed two plasmids; one contains wild-type ADH3 sequence in pYES2; another contains ADH3G211A which has a point mutation in ADH3 sequence. We transformed these plasmids and control plasmid without any extra sequence into Saccharomyces cerevisiae. Then we induced ADH3p over-expression with galactose. After induction, we extracted total protein from yeast cells by a sonifier. To quantify the ADH3p and ADH3pG211A being induced, SDS-PAGE was carried out and specific activity was measured. Then stain yeast mitochondria by MitoTracker Green FM and observe the morphological changes under fluorescence microscopy. We find the proportion of network mitochondria is decreased, and that of particulate mitochondria is increased. We also used FACS to measure fluorescence strength. The fluorescence strength of the ADH3p over-expressing strain is stronger than the control.

目錄
1 中文摘要………………………………………………………………………………I
2 英文摘要………………………………………………………………………………II
3 前言……………………………………………………………………………………1
4 材料和方法
4.1 ADH3G211A-pYES2,ADH3G211A-GFP 點突變……………………………………3
4.2 酵母菌勝任細胞的製備與轉形作用…………………………………………… 5
4.3 目標蛋白質的表現………………………………………………………………7
4.4 粗抽蛋白質的製備………………………………………………………………8
4.5 分析粗抽蛋白質( SDS-PAGE 膠體電泳法) ………………………………… 9
4.6 比活性測量………………………………………………………………………11
4.7 利用螢光顯微鏡看粒線體螢光的變化…………………………………………12
4.8 FACS (Fluorescence-activated cell sorting)測量螢光強度…………………… 13
5 結果
5.1 使用SDS-PAGE 分析ADH3 是否有誘導出來……………………………… 14
5.2 染色後使用螢光顯微鏡直接觀察粒線體形態上的變化…………………… 16
5.3 FACS 分析……………………………………………………………………… 17
5.4 測量誘導出的蛋白質是否擁有正常的活性……………………………………19
6 討論……………………………………………………………………………………20
7 附錄
7.1 pYES2 特徵圖……………………………………………………………………21
7.2 ADH3G211A-pYES2 定序結果圖…………………………………………………22
7.3 ADH3G211A-GFP 定序結果圖……………………………………………………24
7.4 ADH3G211A-pYES2 轉譯結果圖…………………………………………………26
7.5 ADH3G211A-GFP 轉譯結果圖……………………………………………………27
7 參考文獻………………………………………………………………………………28

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