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    Title: I 菸草鑲嵌病毒之甲基轉移酶表現、純化與活性探討 II 啤酒酵母菌GPD1p過度表現對粒線體形態的影響
    Other Titles: I Study of tobacco mosaic virus : expression, purification and activity of methyltransferase II The effect of overexpressing saccharomyces cerevisiae GPD1p on mitochondrial morphology
    菸草鑲嵌病毒之甲基轉移酶表現、純化與活性探討
    啤酒酵母菌GPD1p過度表現對粒線體形態的影響
    Study of tobacco mosaic virus : expression, purification and activity of methyltransferase
    Effect of overexpressing saccharomyces cerevisiae GPD1p on mitochondrial morphology
    Authors: 呂毓鴻;Lu, Yu-Hung
    Contributors: 淡江大學化學學系碩士班
    陳銘凱;Chern, Ming-Kai
    Keywords: 菸草鑲嵌病毒;甲基轉移酶;粒線體形態;甘油-3-磷酸脫氫酶;Tobacco mosaic virus;methyltransferase;mithchondrial morphology;glycerol-3-phosphate dehydrogenase
    Date: 2013
    Issue Date: 2014-01-23 13:44:40 (UTC+8)
    Abstract: Ⅰ 菸草鑲嵌病毒之甲基轉移酶表現、純化與活性探討
    菸草鑲嵌病毒 (Tobacco mosaic virus) 是植物病毒最常見之一,其基因體所轉譯出大小約 126 kDa 的蛋白具有甲基轉移酶活性,近年來有許多研究皆著重於病毒的使宿主核酸甲基化,對於宿主蛋白質甲基化尚未有人研究。本研究利用大腸桿菌異源重組表現 his-tag 異源蛋白質,初期測試中,以降溫、添加界面活性劑等,經過許多次重新摺疊測試,均以不溶解的包涵體 (inclusion body) 存在。由文獻紀載 GST 融合蛋白促進蛋白質正確摺疊,因此以 pGEX4T-1 為載體,建構 pGEX4T-1-MT 成功表現可溶性蛋白質。將 GST 融合蛋白經由 GSH column 純化後與菸草粗蛋白和 Hot-SAM 反應。實驗結果顯示,在感染病毒之菸草比未感染菸草多出較明顯的受質訊號。因此以大腸桿菌異源表現 MT蛋白質可能具有活性且能讓宿主蛋白質甲基化。
    Ⅱ 啤酒酵母菌 GPD1p 過度表現對粒線體形態的影響
    先前的研究發現啤酒酵母菌 (Saccharomyces cerevisiae) 中 ADH3p 對粒線體形態所造成的影響,其原因為蛋白質活性影響 NADH/NAD+ 有關。由文獻指出 GPD1p 合成甘油過程中並將 NADH 氧化成 NAD+,因此本研究建構 pYes2-GPD1-GFP 轉殖入啤酒酵母菌中,觀察過度表現 GPD1p 是否影響粒線體形態。實驗結果顯示,誘導後 pYes2-GPD1-GFP 其 83% 粒線體呈現 fragmented 狀;未誘導 pYes2-GPD1-GFP 與 pYes2 的粒線體皆呈現 tubular 狀。測試將培養基中葡萄糖以甘油取代的條件下培養,其 pYes2-GPD1-GFP 與 pYes2 的粒線體皆呈現 fragmented 狀,結果表示粒線體形態改變會受 NADH/NAD+ 所影響。
    PartⅠ
    Tobacco mosaic virus is the most common virus in the plant. The genome of this virus can be translated to protein which contains methyltransferase activity. In the past, a lot of researches focused on the viral nucleic acid methylation of the host. The host protein methylation has not yet been studied. In this study, we used recombinant E. coli to express heterologous protein. At first, E. coli. expressed the his-tag protein with inclusion body. We tried a number of conditions, such as cooling or adding surfactants, but it still expressed the protein with inclusion body. Some studies have found that GST tag could help protein fold with correct form. Therefore, we used pGEX4T-1 as a carrier vector to construct pGEX4T-1-MT and finally expressed the protein present in the supernatant. We purified the GST fusion protein with GSH column and reacted with Hot-SAM and tobacco crude protein. Experimental results showed that it had more substrate signal in the infected plants than uninfected plants. Therefore, E. coli can express heterologous proteins with activity and may allow host protein methylation.
    PartⅡ
    Previous studies have found that ADH3 protein in Saccharomyces cerevisiae affects morphology of mitochondria. The putative reason is that ADH3 protein can influence NADH/NAD+ ratio. It has been shown that GPD1 protein could synthesize glycerol and oxidize NADH to NAD+. Therefore, we constructed pYes2-GPD1-GFP and transformed it to S. cerevisiae. Experimental results show that after we induced the construct gene, pYes2-GPD1-GFP, the mitochondrial morphology of S. cerevisiae appeared fragmented for 83 percent. With non-induced genes, pYes2-GPD1-GFP and pYes2, the mitochondrial morphology of S. cerevisiae appeared tubular. When we replaced the medium glucose to glycerol, we found that both constructs, pYes2-GPD1-GFP and pYes2, made mitochondrial morphology to patterns appear fragmented. The result showed that the morphology of mitochondria may be influenced by NADH/NAD+ ratio.
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