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研究生:韓宇聰
研究生(外文):Yu-Tsung Han
論文名稱:竹嵌紋病毒RNACappingEnzyme上之特定胺基酸對其Guanylyltransferase活性的影響
論文名稱(外文):Critical Residues of BaMV RNA Capping Enzyme Involved In Guanylyltransferase Activity
指導教授:孟孟孝
指導教授(外文):Meng-Hsiao Meng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:41
中文關鍵詞:竹嵌紋病毒
外文關鍵詞:RNA Capping EnzymeGuanylyltransferase
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  • 被引用被引用:5
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在台灣,竹嵌紋病毒(bamboo mosaic virus) 感染許多綠竹和麻竹等作物而不利於竹子的生產及經濟價值。這病毒是屬於potexvirus group,基因體全長約6.4 kb。其第一個轉譯架構(ORF1)轉譯出約155-kDa大小的複製酵素(replicase),其功能與病毒的複製及基因體5-cap結構形成有關。其中capping酵素的位置經過先前之實驗發現是位於複製酵素的N端,具有guanylyltransferase及methyltransferase的活性。為了確定此病毒capping酵素上對於 GTP 及AdoMet 結合的位置和影響guanylyltransferase及methyltransferase活性的主要胺基酸,我們利用定點突變(site-directed mutagenesis),針對capping酵素上的17個胺基酸做突變,將這些17個經過突變的核酸序列接上表現載體pYES2,並送入酵母菌(Saccharomyces cerevisiae,INVSC1)內,利用半乳糖誘導方式,進行重組蛋白質的大量表現,並進一步分離出含病毒capping enzyme的膜蛋白質且純化出一部份的突變蛋白作guanylyltransferase活性分析。由突變蛋白質的活性分析結果,HH68A、HR125A及HY213A的突變完全破壞了capping enzyme 的guanylyltransferase 活性,而HH66A、HK121A、HD122A、HD310A、K344A及W406A,對guanylyltransferase活性也有很大的影響,HK218A、HC234A、W377A、K389A及K409A對guanylyltransferase活性影響不大或沒有影響。而未來的工作除了純化其餘的突變蛋白質,並要更進一步分析突變蛋白質的methyltransferase活性,以確定病毒的capping酵素上影響methyltransferase及guanylyltransferase活性的重要胺基酸位置及其催化的分子機制。
Bamboo mosaic virus (BaMV), which primarily infects member of the Bambusoideae, has a flexuous rod-shaped morphology with length 470 to 580 nm. Open reading frame 1 (ORF1) of BaMV encodes a 155-kDa polypeptide that is a replicase involved in the replication and formation of the cap structure at the 5end of the viral genome. Previouse study has demonstrated that the N-terminus of the replicase is the capping enzyme of BaMV having methyltransferase and guanylyltransferase activities. In order to define the critical amino acid residues that are involved in the GTP and SAM binding and participate in the catalytic activity of the capping enzyme, seventeen site-directed mutant proteins were generated and expressed in Saccharomyces cerevisiae. The mutated viral proteins were expressed under the induction of galactose and the cells were disrupted by vortexing with glass beads. The recombinant viral capping enzymes were isolated from yeast membrane fraction, and some of them were subsequently solubilized by Sarkosyl-containing buffer and purified by Ni-NTA chromatography. The activity assays show that mutated proteins HH68A、HR125A and HY213A had no detectable guanylyltransferase activities, while HH66A, HK121A, HD122A and HK344A had reduced guanylyltransferase activities to very low levels. Some mutant proteins (HK218A, HC234A, K389A and K409A) were as active as wild-type enzyme, and some (HD310A, W377A, K389A and W406A) had activity reduced moderately. Purification and activity analysis of the rest mutant proteins in the future should help us to understand the structure-function relationship of the BaMV capping enzyme.
目 錄
壹、中文摘要…………………………….1.
貳、英文摘要…………………………….2.
參、前言………………………………….3.
肆、材料與方法………………………….7.
伍、結果………………………………….13.
陸、討論………………………………….18.
柒、附圖………………………………….21.
捌、參考文獻…………………………….39.
參考文獻
李宜佳。1997。竹嵌紋病毒基因體轉譯架構ORF1產物之表現、純化及分 析。國立中興大學農業生物科技學研究所碩士論文。
陳怡君。1999。竹嵌紋病毒複製酵素N端區域的功能分析。國立中興大學農業生物科技學研究所碩士論文。
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