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研究生:蔡佳昇
研究生(外文):Chia Sheng Tsai
論文名稱:竹嵌紋病毒類解旋酵素之特定胺基酸對酵素活性及病毒於白蔾內存活的影響
論文名稱(外文):Critical Residues of the Helicase-like Domain of Bamboo Mosaic Virus Replicase Involved in Enzymatic Activities and Viral Proliferation in Chenopodium quinoa
指導教授:孟孟孝
指導教授(外文):Menghsiao Meng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:61
中文關鍵詞:竹嵌紋病毒類解旋酵素白蔾核苷酸水解酵素核糖核酸5’端三磷酸水解酵素
外文關鍵詞:bamboo mosaic virusBaMVreplicasehelicase-like domainNTPaseRNA 5'-triphosphatasequinoa
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竹嵌紋病毒(Bamboo mosaic virus)為單股正意的絲狀RNA病毒,含有五個轉譯架構(open reading frames, 簡稱ORFs),其中第一轉譯架構(ORF1)可轉譯出一分子量為155 kDa的複製酵素(replicase),經胺基酸比對結果發現複製酵素中間區段有多個保留性區域,推測為一類解旋酵素。先前的研究指出類解旋酵素區段截斷表現出來的蛋白質具有核苷酸水解酵素(ATPase)及RNA 5’端三磷酸水解酵素【RNA 5’-triphosphatase(RNA 5’-TPase)】的活性,突變酵素Q826A及D858A具有部分的酵素活性,其餘的五個突變酵素L543P、D702A、D730A、R855A及GKS-GAA均喪失大部分活性。為了瞭解具有活性的突變酵素與野生型酵素其ATPase與RNA 5’-TPase的差異性,實驗分別求出了在20℃、5分鐘的反應條件下各個酵素之ATPase和RNA 5’-TPase的反應速率,藉此比較兩者酵素活性上的差異。結果發現,類解旋酵素在低受質濃度(1 μM)的情況下,酵素水解ATP和水解RNA 5’終端磷酸分子似乎有一致性的趨勢,亦即一方活性之下降連帶另一方也跟著下降,因此推測類解旋酵素ATPase和RNA 5’-TPase的活性應有使用到重疊的活化中心。另外本實驗想要證實 ATPase與RNA 5’-TPase受損的定點突變株是否確實影響病毒在植物體內的存活,因此重新構築了帶有綠螢光蛋白質基因(GFP)的病毒突變株進行植物的接種,利用生體內(in vivo)的分析結果與生體外(in vitro)的分析做一比較。有研究指出,類解旋酵素靠近 N 端位置的白胺酸(leucine)如果置換成脯胺酸(proline)會大幅降低病毒RNA在植物細胞內的複製能力(Huang and Tsai, 1998)。為了探討此一胺基酸在竹嵌紋病毒複製過程中的重要性,實驗上另構築了一定點突變株(L543A),於生體外分析其ATPase與RNA 5’-TPase的活性;生體內則與其他的病毒突變株一起接種於植物做分析。經由螢光顯微鏡的觀察發現,定點突變病毒株L543P、D702A、D730A、Q826A、R855A以及GKS-GAA均未觀察到螢光的存在,顯示病毒無法存活於植物體內,而D858A則與野生型病毒株一樣能存活於植物體內。另外突變病毒株L543A也可觀察到等同於野生型病毒株的螢光,顯示其存活亦不受影響。由此結果推測,類解旋酵素上重要的保留性胺基酸對病毒的存活具有關鍵性的影響;另外類解旋酵素靠近N端位置的白胺酸取代成脯胺酸亦影響了複製酵素的功能。將來將針對L543A及L543P這兩個突變酵素做圓二色光譜(circular dichroism)的分析,以進一步比較兩者蛋白質二級結構的差異,進而瞭解影響酵素功能的原因。

Bamboo mosaic virus (BaMV) is a positive-stranded RNA virus with five open reading frames (ORFs). ORF1 encodes a 155-kDa replicase that contains a helicase-like domain according to the existence of several conserved featured motifs of helicase. Previous studies showed that the helicase-like domain exhibits both ATPase and RNA 5’-triphosphatase activities, and substitution of alanine for conserved amino acids such as D702, D730, R855, and the GKS motif, and of proline for L543 resulted in the inactivation of the enzymatic activities, while mutation at conserved amino acid Q826 or D858 decreased the activities by ~90 and ~80%, respectively. In order to understand the kinetic details of the wild-type enzyme, Q826A and D858A, the activities of ATPase and RNA 5’-triphosphatase were determined at 20℃ in this study. The results showed that the residual ATPase and RNA 5’-triphosphatase activities are similar under the suboptimal substrate concentrations for each individual mutant, indicating that ATPase and RNA 5’-triphosphatase reactions are carried out at overlapped active sites. Furthermore, to know whether the mutations crippling ATPase and RNA 5’-triphosphatase activities indeed affect the viral proliferation in plant cells, we have introduced mutations into a green fluorescent protein-carrying BaMV genome (Lin et al., 2004) and inoculated them into plant leaves in this study. Besides the aforementioned mutations, the mutation of L543 to alanine was also introduced based on the previous finding that mutation of L543 to proline severely reduced the viral RNA accumulation in protoplast (Huang and Tsai, 1998). The in vitro kinetic behavior and in vivo proliferation of L543A were also assayed to understand the function of L543. Under microscopic observation, the mutant viruses did not proliferate in plant except L543A and D858A according the exhibition of green fluorescence, and these results are consistent with the previous in vitro activity assays. In summary, we have identified some of the critical residues of the helicase-like domain for viral proliferation, and found that L543 itself may not be so important as to severely reduce the viral proliferation. In the future we will investigate the secondary structures of L543A and L543P by circular dichroism for realizing their different effects on the viral survival.

總目錄
正文目錄.....................................................Ⅱ
圖目錄.......................................................Ⅴ
表目錄.......................................................Ⅵ
附錄目錄.....................................................Ⅶ
中文摘要.....................................................Ⅷ
英文摘要.....................................................Ⅹ
正文目錄
第一章、前言
竹類與竹嵌紋病毒..............................................1
竹嵌紋病毒的前人研究..........................................2
核酸解旋酵素的研究............................................3
竹嵌紋病毒的類解旋酵素........................................5
本實驗的目的..................................................7
第二章、材料與方法
第一節、竹嵌紋病毒類解旋酵素之表達及純化......................9
壹、蛋白質表達質體的構築..................................9
貳、酵素大量製備及純化....................................9
一、菌體之培養........................................9
二、蛋白質的萃取.....................................10
三、蛋白質的變性與重新摺疊...........................11
四、金屬親合性管柱層析...............................11
五、陰離子交換管柱層析...............................12
參、蛋白質電泳分析法.....................................13
肆、西方墨點法...........................................14
一、蛋白質電泳轉移...................................14
二、免疫呈色反應.....................................14
第二節、細胞外轉錄作用(in vitro transcription).............15
壹、模板DNA之製備........................................15
貳、受質RNA之製備........................................16
第三節、類解旋酵素之活性分析.................................17
壹、核苷酸水解酵素(ATPase)活性分析.....................17
一、溫度對酵素穩定性的影響測試.......................17
二、酵素動力學分析受質範圍選擇.......................17
三、酵素連結法之推算方法.............................18
貳、核糖核酸5’端三磷酸水解酵素 (RNA 5’-triphosphatase)
活性分析.....................................................19
第四節、建構含有類解旋酵素核酸突變點之病毒全長cDNA於
細胞活體內分析...............................................20
壹、質體的構築...........................................21
貳、大腸桿菌Top10F’之轉型作用(transformation)...........22
一、大腸桿菌勝任細胞(competent cell)之製備...........22
二、轉形作用.........................................22
參、大量質體DNA的製備(plasmid maxipreparation).........23
肆、植物接種.............................................24
第三章、結果
第一節、類解旋酵素的表達與純化...............................26
第二節、類解旋酵素的活性分析.................................27
壹、核糖核酸5’端三磷酸水解酵素(RNA 5’-triphosphatase)
活性分析.....................................................27
貳、核苷酸水解酵素(ATPase)活性分析.....................29
第三節、核糖核酸5’端三磷酸水解活性與核苷酸水解活性的比較....29
第四節、帶有類解旋酵素核酸突變點之病毒全長cDNA於細胞活
體內的分析...................................................30
第四章、討論
類解旋酵素上之特定胺基酸突變對酵素活性的影響.................32
類解旋酵素特定胺基酸的突變與竹嵌紋病毒的存活.................35
參考文獻.....................................................56
圖目錄
圖一、竹嵌紋病毒基因體及其轉譯蛋白質圖示.....................37
圖二、竹嵌紋病毒類解旋酵素與其他alphavirus-like superfamily
的類解旋酵素胺基酸序列比對...................................38
圖三、酵素連結法的反應途徑...................................39
圖四、以內插法計算單位時間內RNA 5’終端被水解的分子數........40
圖五、質體pCBG示意圖及病毒cDNA感染植物後的作用機制...........41
圖六(A.)、質體構築流程.....................................42
圖六(B.)、質體構築流程.....................................43
圖七、竹嵌紋病毒類解旋酵素蛋白質的表達與純化.................44
圖八、竹嵌紋病毒類解旋酵素RNA 5’-triphosphatase的活性.......45
圖九、測試不同溫度處理對酵素穩定性的影響.....................46
圖十、竹嵌紋病毒類解旋酵素ATPase的活性.......................47
圖十(續)、竹嵌紋病毒類解旋酵素ATPase的活性.................48
圖十一、接種竹嵌紋病毒cDNA的白蔾葉片於螢光顯微鏡下觀察結果...49
圖十二、接種後第十八天植物葉片的外觀.........................50
表目錄
表一、病毒解旋酵素的三個superfamilies保留性胺基酸
序列的比較...................................................51
表二、PCR所使用引子之序列....................................52
表三、竹嵌紋病毒類解旋酵素RNA 5’-TPase與ATPase
的活性比較...................................................53
表四、竹嵌紋病毒類解旋酵素ATPase活性分析.....................54
表五、竹嵌紋病毒類解旋酵素的特性整理.........................55
附錄目錄
縮寫表...................................................附錄一

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