臺灣博碩士論文加值系統

竹嵌紋病毒歸屬於馬鈴薯X病毒群，具單股正極的RNA基因體，含有五個轉譯架構(open reading frame,簡稱ORF)。其寄主範圍狹窄，主要感染竹類(Bambusoideae)、少數單子葉植物及菸草。第一轉譯架構(ORF1)可轉譯出一分子量為155 kDa的複製酵素(replicase)，從N端至C端依序是戴帽酵素(capping enzyme)、似RNA解螺旋酵素 (RNA helicase-like domain)及聚合酵素(RNA-dependent RNA polymerase, RdRp)三個功能性區域。在前兩個功能性區域間，有約110個胺基酸組成的高親水性區(hydrophilic region, HR)。而proline-rich stretch 則位於後兩個功能性區域間。為了探討此高親水性區在病毒複製過程中可能扮演的角色及尋找參與病毒複製過程的細胞蛋白質因子，我們試圖以高親水性區及聚合酵素作為釣餌蛋白質(bait protein)，利用酵母菌雙雜交篩選法(yeast two-hybrid screen)，從菸草葉子的cDNA基因庫中找尋能與釣餌蛋白質相互作用的因子。首先，先將高親水性區及聚合酵素構築於載體上，並測試此兩個蛋白質是否適合此系統，結果顯示聚合酵素適用，但是高親水性區本身會活化報導基因，因此不適合作為釣餌蛋白質。為了避免產生此非專一性活化，我們將高親水性區序列做不同的刪除(deletion)，最後則以去除N端33個胺基酸的截斷蛋白質(HRΔN)作為釣餌蛋白質。在此同時，我們也著手進行菸草葉子cDNA基因庫的構築並得到3.5×104個轉型株。以HRΔN作為釣餌蛋白質篩選菸草葉子cDNA基因庫時，有26顆菌落可在缺乏histidine培養基中生長(His+)，並有β-galactosidase活性(LacZ+)。然而，將篩選到之質體送回能夠表現HRΔN之酵母菌，HIS3與LacZ二個報導基因並不被開啟，也就是說這26個轉型株皆為偽陽性，造成此結果之原因可能是高親水性區的N端33個胺基酸，是交互作用的重要區域。另一方面，以複製酵素作為釣餌蛋白質時，則有582顆菌落呈雙陽性反應(His+LacZ+)。利用定量分析法測試β-galactosidase活性，得到47個活性較強之轉殖株，將篩選到之質體送回能夠表現RdRp之酵母菌中，25個clones與RdRp具有專一性交互作用，將這些clones解序之後，與蛋白質資料庫比對，發現可將這些蛋白質分成四類：包括光合作用相關之蛋白質、壓力誘導產生之蛋白質、ubiquitin-proteasome pathway相關之蛋白質及未知功能之蛋白質。未來，我們將以其他生化分析法來確認這些交互作用。而這些與RdRp交互作用之蛋白質如何影響植物之生理功能或是調控病毒的複製，仍需進一步研究探討。

Bamboo mosaic virus (BaMV), a member of the potexvirus group, contains a single-stranded, positive sense RNA genome with five open reading frames (ORFs) and infects primarily members of the Bambusoideae, few other monocotyledonous plants and Nicotiana benthamiana. ORF1 encodes a 155-kDa replicase divided into capping enzyme, RNA helicase-like domain, and RNA-dependent RNA polymerase (RdRp) on the order of from N to C termini. Between the first two domains is a highly hydrophilic region (HR) with approximate 110 amino acids, while a proline-rich stretch sits between the latter two domains. To investigate the potential role of the hydrophilic region and search for cellular proteins involved in the viral replication, yeast two-hybrid screening against a leaf cDNA library of N. benthamiana is being employed by using the hydrophilic region and RdRp as bait. At first, the bait plasmids were constructed. RdRp was a well-behaved bait, but the hydrophilic region itself can transactivate the reporter genes (HIS3 lacZ). To avoid the nonspecific activation activity, the hydrophilic region was variously truncated, and finally the one with 33 amino acids deleted at N terminus (HRΔN) was used as bait in yeast two-hybrid screen. In the meanwhile, a leaf cDNA library containing 3.5×104 clones of N. benthamiana was constructed. Using HRΔN as bait to screen the leaf cDNA library, 26 colonies could grow up on minimal medium that lacked histidine (His+) and showed β-galactosidase activity (LacZ+). However, when putative interactors were retrieved and retested the ability of the two-hybrid proteins to interact specifically by retransforming to yeast, both of HIS3 and LacZ reporter genes could not be activated. It meant that all of 26 clones were false positive. This may result from the deletion of the N terminus of HR. On the other hand, 582 double-positive colonies (His+ LacZ+) were obtained when RdRp was used as bait. Using quantitative assay of β-galactosidase activity, 47 clones showed stronger interaction. By retrieving these putative interactors and retransforming to yeast to test the specific interaction, 25 clones can specifically interact with RdRp. Then the nucleotide sequences of these clones were determined and their deduced amino acid were searched in database. As a result, their encoded proteins have homology with four class proteins including photosynthesis related proteins, stress induced proteins, ubiquitin-proteasome pathway related proteins and unknown function proteins. In the future, biochemical assay will have to be performed to confirm these interactions. And how these proteins interacted with RdRp to affect the growth of the host plant or regulate virus replication? They also have to be further investigated.

目錄
中文摘要…………………………………………………………………………….....I
英文摘要…………………………………………………………………...………...III
目錄…………………………………………………………………………………...V
圖目錄……………………………………………………………………..……......VII
表目錄…………………………………………………………………………...…VIII
附錄目錄……………………………………………………………………………..IX
第一章、 前言…………………………………………………………………………1
第二章、 材料與方法………………………………………………………….……...6
第一節、菌株、載體及培養基……………………………………………………6
第二節、質體的構築………………………………………………………………7
第三節、大腸桿菌(E. coli)質體DNA之抽取…………………………………….8
第四節、大腸桿菌Top10F’之轉型作用(transformation) …………………………9
第五節、酵母菌L40之轉型作用…………………………………………………10
第六節、酵母菌蛋白質的萃取…………………………………………………..10
第七節、西方墨點法………………………………………………………………11
第八節、總體RNA (total RNA) 之萃取…………………………………………12
第九節、mRNA之純化…………………………………………………………..12
第十節、cDNA之合成……………………………………………………………13
第十一節、純化大量大腸桿菌質體DNA……………………………………….16
第十二節、大規模酵母菌轉型作用(Large-scale library transformation)……….17
第十三節、β-galactosidase活性分析…………………………………………….18
第十四節、酵母菌質體DNA的萃取…………………………………………….19
第三章、 結果……………………………………………………………………..…20
第一節、 質體的構築與非專一性活化的測試……………………………..…20
第二節、cDNA基因庫的製備…………………………………………………23
第三節、酵母菌雙雜交篩選法之執行………………………………………..25
第四章、 討論………………………………………………………………………..28
參考文獻…………………………………………………………………………..…35
圖…………………………………………………………………………………..…39
表…………………………………………………………………..…………………51
附錄…………………………………………………………………………………..56
圖目錄
圖一、酵母菌表現LexA-RdRp及LexA-HR融合蛋白質。…………………………39
圖二、利用β-galactosidase活性分析，測試HR與RdRp
非專一性的活化報導基因。…………………………………………………40
圖三、HR的不同截斷蛋白質。………………………………………………….…41
圖四、酵母菌表現LexA-HRΔN及LexA-HRΔC融合蛋白質。……………..…..42
圖五、利用β-galactosidase活性分析，測試HRΔN與HRΔC
非專一性的活化報導基因。………………..……………………………….43
圖六、以alkaline agarose gel電泳分析第一股與第二股cDNA合成情形。.………44
圖七、以gel filtration分離cDNA。………………………………………………..45
圖八、以瓊膠電泳分析確認cDNA有接入載體中。…………………………..…46
圖九、測試以HRΔN為釣餌蛋白質進行酵母菌雙雜交法篩
選到之酵母菌轉型株的HIS3與LacZ兩種報導基因
的表現。………………………………………………………………………47
圖十、測試以RdRp為釣餌蛋白質進行酵母菌雙雜交法篩選
到之酵母菌轉型株的HIS3與LacZ兩種報導基因的表
現。……………………………………………………………………………48
圖十一、β-galactosidase定量分析。………………………………………………49
圖十二、測試篩選得到之酵母菌轉型株非專一性的活化HIS3
與LacZ兩種報導基因。………………………………………………...…50
表目錄
表一、參與病毒RNA-dependent RNA合成的細胞蛋白質的模式………………..51
表二、參與病毒RNA合成的細胞蛋白質的分類………………….……………….52
表三、以HRΔN為釣餌蛋白質執行酵母菌雙雜交法篩選之結果…………………53
表四、以RdRp為釣餌蛋白質執行酵母菌雙雜交法篩選之結果…………………54
表五、酵母菌雙雜交法篩選到之clones定序結果…………………………………55
附錄目錄
附錄一、竹嵌紋病毒基因體架構…………………………………….……………..56
附錄二、酵母菌雙雜交系統之模式圖………………………………….…………..57
附錄三、質體pHybLex/Zeo之圖譜與特徵…………………………….…………..58
附錄四、質體pYESTrp2之圖譜及特徵…………………………………..…….…..59
附錄五、合成cDNA之流程圖………………………………………………………60

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