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研究生:陳璇
研究生(外文):I-Hsaun Chen
論文名稱:竹嵌紋病毒核酸基因體3´端非轉譯區中ABCdomain功能之探討
論文名稱(外文):Functional Analyses of Cloverleaf-like Structure in the 3´ Untranslated Region of Bamboo Mosaic Potexvirus Genomic RNA
指導教授:蔡慶修蔡慶修引用關係
指導教授(外文):Ching-Hsiu Tsai
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
中文關鍵詞:竹嵌紋病毒端非轉譯區
外文關鍵詞:Bamboo mosaic virusuntranslated region
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竹嵌紋病毒 (bamboo mosaic virus, BaMV),屬於potexvirus group,為一正極性RNA基因體病毒,全長不包括3´端的poly(A)部份有6366個核苷酸。在其基因體3´端非轉譯區已被證實存在有一特殊的三級結構,這三級結構顯示有三個連續的stem-loops稱之為ABC domain,接著為一major stem-loop之後則為一pseudoknot的結構。在先前的研究結果顯示,若將ABC domain的序列同時移除,會使得外鞘蛋白在Nicotiana benthamiana原生質體中的累積量下降至15%,猜測ABC domain可能與BaMV RNA 的複製有關,因此本實驗就針對個別移除ABC domain中stem-loop A、B或C來比較其複製效率並進一步的探討其可能的重要性。而由實驗結果發現,個別移除stem-loop A對病毒的複製效率影響並不大,但若個別移除stem-loop B或C則外鞘蛋白或是正股RNA在原生質體中的累積量明顯的降低,顯示stem-loop B和C對病毒的複製有較大的影響。若將病毒RNA直接接種在植物體上則發現,移除stem-loop A的突變株在接種葉上可以和野生型有相當的複製效率,但在系統葉上則明顯的下降許多,顯示stem-loop A是否可能與病毒在植物體長距離移動的因子有關。而移除stem-loopB或C的突變株,雖然在原生質體上的複製效率就很差但在接種葉上仍可偵測得到病毒的外鞘蛋白而在系統葉上就完全偵測不到,猜測stem-loop B和C也可能與病毒在植物體長距離移動的因子有關。而由於大量表現病毒複製酵素C端區域polymerase已證實可專一性的結合在D和E domain上,因此推測病毒複製酵素中間區域helicase-like domain是否會結合在ABC domain上,並且經由這種結合的關係而使得病毒在原生質體上的複製有所差異。經由UV cross-linking 證實,helicase-like domain的確會和ABC domain結合,但並無法利用競爭的方式在UV cross-linking上看出其對stem-loops A、B與C之間的差異性,推測可能是在我們所使用UV cross-linking的條件下無法來分辨。另外,由於ABC domain會影響到病毒的複製而且也可能跟病毒的長距離移動有關,因此猜測是否會有寄主內的因子來參與,所以試著從健康植物的萃取物中找出可能影響的因子。利用UV cross-link初步觀察到,從健康植物的萃取液中似乎存在有可和ABC domain序列發生交互作用的蛋白,但這蛋白是否能專一性的辨識以及牽涉病毒在植物體長距離移動則是值得再做進一步的探討。
Bamboo mosaic potexirus (BaMV) is a single-stranded positive-sense RNA virus. It has been demonstrated that the 3´untranslated region (UTR) of BaMV RNA could fold into a tertiary structure comprising a three-stem-loop cloverleaf-like domain, a major stem-loop domain, and a pseudoknot designated ABC, D, and E domain, respectively. In previous study, the coat protein accumulation of mutant with ABC deletion was only 15% that of wild type in protoplasts. It was suggested that ABC domain might play a role on the viral RNA replication. In this study we would like to distinguish which stem-loop of ABC domain is the major determinant on the viral RNA replication. Three independent mutants were created with deletion of each stem-loop and inoculated into protoplasts and plants of Nicotiana benthamiana. Results showed that the accumulation level of coat protein and viral RNAs of mutant with stem-loop A deletion was similar to that of wild type in protoplasts and inoculated leaves but far less in systemic leaves. It was revealed that stem-loop A was not a key structure on the replication of BaMV RNA but might be involved in viral long-distance movement. Mutant with stem-loop B or C deletion had major effect on the accumulation of viral coat protein and RNAs similar to that of ABC deletion in protoplasts and in systemic leaves. It suggested that stem-loop B and C were the key structure on the viral RNA replication and might also involved in viral long-distance movement. It had been demonstrated that the truncated polymerase domain of BaMV ORF1 could specifically interact with D and E domains of the 3´ UTR (Huang et al., 2001). We are wondering whether the connection of polymerase domain with helicase-like domain of ORF1 may relate to the connection of D and E domains with cloverleaf-like domain of the 3´ UTR and by the way involved in viral replication. In UV cross-linking experiment, the BaMV helicase was demonstrated to specifically interact with ABC domain, but could not distinguish each stem-loop involved in RNA replication in our condition. Besides, we also discovered a possible factor derived from cytoplasmic extracts (S100) of uninfected N. benthamiana could interact with ABC domain. But it still needed to be cleared out whether this factor is involved in viral RNA replication as binding to stem-loops B and C or in long distance movement as interacting with ABC domain.
壹、中文摘要----------------------------------------------1
貳、英文摘要----------------------------------------------3
參、前人研究----------------------------------------------5
肆、內文-------------------------------------------------10
介紹(Introduction)-----------------------------------10
材料與方法(Materials and Methods)--------------------13
結果(Results)----------------------------------------20
討論(Discussion)-------------------------------------25
圖表與說明(Figures and legends)----------------------28
參考文獻(Reference)----------------------------------39
伍、結論-------------------------------------------------46
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