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研究生:朱秋慧
研究生(外文):CHIU-HUI, CHU
論文名稱:竹嵌紋病毒3'端非轉譯區功能之研究
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:英文
論文頁數:48
中文關鍵詞:竹嵌文病毒3' 非轉譯區
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竹嵌紋病毒 (bamboo mosaic virus, BaMV) 與馬鈴薯病毒 (potato virus X, PVX)皆屬於馬鈴薯病毒X群 (potexvirus) ,為單鏈正股核醣核酸病毒,其基因體3´端均含一段非轉譯區 (untranslated region, UTR) ,其中BaMV之3´UTR可分為ABC,D,E三個區域 (domains) ,分別由四個stem-loops及一個pseudoknot組成;而PVX之3´UTR經mfold預測含3個stem-loops的結構。在生體外的複製實驗 (in vitro replication), PVX 3´UTR能被BaMV replicase辨識,合成負股。欲知PVX 3´UTR是否亦能於生體內 (in vivo) 在植物細胞內複製,於是以PVX 3´UTR分別取代不同區域BaMV 3´UTR之8個BaMV突變株 (mutants) (BaMV/P, -/ABCP, -/ABCDP, -/ABCD*P, -/PDE, -/PE, -/ABCPE和-/PABCDE) 進行實驗,以了解PVX 3´UTR扮演的角色及BaMV 3´UTR各區域之重要性。於Nicotiana benthamiana原生質體 (protoplast)及植株的接種實驗證實,在生體內 (in vivo) 除BaMV/PABCDE此突變株外,所有突變株皆無法複製且在植物體無病徵產生。 此結果顯示,生體內 (in vivo) BaMV 3´UTR不可被PVX 3´UTR取代,且其上各區域不可分割。由生體外複製實驗可以得知各突變株被BaMV replicase辨識與結合的效率。其中rABCP之效率可達80% (以r138 40A為100%) ,顯示PVX 3´UTR在生體外可取代BaMV 3´UTR之DE區域。但因為此突變株無法在植物體 (in vivo) 觀察到複製及病徵,顯示3´ UTR的完整性影響病毒在植物體內的累積。 在RdRp complex催化效率 (catalytic efficiency) 測試方面,r138/40A、rABCP、rP和rDE的KM及Vmax分別為302、296、533、210 nM及63、21、25、17 min-1。 Vmax/KM以rP最差,為r139/40A (WT) 之四分之一。由此實驗得知,BaMV 3´UTR被PVX 3´UTR任意取代後會嚴重影響病毒在植物體的累積,顯示BaMV 3´UTR在in vivo的功能無法被同屬的PVX 3´UTR取代。然而在in vitro 複製實驗及RdRp complex催化效率實驗得知,PVX 3´UTR可取代DE區域,雖然不影響RdRp complex結合,但影響作用的速率,此因素可能導致病毒無法在植物體累積。

Bamboo mosaic potexirus (BaMV) and potato virus X (PVX) belong to potexvirus group whose genome consists of a single-stranded, positive-sense RNA molecule. In BaMV RNA, the sequence in the 3´ UTR has been demonstrated to interact with the viral RNA-dependent RNA polymerase. In PVX RNA, an eight-nucleotide sequence in the 3´UTR is required for host protein binding and viral RNA multiplication. The 3´ UTR of BaMV RNA can be divided into 3 domains, ABC, D and E and form a tertiary structure comprising four stem-loops and a pseudoknot structure. The structure of the 3´ UTR of PVX RNA has only been predicted by mfold program and shows a simply three-stem-loop structure. In previous study, the 3´ UTR of PVX RNA could be recognized by BaMV replicase, and used as a template to synthesize the minus strand in vitro. In this study, we would like to know whether the 3´ UTR of BaMV RNA could be replaced with the 3´ UTR of PVX RNA in vivo as that was shown in vitro. We have designed eight different mutations with the replacement of the 3´ UTR of PVX RNA at different region of the 3´ UTR of BaMV RNA. Results showed that no mutant could replicate in protoplasts and plants of Nicotiana benthamiana except BaMV/PABCDE. It suggested that the 3´ UTR of BaMV RNA could not be replaced with the 3´ UTR of PVX RNA in vivo. However, in an in vitro replication assay rABCP could reach to 80% that of wild type. It suggested that the 3´ UTR of PVX RNA could functionally replace D and E domains of the 3´ UTR of BaMV RNA in vitro. Results of kinetic study of the RNAs with BaMV replicase showed that the KM and Vmax of r138/40A, rABCP, rP, and rDE is 302, 296, 533, and 210 nM, and 63, 21, 25, and17 min-1, respectively. These results indicated that the replication failure of mutant BaMV/ABCP in protoplasts and plants could be due to the less efficient of replication rate in minus strand RNA synthesis.

目 錄
頁次
壹、中文摘要 -----------------------------------------------1
貳、英文摘要-------------------------------------------------2
參、前人研究-------------------------------------------------3
肆、內文-----------------------------------------------------8
介紹(Introduction) ------------------------------------------8
材料與方法(Materials and Methods)---------------------------11
結果(Results) ----------------------------------------------19
討論(Discussion)--------------------------------------------23
圖表與說明(Figures and legends) ----------------------------25
參考文獻(Reference)-----------------------------------------37
伍、結論----------------------------------------------------48

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