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研究生:朱建勲
研究生(外文):Chien-Hsun Chu
論文名稱:RNA結構與金屬離子對日本腦炎病毒 sfRNA生合成之影響
論文名稱(外文):RNA structure and metal ion involved in biogenesis of the sfRNA of Japanese encephalitis virus
指導教授:張瑞宜張瑞宜引用關係
指導教授(外文):Ruey-Yi Chang
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:56
中文關鍵詞:日本腦炎病毒sfRNA生合成
外文關鍵詞:Japanese encephalitis virusbiogenesis of the sfRNA
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  所有蟲媒病毒傳播之黃質病毒屬病毒包括日本腦炎病毒(Japanese encephalitis virus, 簡稱JEV)皆會產生一段非編碼RNA,與3’-端未轉譯區中高度保留區片段相同,稱之為sfRNA。本實驗室之前研究顯示sfRNA有可能由病毒負股基因體轉錄出來,且在sfRNA起始點發現一段相似於3' cyclization sequence (簡稱CYC)的序列,稱為CYC-like motif,此序列高度保守於JEV序列中並與sfRNA之生合成有關。為了探究sfRNA的生合成機制,本研究以JEV負股nt 10431-10566 RNA為模板,建立活體外RNA-dependent RNA polymerase酵素活性分析(in vitro RdRp assay)最佳化條件,實驗證明最佳化所需條件為pH 8.0, 25 mM potassium glutamate, 100 mM NaCl, 5 mM MnCl2及5 mM MgCl2;並發現產物有兩種大小,分別為直接合成出模板全長的RNA (de novo synthesis)以及轉錄(transcription)出較短的產物。增加Mn2+可以增加轉錄的效率。以定點突變分析RNA序列與結構之重要性,發現將JEV(-)10431-10566 模板中CYC-like motif刪除(Δ10447-10459 )後,即無產物合成;刪除下游序列(Δ10516-10566)雖降低產量但不影響RNA生合成;而破壞stemloop 4之結構即無de novo synthesis產物之生成,但不影響transcription產物,說明CYC-like motif及stemloop 4結構對於RNA的生合成有絕對的必要性。本研究結果證明JEV sfRNA極有可能為轉錄產生,其中CYC-like motif扮演轉錄時啟動子的角色。
  All arthropod-borne flaviviruses including Japanese encephalitis virus (JEV) produce short fragment noncoding RNA (sfRNA) collinear with highly conserved sequences of the 3’-untranslated region in the viral genome. Previous studies in our laboratory found that the 5’-end of the sfRNA contains cyclization-like sequences (CYC-like motif) that are highly conserved among all JEV isolates suggesting a correlation with the sfRNA biogenesis. In this study, I optimized in vitro RNA-dependent RNA polymerase assay with recombinant JEV RdRp protein using JEV(-)10431-10566 (containing the CYC-like motif and the stemloop 4 structure) as RNA template revealing that pH 8.0, 25 mM potassium glutamate, 100 mM NaCl, 5 mM MnCl2, and 5 mM MgCl2 is the best condition. Two major products, the de novo synthesized full-length product and the CYC-like motif initiated transcription product, were found. Increasing the amount of manganese ion enhanced the transcription product. Deletion of the CYC-like motif (Δ10447-10459) abolished RNA synthesis, while deletion of downstream sequences (Δ10516-10566) of the CYC-like motif had minor effect on both de novo synthesis and transcription. Furthermore, mutation disrupted the stemloop 4 structure slightly affected transcription but impaired de novo synthesized products. Our results indicated that the JEV sfRNA can be synthesized by viral RdRp from antigenome and the CYC-like motif may function as a promoter for sfRNA transcription.
目錄.................................................I
第一章 前言...........................................1
1. 日本腦炎簡介.......................................1
2. 日本腦炎病毒基因體及蛋白質............................2
3. NS5 的功能以及酵素活性..............................3
4. 影響黃質病毒屬病毒複製之重要RNA結構....................4
5. JEV sfRNA之結構與功能..............................7
6. 黃質病毒sfRNA生合成機制.............................8
7. 正股RNA病毒次基因體的生合成機制.......................9
8. 研究目的..........................................12
第二章 材料與方法.....................................13
1.本實驗所使用之引子列於Table 1........................13
2.本實驗所使用之質體整理於Table 2,所構築之載體詳述如下.....13
3.RNA-dependent RNA polymerase製備..................15
4. In vitro RdRp assay.............................19
6. 日本腦炎病毒3’端非轉譯區段Sequence Logo建置..........23
第三章 結果.........................................25
1. 建立in vitro RdRp assay分析系統 ...................25
2. 分析RdRp酵素活性最佳化條件..........................26
3. JEV CYC-like motif具高度相似性並會影響RNA轉錄作用....28
第四章 討論.........................................31
1.In vitro RdRp最佳化條件............................31
2.CYC-like motif可能合成sfRNA所需辨識之啟動子..........32
3.黃質病毒環化與sfRNA生合成之關係......................34
第五章 文獻.........................................37
第六章 表圖.........................................43
附件................................................55

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