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研究生:陳薇
研究生(外文):Wei Chen
論文名稱:竹嵌紋病毒衛星核酸轉譯蛋白質P20之生物功能及其對衛星核酸活性之影響
論文名稱(外文):Characterization of Bamboo Mosaic Potexvirus Satellite RNA (SatBaMV) Encoded P20 Protein and Its Effects on SatBaMV Expression
指導教授:徐堯煇
指導教授(外文):Yau-Heiu Hsu
學位類別:博士
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:2003
畢業學年度:91
語文別:中文
中文關鍵詞:竹嵌紋病毒衛星核酸長距離移動
外文關鍵詞:bamboo mosaic virussatellite RNAsatBaMVlong distance movement
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竹嵌紋病毒(Bamboo mosaic virus,簡稱 BaMV)之衛星核酸(satellite RNA of BaMV,簡稱satBaMV RNA),全長836個核苷酸(poly(A) 不計),可轉譯一 20kDa的蛋白質,簡稱為 P20。自感染的竹類中分離出之衛星核酸分離株BSF4,會隨著 BaMV的S分離株(BaMV-S)在菸草中系統性移動。前人研究顯示,P20並非衛星核酸包被用的鞘蛋白亦非其複製所必需,而可能與病毒的移動有關。本實驗中以竹嵌紋病毒 BaMV-S與衛星核酸 BSF4混合接種於菸草(Nicotiana benthamiana)與白藜(Chenopodium quinoa)上,於不同的時間點收集葉片,偵測衛星核酸的核酸及蛋白質的累積量,結果發現其核酸的累積與P20的表現於感染前期同屬快速累積,但到後期 RNA的增加趨於緩和時,P20的累積則開始減少;此外,隨著接種時間的增加,一些與 P20有血清相關性的短肽可被偵測到,其中有一分子量為16 kDa的蛋白質 (P16) 穩定性高於 P20;為了進一步瞭解 P16與 P20的相關性,以P20、P20N端及P20C端 (各15個氨基酸) 的抗血清進行偵測二者之關聯性,並以 2D電泳分析二者之 pI值,結果推論 P16應為P20去除其C端部分氨基酸的衍生物。前人研究顯示,P20的 N端包含有許多帶正電的氨基酸與核酸的結合能力有關;而 C端有被轉譯後修飾的區間,故而構築蛋白質N與C端序列刪減之衛星核酸突變株,N端序列刪減的突變株計有刪減至15、19、42、62、84、106、145個氨基酸者共七株,其中序列刪減至15、19及42個氨基酸的突變株與野生株 BSF4在植物中的表現相似(80%以上),而刪減至84個氨基酸時,接種葉中核酸的累積只剩下野生株的 20-30%,而系統葉則已少於10%;若刪減至106及 145個氨基酸,接種葉中核酸的累積已少於 5%,且系統葉中無法偵測到核酸的累積;在C端序列刪減的突變株方面則有轉譯至第 166、150、134個氨基酸共三種突變株,結果轉譯刪減至第 150個氨基酸或更多時,衛星核酸於系統葉中的累積降低至 10%以下;以定區間的方式刪除包括第150-166、134-150、及 134-166氨基酸序列發現,其中刪降第150至166個氨基酸之間的區域,可使衛星核酸於系統葉中的累積降低至 10%以下;這表示該區域的氨基酸在衛星核酸之長距離移動上扮演重要的角色。為了近一步驗證 P20之功能故以 BaMV攜帶及轉型植物表現 P20二種方式進行互補試驗 (trans- complementation test),由 BaMV載體所表現的 P20確可於接種植物的接種葉及系統葉中偵測到蛋白的表現,其表現量與接種 BSF4並無明顯差異,而轉型了 P20基因的轉殖植物單株 13-6亦可偵測到 P20的累積,但表現的 P20於電泳分析中的位置亦略低於 BSF4所表現者;此二系統均可提高雜合的衛星核酸 BSCAT,轉譯架構被置換為 CAT基因的 satBaMV RNA在系統葉被測到的機會,可見 P20對衛星核酸本身的長距離移動確實具有幫助之能力,這對改良以衛星核酸為基底的病毒載體表現量有具體之幫助。
The satellite RNA associated with Bamboo mosaic virus (BaMV), designated satBaMV RNA, encodes a 20 kDa nonstructural protein (P20) that binds preferentially to satBaMV RNA. The molecular characterization and functional analyses of P20 were performed in this study. The expression profile of P20 was analyzed in Nicotiana benthamiana and Chenopodium quinoa. In the early stage of infection, a positive correlation was observed between the rapid accumulation of satellite RNA and P20 with respect to the inoculation time. As the accumulation of satBaMV RNA approached plateau, the amount of P20 decreased and few smaller proteins serologically related to P20 were observed in the western blot analyses. A 16 kDa protein (P16) is the most stable among the small proteins. Centrifugal fractionation revealed that the P20 was distributed to every sub-cellular fraction, while the P16 was mostly found in the PE275 fraction. Two-dimensional gel electrophoresis and western blot analyses using antisera against the N- or C-terminal 15 amino acids of P20 revealed that P16 was derived from P20 with C-terminal truncation. Serial deletions at the N-, C-termini and internal deletions of P20 were constructed to investigate the biological functions. The results of inoculation assays indicated that P20 is involved in the movement of satBaMV RNA and that the region between amino acid positions 150 to 166 is required for efficient systemic movement of satBaMV RNA. The biological functions of P20 and its potential in improving the efficiency in systemic movement of the satBaMV-based vector system were further confirmed by the trans-complementation experiments. Two approaches were used to supply P20 in trans: 1.) transient expression of P20 from a recombinant BaMV vector; and 2.) stable expression of P20 in transgenic plants. The results confirmed that P20 is responsible for the systemic movement of satBaMV RNA and demonstrated that the biological functions could be complemented in trans. In all, this study established the molecular characteristics of P20, revealed the roles of P20 in the systemic movement of satBaMV RNA and demonstrated the applications in the design of satBaMV-based vector system.
中文摘要 ----------------------------------------------------- 1
Abstract ----------------------------------------------------- 3
緒言 ----------------------------------------------------- 5
Chapter I Time Course Expression of BaMV Associated Satellite RNA (SatBaMV) and Evidence of Proteolytic Processing of SatRNA Encoded Protein
中文摘要----------------------------------------------------13
Abstract ---------------------------------------------------14
Introduction------------------------------------------------15
Materials and Methods---------------------------------------19
Results-----------------------------------------------------29
Discussion -------------------------------------------------34
Chapter II The Effects of Mutations of P20 on Replication and Movement of SatBaMV RNA
中文摘要----------------------------------------------------38
Abstract ---------------------------------------------------39
Introduction -----------------------------------------------40
Materials and Methods---------------------------------------43
Results ----------------------------------------------------48
Discussion -------------------------------------------------54
ChapterIII Functional Analysis of P20 by trans- complementation test with P20 Expressed by BaMV Vector or Transgenic Plants
中文摘要----------------------------------------------------57
Abstract----------------------------------------------------58
Introduction -----------------------------------------------59
Materials and Methods---------------------------------------62
Results ----------------------------------------------------64
Discussion--------------------------------------------------68
REFERENCES ---------------------------------------------------73
TABLES -----------------------------------------------------T
FIGURES -----------------------------------------------------F
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