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研究生:王冠智
研究生(外文):Guan-Zhi Wang
論文名稱:竹嵌紋病毒衛星核酸蛋白P20與粒線體關係之研究
論文名稱(外文):Association of Bamboo mosaic virus satellite RNA encoded P20 with mitochondria
指導教授:徐堯煇
指導教授(外文):Yau-Heiu Hsu
學位類別:碩士
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:2008
畢業學年度:96
語文別:中文
論文頁數:61
中文關鍵詞:竹嵌紋病毒衛星核酸粒線體
外文關鍵詞:satBaMVP20mitochondriaconfocal
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壹、中文摘要

竹嵌紋病毒 (Bamboo mosaic virus, BaMV),為彎曲絲狀外型,單股正極性的RNA病毒。有些病毒分離株攜帶有衛星核酸 (satellite RNA, satBaMV),其複製與包被必須完全依賴BaMV的協助。satBaMV RNA全長為836個核
Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. The satellite RNA (full-length 836 nt) associated with BaMV, designated as satBaMV RNA, encodes a 20 kDa nonstructural protein (P20). Using several programs to predict the subcellular locatizaion of P20, we found transmembrane domain and mitochondrial targeting signal (MTS), typical viral proteins that can localize on mitochondria. We used discontinuous percoll gradient to isolate mitochondria of Nicotiana benthamiana. The mitochondrial fraction was confirmed by a mitochondrial specific dye Mitotracker Orange CMTMRos and antibody of cytochrome c. Using western blot analysis P20 was detected in mitochondrial enriched fraction from satBaMV transgenic line 2-6 that was inoculated with BaMV. Protoplasts were prepared from inoculated leaves of N. benthamiana co-infected with BaMV-S viral RNA and in vitro transcripts of pBSGFP, pBSGFPP20 and pBSP20GFP, respectively. By Confocal microscopy, protoplasts from pBSGFPP20 infected leave, similar to pBSGFP displayed the green fluorescence which scattered in cytoplasm. The protoplasts contained P20GFP had obvious GFP signal that co-localized and surrounded with mitochondria of diameter at least 2μm at low expression, but the fluorescence were too strong to recognize if P20GFP had transported into mitochondria. When P20GFP gradual accumulated, the phenomenon which green fluorescence co-localize with mitochondria was lost and the 2μm diameter mitochondria were disappeared. The Protease K protection assay showed the mitochondrial membrane could be preserved completely 7 days after isolation. However P20 within mitochondrial fraction from N. benthamiana line 2-6 challenged with BaMV-S could not be protected by mitochondrial membrane after Protease K treatment. No appearance of products once MTS predicted by MitoProt II were cleaved as most of mitochondrial import mechanism. Our data support a hypothesis that P20 of satBaMV interacted with mitochondria that may be just at early phase of
viral life cycle.
目 錄
壹、中文摘要
貳、英文摘要
參、前言-----------------------------------------------------------------------------1
一、竹嵌紋病毒及其衛星核酸--------------------------------------------1
二、典型的病毒粒線體標定蛋白-----------------------------------------2
三、粒線體功能與特性-----------------------------------------------------3
(一)粒線體蛋白質的轉運機制--------------------------------------3
(二)雷射共軛焦顯微鏡視野下的粒線體外觀--------------------5
肆、材料與方法--------------------------------------------------------------------7
一、電腦分析竹嵌紋病毒之衛星核酸序列-----------------------------7
二、菸草粒線體純化之方法-----------------------------------------------7
三、西方墨點法分析--------------------------------------------------------9
四、質體構築和生體外轉錄實驗---------------------------------------10
五、竹嵌紋病毒viral RNA之抽取和接種試驗----------------------11
六、原生質體的製備------------------------------------------------------12
七、粒線體染色-----------------------------------------------------------13
八、雷射共軛焦顯微鏡螢光---------------------------------------------14
九、Protease K處理實驗-------------------------------------------------14
伍、結果---------------------------------------------------------------------------15
一、蛋白質P20 N端具粒線體定位序列------------------------------15
二、修正後的不連續性Percoll梯度離心去有效分離菸草的二
粒線體-----------------------------------------------------------------16
三、竹嵌紋病毒衛星核酸轉基因菸草的粒線體含P20蛋白------18
四、原生質體粒線體染劑測試與葉綠體自體螢光現象------------19
五、雷射共軛焦顯微鏡觀察P20在細胞內的座落位置------------20
六、P20嵌入粒線體膜之分析試驗------------------------------------22
陸、討論--------------------------------------------------------------------------24
柒、參考文獻---------------------------------------------------------------------30
捌、圖表及附錄------------------------------------------------------------------43
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