雙生病毒為雙球型病毒顆粒包裹單股環狀DNA的植物病毒，其中豆類金黃嵌紋病毒屬（Begomovirus）之病毒為經由粉蝨傳播感染雙子葉植物之雙生病毒通常會對植物造成金黃色的病癥，是熱帶及亞熱帶地區之主要植物病毒。當雙生病毒進入已經停在G0/G1 phase的植物細胞時，如何開始其核酸合成及複製是一件待解的問題。雙生病毒已被發現可以在原核生物中複製。而我們實驗室裡也在E. col.及phage M13系統中發現雙生病毒的單股環狀基因體。由於植物細胞中，葉綠體是類似於原核系統的胞器，因此提出一個假說：在植物中，葉綠體可以提供雙生病毒進行複製的場所，也因此造成植物金黃色的病徵。本研究即以霍香薊黃脈病毒 (ageratum yellow vein virus，AYVV) 及番茄捲葉病毒（tomato leaf curl virus，TLCV）為材料探討在雙子葉植物葉綠體中複製的可能性。利用南方墨點分析法及DNase切割等方法在感病霍香薊及感病番茄所純化的完整葉綠體中偵測到雙生病毒單股環狀的基因體，但並沒有偵測到負股的存在，可能是TLCV負股基因體在完整葉綠體中含量並不高或並不存在。在蛋白質層面，本研究自台灣不同地區收集感病霍香薊、番茄、石薯之雙生病毒株系，將其外鞘蛋白基因選殖並定序，之後利用生物資訊學的分析，發現所測試的台灣地區與世界各地雙生病毒外鞘蛋白上具有可能之葉綠體導向序列（chloroplast transit peptides， cTP），為了確定此序列的生物性功能，使用重組的方式將此cTP接合在綠色螢光蛋白GFP的N端，並建構在由CaMV 35S promoter帶領的pCBSGFP載體中。之後利用接種的方式使其進入菸草 (N. benthamiana)的植物中，再以螢光顯微鏡直接觀察接種葉的螢光來判斷cTP的生物功能，惟目前仍無法明確區別，而以西方墨點法在葉綠體中亦無偵測到AYVV或TLCV之外鞘蛋白。因此在本研究中發現TLCV及AYVV的基因體會進入葉綠體中，然而其進入葉綠體後會進行哪些活動以及對於雙生病毒在感染植物有何影響仍需再進一步的探討。

Begomoviruses are whitefly-transmitted geminiviruses that cause significant damage to dicotyledonous plants. Begomoviruses usually inflict bright yellowing symptoms on infected plants. The question how geminivirus initiated DNA synthesis and replication in differentiated cells that are in G0/G1 phase remain to be elucidated. It had been shown that geminivirus could replicate in prokaryotic organisms. Single-stranded circular DNAs of geminivirus were also found in Escherichia coli/phage M13 system in our laboratory. Since the plastids resemble prokaryotic system in plant cells, it is hypothesized that plastids may also support the replication of geminiviruses in plants, which may lead to the damage of plastids and result in the bright yellowing symptoms. The purpose of this study is to study the replication of ageratum yellow vein virus (AYVV) and tomato leaf curl virus (TLCV) in plastids of dicotyledonous plants. AYVV DNA and TLCV DNA genome genome, but not complementary-strand DNA, were detected in intact plastids of virus-infected plants by Southern-blot hybridization with a series of probes specific to TLCV DNA genome. Various AYVV or TLCV isolates were collected from different regions in Taiwan. The respective coat protein (CP) genes were cloned and sequenced. At the protein level, by means of the bioinformatics analyses, it is found that geminivirus coat proteins from many regions of Taiwan and the world may contain chloroplast transit peptides (cTP). In order to confirm the biological function, the putative viral cTP was fused to the N-terminal of GFP and cloned into the pCBSGFP vector under the control of CaMV 35S promoter. The constructs were inoculated into N. benthamiana and analyzed directly by fluorescence microscopy to confirm the biological functions of the putative cTP. However, no significant GFP signals were observed in chloroplasts by fluorescence microscopy. Western blot analyses further suggested that TLCV may not enter the chloroplasts. Therefore, in this study, it is found that TLCV and AYVV DNA genome could enter the chloroplast, but further studies are needed to elucidate the actual functions.

中文摘要----------------------------------------------------------------------------------------2
英文摘要----------------------------------------------------------------------------------------3
壹、 前言----------------------------------------------------------------------------------------4
貳、 前人研究----------------------------------------------------------------------------------7
一、 雙生病毒簡介----------------------------------------------------------------------7
二、 在感病植物的plastid中發現雙生病毒的單股環狀DNA及雙球形病毒顆粒------------------------------------------------------------------------------------15
三、 雙生病毒利用原核系統進行DNA複製及相關之研究-------------------15
參、 材料與方法------------------------------------------------------------------------------18
一、 試驗材料---------------------------------------------------------------------------18
二、 試驗方法---------------------------------------------------------------------------18
1. 外鞘蛋白基因選殖---------------------------------------------------------18
2. 花蓮吉安番茄捲葉病毒(Tomato leaf curl virus)外鞘蛋白多株抗體之製備---------------------------------------------------------------------------21
3. 病毒的純化------------------------------------------------------------------23
4. 感病菸草上雙生病毒基因體全長的選殖------------------------------24
5. 植物葉綠體之分離---------------------------------------------------------25
6. 葉綠體內雙生病毒核酸及外鞘蛋白的分析---------------------------25
7. 外鞘蛋白上cTP的預測與其生物性測試------------------------------28
肆、 結果---------------------------------------------------------------------------------------31
伍、 討論---------------------------------------------------------------------------------------39
陸、 參考文獻---------------------------------------------------------------------------------43
圖------------------------------------------------------------------------------------------------57
表------------------------------------------------------------------------------------------------80

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