Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89325
標題: 建立與新德里番茄捲葉病毒移動蛋白相互作用之植物蛋白純化系統進行機械接種特性之研究
Establishing the purification system for the host proteins interacting with Tomato leaf curl New Delhi virus movement protein for the functional study of the movement protein on mechanical transmissibility
作者: Shu-Hui Lin
林書卉
關鍵字: 新德里番茄捲葉病毒
移動蛋白
親和性純化
機械傳播特性
機械傳播
Tomato leaf curl New Delhi virus
movement protein
affinity purification
mechanical transmissibility
mechanical transmission
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摘要: 雙生病毒 (Geminiviruses) 分佈遍及全球危害多種重要經濟作物,造成每年數百億美元以上的經濟損失,是目前防治植物病毒病害最棘手的病毒群之一,其中隸屬於菜豆黃金嵌紋病毒屬 (Begomovirus) 的新德里番茄捲葉病毒 (Tomato leafcurl New Delhi virus, ToLCNDV) 嚴重危害熱帶及亞熱帶地區的番茄栽培區,為番茄生產的主要限制因子。ToLCNDV 的基因組由兩個單股環狀 DNA 所組成,分別為轉譯鞘蛋白、複製及轉錄相關蛋白的 DNA-A 及轉譯移動相關蛋白的 DNA-B。本實驗室於 2007 年自東方型甜瓜 (oriental melon) 分離得到一個可藉由機械接種傳播至原寄主作物上的 ToLCNDV 東方型甜瓜分離株 (ToLCNDV-OM),然而多數的 begomoviruses 無法機械傳播,為了找出 ToLCNDV 影響機械接種的關鍵因子,本研究室以分離自胡瓜且無法機械傳播的 ToLCNDV 胡瓜分離株 (ToLCNDV-CB)作為對照 經過基因體互換與移動蛋白 (movement protein, MP) 基因重組的接種試,驗後,發現 MP 是 ToLCNDV-OM 具有機械傳播能力的關鍵因子。本研究以ToLCNDV-OM 及 ToLCNDV-CB 兩相對照下,首先在菸草上進行 TAP-tag 標的之ToLCNDV MP 的短暫表現初步測試後,搭配細胞成分的區分與界面活性劑的萃取,建立 ToLCNDV MP 可溶性膜蛋白複合體的活體內批量法之親合性純化 (invivo batch affinity purification) 系統,並利用其找到約 47 kDa,疑似與ToLCNDV-OM MP 形成複合體的菸草蛋白 P47,經過質譜分析鑑定確認蛋白身分,後續可以進一步分析該植物蛋白與 ToLCNDV-OM 和 ToLCNDV-CB MP 之間交互作用的差異,以探討其對 ToLCNDV 機械傳播特性之影響。藉由蛋白複合體的純化與質譜身分鑑定,有助於探究蛋白的未知功能,而本研究所建立的純化系統,有應用於純化其他膜蛋白複合體的潛力。
Plant-infecting geminiviruses cause considerable crop losses and are threatening commercial crop production worldwide. The virus contains circular single-stranded DNA genome encapsidated in geminate particles. Tomato leaf curl New Delhi virus (ToLCNDV), a geminivirus that severely affects tomato production, contains bipartite genome, designated as DNA-A and DNA-B. DNA-A encodes the coat protein, the replication-associated proteins, and the proteins involved in gene expression. DNA-B encodes the proteins responsible for the virus movement. Generally, most ToLCNDV isolates are not mechanically transmissible to its natural hosts, but a ToLCNDV oriental melon (ToLCNDV-OM), an isolate identified by molecular virology laboratory at National Chung Hsing University in 2007 is mechanically transmissible. Our previous study showed that ToLCNDV-OM movement protein (MP) is the critical factor for its mechanical transmissibility. In this study we established a purification system that combines tissue fractionation by differential centrifugation, surfactant extraction and batch affinity purification. We also found an unidentified Nicotiana protein, P47 that may specifically interact with ToLCNDV-OM MP but not with ToLCNDV-CB MP via this soluble membrane protein complex purification system. Further study on the analysis of P47 by using the liquid chromatography–mass spectrometry is needed to confirm this specific interaction and its significance to the mechanical transmissibility of ToLCNDV. The affinity purification combined with the mass spectrometry is one of the reliable methods for the isolation of the protein complex and the analysis of the interaction patterns can provide hints for the unknown functions of a protein. The purification system established here has also the potential for other membrane protein complex purification.
URI: http://hdl.handle.net/11455/89325
文章公開時間: 10000-01-01
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