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研究生:郭冠容
研究生(外文):Guan-Rung Kuo
論文名稱:雙生病毒C4蛋白與寄主葉綠體產氧蛋白之交互作用與功能分析
論文名稱(外文):Analyses of Interactions and Functions of Begomovirus-encoded C4 Proteins and Host-encoded Chloroplast Oxygen Evolving Protein
指導教授:胡仲祺
口試委員:鄧汀欽黃秀珍
口試日期:2016-07-01
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:2016
畢業學年度:104
語文別:中文
論文頁數:77
中文關鍵詞:雙生病毒C4蛋白葉綠體產氧蛋白
外文關鍵詞:BegomovirusC4 ProteinsChloroplast Oxygen Evolving Protein
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雙生病毒屬於單股環狀DNA植物病毒,由雙球形病毒顆粒包裹其基因體,使感染的植物出現嚴重捲葉、黃脈等病癥,對熱帶及亞熱帶地區造成農業、經濟的重大損害。本實驗室先前研究發現霍香薊黃脈病毒(Ageratum Yellow Vein Virus, AYVV)感染圓葉菸草(Nicotiana bennthamiana)後,會造成葉片上捲;感染番茄捲葉病毒(Tomato Leaf Curl Virus, TLCV)則產生葉片下捲之病徵。經由病毒基因交換重組實驗發現雙生病毒的C4蛋白為病徵決定因子,影響葉片捲曲。並透過遠西方墨點法(Far-western blot analysis)發現病毒的C4蛋白會和N. bennthamiana中數個寄主因子產生交互作用。其中葉綠體光合作用產氧蛋白(Chloroplast photosynthetic oxygen-evolving protein, CPOEP)透過酵母菌雙雜交技術(Yeast two hybrid)分析,發現和病毒的C4蛋白具有交互作用。因此,本研究的目的即為深入探討雙生病毒的C4蛋白與CPOEP之間的交互作用,並分析其可能的功能。本研究將CPOEP分成三個區域(domain),利用酵母菌雙雜交技術研究AYVV、TLCV的C4蛋白和CPOEP三個區域具有交互作用的位置。同時利用病毒誘導基因靜默法(Virus induced gene silencing, VIGS)將N. bennthamiana的CPOEP蛋白表現量降低,並接種病毒觀察植物表現型的變化。根據酵母菌雙雜交結果推測,CPOEP與AYVV的C4蛋白中段與C端有較強的交互作用。此外,本研究也發現AYVV病毒C4蛋白本身可能具有promoter Binding domain的功能,未來將會利用Southwestern dot blotting技術,針對酵母菌報導基因之上游啟動子序列和病毒的C4進行交互作用分析。而經由測試CPOEP基因之不同片段序列,本研究也成功的利用VIGS方法將N. benthamiana的CPOEP基因表現量抑制到原本的約16%,並將AYVV或TLCV接種至CPOEP表現量降低的植株中以觀察CPOEP基因對於捲葉病徵的影響,目前實驗仍進行中。期望能藉由分析雙生病毒C4蛋白與寄主蛋白間的交互作用,以了解雙生病毒對植物捲葉病徵影響的機制,並進而能應用於雙生病毒病害管理策略的研發。

Begomoviruses are whitefly-transmitted, single stranded circular DNA viruses that often cause severe leaf curling symptoms on infected plants. Previous studies in our laboratory revealed that Nicotiana benthamiana plants infected by Ageratum yellow vein virus (AYVV) and Tomato Leaf Curl Virus (TLCV) display distinct upward and downward curling symptoms, respectively, which were further demonstrated to be modulated by the viral-encoded C4 protein. In addition, several host factors, including Chloroplast photosynthetic oxygen-evolving protein (CPOEP), have been identified to interact with viral C4 proteins by Far-western blot analysis and yeast-two hybrid analysis. Thus, the objectives of this study is to analyze the molecular interactions between the virus-encoded C4 proteins and host-encoded CPOEP and to explore the possible functions. In this study, the CPOEP gene was split into three parts to determine the domains interacting with virus C4 protein by yeast-two-hybrid analysis. In addition, the expression of CPOEP gene in N. benthamiana was knocked down by using virus induced gene silencing (VIGS), followed by the infection with AYVV or TLCV by agroinfiltration to characterize the effects on the symptom expression in the plants. The result of yeast two-hybrid analyses indicated that the middle and C-terminal regions of CPOEP interacted differentially with the C4 proteins of AYVV and TLCV. It was also found that virus C4 protein might contain promoter binding domain function. In addition, the expression of CPOEP in N. benthamiana was successfully knocked down to about 16% of the original level, and its effects on leaf curling symptom development is currently underway. Through these studies, it is anticipated that the mechanisms by which geminiviruses modulate the symptom development could be revealed and applied in the development of effective management strategies for diseases caused by geminiviruses.

致謝i
摘要 ii
Abstract iv
前言 1
一、 雙生病毒簡介 1
(一) 經濟重要性 1
(二) 分類地位 2
(三) 基因體結構 3
(四) C4蛋白與宿主交互作用 5
二、 宿主因子葉綠體產氧蛋白簡介 6
(一) 葉綠體產氧蛋白 6
(二) 葉綠體產氧蛋白與病毒之交互作用 8
三、 實驗室先前研究 9
四、 實驗目的 10
材料與方法 11
一、 試驗材料 11
二、 試驗方法 11
(一) 酵母菌雙雜交試驗 11
(二) C4蛋白與Gal4 UAS序列南方西方墨點法 16
(三) 蛋白質交互作用沉澱試驗 (Pull-down assay) 18
(四) TLCV-C4與AYVV-C4抗體免疫吸附純化 20
(五) C4基因過表現(over-expression)試驗 21
(六) 利用VIGS誘導CPOEP基因靜默 23
結果 28
一、 酵母菌雙雜交試驗 28
(一) CPOEP與病毒C4蛋白功能區域分析 28
(二) C4 Binding Domain功能探討 29
二、 C4蛋白南方西方墨點法分析(South-western blot analysis) 30
三、 蛋白質交互作用沉澱試驗 31
四、 C4抗體免疫純化實驗 31
五、 C4基因過表現試驗 32
六、 利用病毒載體系統誘導CPOEP基因靜默 32
(一) CPOEP片段基因靜默效率分析 33
(二) CPOEP基因靜默後接種病毒 34
討論 36
一、 雙生病毒C4蛋白的生物功能 36
二、 利用病毒載體系統誘導CPOEP基因靜默 40
參考文獻 43
圖表與附錄 48


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