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標題: 協助竹嵌紋病毒在寄主植物中移動相關蛋白之功能機制研究
Studies of the Functional Mechanisms of Protein Factors Assisting the Movement of Bamboo Mosaic Virus in Host Plant
作者: 張邦彥
關鍵字: 生物技術, 植物保護類;基礎研究;Bamboo mosaic virus;竹嵌紋病毒;三重疊基因區;細胞間移動;系統性移動;同源多聚體;蛋白交互作用;triple gene block;cell-to-cell movement;systemicmovement;protein-protein interaction
中文摘要竹嵌紋病毒(Bamboo mosaic virus, 簡稱BaMV)基因體上之三重疊基因區(triple gene block, TGB)所轉譯的三個蛋白,TGBp1、TGBp2 和TGBp3,參與病毒核酸於寄主植物中的轉移,不過其詳細機制,仍有待釐清。TGBp3 為一分子質量約為6 kDa,且含有一個穿膜區域的膜蛋白。相較於TGBp1 和TGBp2,TGBp3 之生產、純化及抗體製備,均相當不易,因此一直缺乏此蛋白之拓蹼及生化特性分析結果。根據TGBp3 蛋白之胺基酸序列比對分析結果,推測TGBp3蛋白的C 端,具有一個高度保留的(Cys –x5 –Gly –x6~9 –Cys)motif。我們的預備研究結果顯示,TGBp3 的確是一個穿膜蛋白,且其所擁有的這個motif 上的兩個高度保留的cysteine 胺基酸 (Cys-31 和Cys-46),分別或同時以Ala 取代,雖不影響病毒核酸之複製,但卻明顯抑制病毒核酸在植物細胞間的移動。本計劃第一年,我們除了想探討TGBp3 蛋白在膜上之拓蹼特性外,還想進一步探討TGBp3之Cys-to-Ala 突變,如何影響病毒核酸在植物細胞與細胞間之移動。竹嵌紋病毒之TGBp2 亦為一穿膜蛋白,其C 端含有兩個保留性(Cys-109和Cys-112)。我們的研究發現此兩個保留性cysteines,個別或同時以Ala 取代,會造成突變型病毒核酸於葉柄韌皮部內移動受抑制。另外,不論是以野生型或突變型病毒感染的植物膜狀成分樣品為材料,都可以在電泳膠片上偵測到以單體、雙體、三體及四體形式存在的TGBp2 蛋白。本研究計劃第二年,乃希望進一步瞭解TGBp2 是否會藉由其C 端之保留性cysteines 自我作用,形成同源多聚體,且此多聚體之形成是否為病毒核酸進行系統性移動所必需。另外,我們也想瞭解在寄主韌皮部中是否有特定蛋白會與TGBp2 結合,協助病毒核酸移動。本計劃第三年則在分析及鑑定能夠與TGBp3 發生交互作用,並協助病毒核酸在植物細胞間移動的蛋白夥伴。可能與TGBp3 交互作用,並協助病毒核酸在細胞間移動之蛋白夥伴,可能包括有病毒基因體本身所解譯的病毒殼蛋白和其它TGB 蛋白、以及植物宿主所生產的未知蛋白。在本年度研究中,我們將把重點放在TGBp3 與TGBp2 蛋白間的交互作用。此外,我們也將搜尋在細胞膜狀構造中能和TGBp3 交互作用的宿主蛋白。此研究結果將有助闡明TGBp3 在BaMV感染植物宿主過程中,如何發揮其功能協助病毒核酸之轉移。

AbstractTGBp1, TGBp2 and TGBp3 encoded by the triple-gene-block (TGB) of Bamboomosaic virus (BaMV) genome are required for virus movement. However, the detailfunctional mechanisms of the three proteins remain unclear. The 6-kDa TGBp3 wasproposed to be an integral membrane protein with a transmembrane helix. Comparingwith TGBp1 and TGBp2, the production, purification and antibody preparation ofTGBp3 are much more difficult. Thus, the topological and biochemical properties ofTGBp3 are lacking. On the basis of amino acid sequence alignment analysis of knownTGBp3, it was predicted that TGBp3 has a conserved Cys -x5 -Gly -x6~9 -Cys motif.Our preliminary data revealed that the BaMV TGBp3 is indeed an integral membraneprotein and that the Cys-to-Ala substitution of either one or both of the two conservedcysteine residues (Cys-31and Cys-46) of TGBp3 would inhibit the cell-to-cellmovement of BaMV in the host plant but not its replication. In the fist year of thisproject, we are aimed to investigate the membrane topology of TGBp3 and to unravelthe mystery of how the Cys-to-Ala substitution of TGBp3 affects the cell-to-cellmovement of BaMV.The TGBp2 is also a transmembrane protein which has two conserved Cysresidues (Cys-109 and Cys-112) at its C-terminal tail. We have found that thereplacement of either one or both of the conserved Cys residues with Ala wouldinhibit the movement of BaMV in the phloem of petiole. Moreover, monomeric,dimeric, trimeric, and tetrameric TGBp2 can be detected when the membrane fractionof N. benthamiana leaves inoculated with the Wt or mutant BaMV containing theCys-to-Ala substitution(s) was electrophoresed on the Tricine-SDS polyacrylamidegel. In the second year of this study, we would like to investigate whether TGBp2 isable to self-interact through the conserved Cys residues and whether the formation ofoligomeric TGBp2 is required for the systemic movement of BaMV. In addition, thephloem proteins which have the potential to interact with TGBp2 to assist thesystemic movement of BaMV will be screened.The goal of the third-year project is to demonstrate how TGBp3 plays its role inassisting the movement of viral RNA during the course of infection. To fulfill thisgoal, we plan to analyze and identify proteins which are able to assist virus movementacross the plant cells by interacting with TGBp3. The proteins which are able tointeract with TGBp3 and assist the cell-to-cell movement of BaMV may include theproteins (such as capsid and other TGBp) encoded by the BaMV genome, and certainmembrane proteins associated with the TGBp3-containing membrane complex andencoded by host plant. In this study, the interaction between TGBp3 and BaMVprotein will be focused on TGBp2. Moreover, we will look for membrane proteins ofthe host, which is able to interact with TGBp3. Hopefully, the results will help us todemonstrate how TGBp3 functions to assist viral movement during the course ofinfection.
其他識別: NSC99-2313-B005-019-MY3
Appears in Collections:生物化學研究所

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