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標題: 竹嵌紋病毒三重疊基因區第二轉譯蛋白C 端半胱胺酸取代突變造成病毒於細胞內移動缺失原因之探討
Studies on the mechanism responsible for the defect of movement of Bamboo mosaic virus caused by mutation(s) of the three cysteine residues in the triple gene block protein 2
作者: 何采凌
Tsai-Ling Ho
關鍵字: 無;no
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竹嵌紋病毒 (Bamboo Mosaic Virus, BaMV) 為單鏈正股 RNA 病毒,其基因體之三重疊基因區 (triple gene block, TGB) 所轉譯出的 TGBp1、TGBp2 和TGBp3,均參與病毒在植物細胞間的移動,因此它們被稱為移動蛋白。TGBp2蛋白具有兩個穿膜區域,其 N-端及 C-端皆位於細胞質中,而 loop region 則位於內質網內腔 (ER lumen)。TGBp2 蛋白 C-端含有 Cys-109 和 Cys-112 等兩個高保留性胺基酸,以及一個非保留性的 Cys 胺基酸,Cys-119。實驗室先前的研究發現,兩個保留性 Cys 胺基酸單獨或同時以 Ala 取代,均會造成病毒於植物細胞間短距離移動的能力下降,長距離移動的能力則完全喪失。本研究旨在探討 TGBp2蛋白 C-端之 Cys 取代突變,導致 BaMV 在植物細胞間移動能力的減弱到底受下列那一種可能因素所影響。一、Cys 取代突變會造成 TGBp2 蛋白本身或接受TGBp3 協助而移動至細胞周邊原生質絲 (plasmodesmata, PD) 的能力喪失 二、;具有 Cys 取代突變的 TGBp2 蛋白會影響 TGBp1 移動到細胞周邊 PD 的能力。

為瞭解第一種可能性是否存在,我以農桿菌注入法,將能於植物細胞中表現野生型或突變型 TGBp2 的質體,或同時表現野生型或突變型 TGBp2 與 TGBp3的質體 送入菸草植物中 並於接種之菸草葉片中 注入 aniline blue fluorochrome,再利用倒立式雷射共軛焦顯微鏡觀察具有 Cys 取代突變的 TGBp2 蛋白是否會影響其本身或其接受 TGBp3 協助移動至細胞周邊 PD 的能力 結果發現不論 TGBp3。存在與否,突變型 TGBp2 本身仍會移往 PD。為瞭解第二種可能性是否存在,我利用農桿菌注入法,將能於植物中同時表現野生型或突變型 TGBp2 和 TGBp3的質體,搭配表現 TGBp1 的質體,送入菸草葉片中,再以倒立式雷射共軛焦顯微鏡觀察 TGBp1 是否可運送至 PD。結果發現 TGBp1 必需在野生型 TGBp2 和TGBp3 同時存在下,才能夠有效地移動至 PD。但是,當 TGBp2 蛋白 C-端的Cys-109 或 Cys-112 發生單點取代突變時,TGBp1 移動至 PD 的能力均會受到明顯影響,但 Cys-119 的單點取代突變,影響則不明顯。

The TGBp1, TGBp2 and TGBp3 proteins encoded by the triple gene block (TGB) of Bamboo Mosaic virus (BaMV), a positive-stranded RNA virus, are required for virus cell-to-cell movement, and therefore are called movement proteins. The TGBp2 is a transmembrane protein with its N- and C-terminal tails exposed to the cytoplasm and the loop region located at the ER lumen. The C-terminal tail of the TGBp2 protein contains two highly-conserved cysteine residues, Cys-109 and Cys-112, and one non-conserved cysteine residue, Cys-119. The previous study in our laboratory has revealed that substitution of either one or both of the highly-conserved cysteine residues for Ala results in the reduction of cell-to-cell movement and severe defect in long-distance movement phenotypes of the virus. This study is aimed to understand the mechanism responsible for the phenomenon. According to the known functional relationship among TGBp1, TGBp2 and TGBp3, two possible hypothesis have been proposed. One is the inability of the mutant TGBp2 to target themselves or in cooperation with TGBp3 to move to plasmodesmata (PD) of cell periphery; the other is the inability of the mutant TGBp2 to assist the targeting of TGBp1 to PD.

Using the agroinfiltration, plasmids which are able to express the wild-type (WT) or mutant TGBp2 or to co-express the WT or mutant TGBp2 and TGBp3 were introduced into the leaves of Nicotiana benthamiana. The infected leaves were examined with confocal microscopy after staining with aniline bule which specifically stains the callose in PD. Our results revealed that both of the WT and mutant TGBp2 proteins are able to move to PD of cell periphery in the presence or absence of TGBp3. Using the same strategy, plasmids which are able to co-express TGBp1 and the WT or mutant TGBp2 as well as TGBp3 were introduced into the leaves of N. benthamiana to examine the PD localization of TGBp1 by confocal microscopy. The results indicate that both the WT TGBp2 and TGBp3 are essential for efficient targeting TGBp1 to the PD. Cys-to-Ala substitution at position 109 or 112 of TGBp2 significantly decreased the PD targeting of TGBp1.
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