Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24040
標題: 竹嵌紋病毒TGBp2與TGBp3 C-端保留性cysteines對此兩蛋白協助病毒在植物中移動之重要性
The Importance of the Conserved Cysteine Residues at the C-terminal Tails of TGBp2 and TGBp3 on the Movement of Bamboo mosaic virus
作者: 曾揚皓
Tseng, Yang-Hao
關鍵字: Bamboo mosaic virus
竹嵌紋病毒
Triple gene block
Phloem
三重疊基因區
韌皮部
出版社: 生物化學研究所
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摘要: 竹嵌紋病毒(Bamboo mosaic virus,簡稱BaMV)屬正股RNA病毒,其基因體之三重疊基因區(triple gene block)所轉譯的三個蛋白,TGBp1、TGBp2和TGBp3,參與病毒核酸於植物細胞間的轉移,因此稱之為移動蛋白。其中TGBp2和TGBp3均為在C-端各有兩個保留性Cys的穿膜蛋白。本論文的第一章乃在探討TGBp2上的這兩個保留性Cys胺基酸(Cys-109和Cys-112)對病毒核酸於植物體中移動的重要性。實驗中,我以定點突變的方式構築三種能夠表現具有單一或雙重Cys-to-Ala取代之TGBp2蛋白的突變型竹嵌紋病毒。這三種病毒皆能夠感染白藜和菸草,並在感病葉中累積鞘蛋白,但其等的感染力相對低於野生型病毒。藉由病毒感染原生質體的方式,分析病毒的複製能力,並且利用螢光顯微鏡觀察突變病毒的移動能力,發現這三種病毒均有正常的複製能力,但它們在細胞間之移動能力,卻至少兩倍低於野生型病毒。可見,這三種突變型病毒感染力之變弱,乃導因於它們移動能力的降低。進一步觀察這三種病毒在植物維管束韌皮部中之長距離移動能力,發現它們於葉柄韌皮部移動效率變差。其中,表現Cys-109, 112-Ala雙重取代之TGBp2的突變型病毒,甚至無法順利從葉脈韌皮部進入葉柄韌皮部中。可見,TGBp2蛋白上之Cys-109和Cys-112為TGBp2協助病毒核酸於植物細胞間的短距離移動所必需外,它們也為TGBp2協助病毒核酸在植物韌皮部內長距離移動所必需。 本論文的第二章則在探討TGBp3上的這兩個保留性Cys胺基酸(Cys-31和Cys-46)對病毒核酸在植物體中移動的重要性。實驗中,我同樣以定點突變的方式構築三種能夠表現具有單一或雙重Cys-to-Ala取代之TGBp3蛋白的突變型竹嵌紋病毒。這三種病毒皆無法使白藜和菸草產生病徵。藉由病毒感染原生質體之分析方式,探討病毒的複製能力,並且利用共軛焦雷射掃瞄顯微鏡,觀察突變型病毒移動能力,發現這三種病毒均能正常複製,但它們卻都喪失在白藜表皮細胞間移動之能力。可見,TGBp3蛋白上之Cys-31及Cys-46為TGBp3協助病毒核酸在感病植物細胞間的短距離移動所必需。
The TGBp1, TGBp2 and TGBp3 encoded by the triple gene block (TGB) of Bamboo mosaic virus (BaMV) genome are required for virus cell-to-cell movement. Both TGBp2 and TGBp3 are transmembrane proteins which have two conserved Cys residues at their C-terminal tails, respectively. In the first chapter of this study, I investigated the importance of the two conserved cysteine residues (Cys-109 and Cys-112) of TGBp2 on the movement of BaMV. Three mutant BaMV, each having either one or both of the conserved cysteine residues in TGBp2 being replaced with alanine, were constructed and then inoculated onto the leaves of Chenopodium quinoa and Nicotiana benthamiana. Infectivity of each of the BaMV mutants was confirmed by the presence of disease symptoms and accumulation of CP in the leaves. However, the infectivity of the mutant BaMV was lower than that of the wild-type (Wt). To investigate the reason (s) causing the discrepancy, I analyzed the replication efficiency and the cell-to-cell movement activities of the Wt and mutant BaMV. The results indicated that the activities of cell-to-cell movement of mutant BaMV are at least two folds lower than that of the Wt, although they replicate with similar efficiency. Thus, the reduced infectivity of the mutant BaMV is attributed to their defects in virus cell-to-cell movement. In addition, I examined the effects of Cys-to-Ala substitutions in TGBp2 on systemic movement of BaMV and found that the three mutant BaMV are unable to move efficiently in the phloem of the petiole or from phloem of vein to the phloem of petiole. Taken together, the two conserved Cys residues (Cys-109 and Cys-112) at the C-terminal tail of TGBp2 are required for TGBp2 to assist both the cell-to-cell and systemic movement of BaMV. In the second chapter of this study, I investigated the importance of the conserved cysteine residues (Cys-31 and Cys-46) of TGBp3 on the movement of BaMV. Three BaMV mutants, each having either one or both of the conserved cysteine residues in TGBp3 being replaced with alanine, were constructed and then inoculated onto the leaves of C. quinoa and N. benthamiana. No disease symptoms were observed on the leaves inoculated with each of the mutant BaMV. To investigate the reason (s) causing the defect of infection, I checked the replication efficiency and the cell-to-cell movement activities of the Wt and mutant BaMV. Our results revealed that replacement of either one or both of the conserved Cys residues with Ala at the C-terminal tail of TGBp3 inhibits the cell-to-cell movement of BaMV severely but not the replication efficiency. Thus, the two conserved Cys residues (Cys-31 and Cys-46) at the C-terminal tail of TGBp3 are required for the functioning of TGBp3 in assisting the cell-to-cell movement of BaMV.
URI: http://hdl.handle.net/11455/24040
其他識別: U0005-1208201016033900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1208201016033900
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