Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35799
標題: P20/GFP融合蛋白對竹嵌紋病毒衛星核酸在細胞間移動的影響
Effect of P20/GFP fusion on cell to cell movement of Bamboo mosaic virus satellite RNA
作者: 劉光仰
Liu, Kuang-Yang
關鍵字: BaMV;竹嵌紋病毒
出版社: 生物科技學研究所
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摘要: 
Satellite RNAs (satRNAs) are subviral agents which depend on helper viruses and host factors for replication, encapsidation and movement. Bamboo mosaic virus (BaMV), a member of the genus Potexvirus, is the only potexvirus that naturally associates with a satRNA, designated satBaMV, which encodes a 20 kDa RNA-binding protein (P20) involved in efficient long-distance movement and replication of satBaMV. The aims of this study were to investigate the abilities of other non-cognate viruses in supporting the replication of satBaMV and to explore the effects of P20 phosphorylaion on the movement of satBaMV. Dimeric satBaMV transgenic plant lines expressing P20 fused with green fluorescent protein (GFP) either at N- or C- terminus were generated. BaMV and two non-cognate viruses, Potato virus X (PVX) and Foxtail mosaic virus (FoMV), were inoculated onto the transgenic plant to monitor their abilities in supporting satBaMV. Through the examination of GFP expressions in the inoculated transgenic plants, it was confirmed that the non-cognate helper FoMV marginally supports the replication of satBaMV, whereas PVX does not. Plasmids harboring GFP fused to the N- or C-terminus of P20 protein with mutations on the serine at amino acid position 11 (S11) to inhibit or mimic the phosphorylation state of P20 were constructed, and there cell-to-cell movement ability were examined in Chenopodium quinoa by fluorescent microscopy. The results revealed that both mutations of P20 reduced the cell-to-cell movement of satBaMV, corroborating the previous finding that phosphorylation of S11 is important for the expression and movement of satBaMV.

竹嵌紋病毒衛星核酸(satllite Bamboo mosaic virus RNA, satBaMV)必須依賴竹嵌紋病毒(Bamboo mosaic virus, BaMV)進行複製、包被及移動。BaMV是目前唯一在自然界被發現具有衛星核酸之馬鈴薯病毒屬之植物病毒,其satBaMV可轉譯出大小為20kDa的蛋白,稱作P20,可結合satBaMV並幫助其進行長距離之移動及複製。satBaMV載體之轉型植物系統,在竹嵌紋病毒接種後,能有效且大量地誘導外源基因的表現。為了進一步探討非同源性病毒是否具有誘導此系統中satBaMV載體的複製能力,本實驗運用兩種以satBaMV為載體之轉型植物品系satP20GFP及satGFPP20,分別是將綠色螢光蛋白(Green fluorescent protein, GFP)構築於satBaMV所含有的P20蛋白之C端及N端,再經由農桿菌(Agrobacterium)轉型系統得到轉型株。首先,為快速獲得具有同源性的F3子代轉型株,建立了以冷光螢光分析儀作為篩選satBaMV載體帶有GFP轉型株之方法。接著,進行BaMV及Potexvirus屬的狐尾草嵌紋病毒(Foxtail mosaic virus, FoMV)和馬鈴薯病毒X (Potato virus virus, PVX)之接種試驗,實驗結果顯示BaMV及FoMV測試病毒都可以放大GFP訊號,但以BaMV病毒的效果最佳。另外,為了運用GFP蛋白為報導基因來驗證P20蛋白第11個胺基酸Serine,經磷酸化後會影響satBaMV於細胞與細胞間的移動,首先將P20蛋白第11個胺基酸突變成Alanine及Aspartic acid的兩個質體pCS11A及pCS11D,同樣地將GFP構築在突變的P20蛋白之N端及C端,分別稱為pCGFPP20-S11A、pCGFPP20-S11D、pCP20GFP-S11A及pCP20GFP-S11D,將上述質體純化並接種於白藜,在接種第5天後利用螢光顯微鏡觀察GFP螢光蛋白分佈,發現上述4個選殖株之GFP蛋白螢光散佈於細胞之大小明顯小於將GFP蛋白分別構築於N端及C端之pCGFPP20及pCP20GFP選殖株,結果顯示P20蛋白的磷酸化確實會影響satBaMV於細胞與細胞間之移動。
URI: http://hdl.handle.net/11455/35799
其他識別: U0005-2008201214050100
Appears in Collections:生物科技學研究所

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