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標題: 參與竹嵌紋病毒核酸基因體正股複製的寄主蛋白鑑定與探討
The Study of Host Factors Involved in the Synthesis of Positive Strand RNA of Bamboo Mosaic Virus
作者: 陳盈君
關鍵字: Bamboo mosaic virus;竹嵌紋病毒;subgenomic RNA;Hsp70;Hsp90;VIGS;次基因體核醣核酸;熱休克蛋白70;熱休克蛋白90;病毒誘導式基因沉默系統
出版社: 生物科技學研究所
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竹嵌紋病毒,隸屬於Flexviridae科中之Potexvirus屬,具有單股正極性核醣核酸的基因體。正股核醣核酸的病毒通常會轉錄出病毒的次基因體核醣核酸來調控病毒的蛋白表現。在本篇的研究,主要在探討是否在竹嵌病毒進行次基因體核醣核酸表現的過程中會有寄主蛋白參與其中。我們利用竹嵌紋病毒1 kb次基因體核醣核酸啟始點附近的互補序列為探針 (sgBa-135 RNA),偵測能與此探針發生交互作用的寄主蛋白是否存在於含有竹嵌紋病毒複製複合體的蔗糖梯度離心萃取液中。經由紫外光誘發的核醣核酸–蛋白質交互作用以及核醣核酸競爭試驗的結果顯示,有兩個分子量分別約81和72 kDa的蛋白質能與此竹嵌紋病毒核醣核酸探針發生專一性的交互作用。此一結果與竹嵌紋病毒負股3''端啟動子為核醣核酸探針時,相同分子量的兩個寄主蛋白也會跟核醣核酸探針發生專一性的交互作用。故推論此兩個寄主蛋白可能與竹嵌紋病毒基因體以及次基因體合成有所相關聯。經由蛋白質的層析純化及質譜儀鑑定結果顯示72-kDa的蛋白質為熱休克蛋白70 (heat shock protein cognate 70, Hsp70), 81-kDa的蛋白質為熱休克蛋白90 (molecular chaperone Hsp90-2, Hsp90)。我們進一步利用病毒誘導式基因沉默系統 (virus-induced gene silencing, VIGS) 來抑制熱休克蛋白70以及熱休克蛋白90的表現,發現抑制熱休克蛋白70以及熱休克蛋白90基因的表現,確實會抑制竹嵌紋病毒鞘蛋白的累積量。綜合上述,熱休克蛋白70與熱休克蛋白90對於竹嵌紋病毒生活史上扮演非常重要的角色。

Bamboo mosaic virus (BaMV), belonging to genus Potexvirus of the family Flexiviridae, is a positive-sense RNA virus. Positive-sense RNA viruses utilize a wide variety of strategies to express their genes in the limited size of small genome. A common strategy used by many viruses is to transcribe subgenomic RNAs. In this study, we focus on the identification of host factors participating in the synthesis of the BaMV 1 kb subgenomic RNA. We used the radioisotope-labeled RNA (sgBa-135; containing the putative promoter sequence for subgenomic RNA synthesis) as probe for UV cross-linking experiment to see if proteins in BaMV RNA-dependent RNA polymerase (RdRp) extracts from infected plants could interact with. Two host factors with molecular mass 72 and 81 kDa were found to specifically interact with sgBa-135. The same two factors were also found previously to interact with the 3''-end of BaMV minus-strand (Ba-77) which is the promoter sequence for genomic RNA synthesis. The mass spectrometry analysis indicated that 72-kDa protein is heat shock protein cognate 70 (Hsp70), and 82-kDa protein is molecular chaperone Hsp90-2 (Hsp90). A virus-induced gene silencing (VIGS) system was used to investigate the involvement of Hsp70 and Hsp90 in BaMV life cycle. Results showed that the reduction of gene expression of either one of these two genes in N. benthamiana caused a reduction in coat protein accumulation of BaMV but not for that of Cucumber mosaic virus. We conclude that these two host factors, Hsp70 and Hsp90, are involved in BaMV infection cycle.
其他識別: U0005-2607200716321600
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