• 作者： 蔡慶修; 鄭再宏
• 作者服務機構： 中興大學遺傳工程中心; 立中興大學農業生物科技學研究所
• 中文摘要： 胡瓜嵌紋病毒為一球形植物病毒，含有三段單股正向核酸基因體。每一段基因體的最末端約兩百個核甘酸左右可以形成一個很特殊的三級結構，這個結構稱為tRNA-like structure並且可以接上一個特定的氨基酸tyrosine。在相類似的雀麥嵌紋病毒（brome mosaic virus）中，這個三級結構已經由實驗確認它的存在，我們引用雀麥嵌紋病毒的三級結構圖譜劃出胡瓜嵌紋病毒核酸基因體末端三級結構的圖譜。為進一步探討植物病毒的複製機制，我們必須要有一個分析病毒核酸基因體複製能力的系統，這個系統又稱為體外複製系統（in vitro replication）。我們將感病與健康的煙草葉片取下均質化後離心，上清液以三次超高速的離心取得組織膜體部份（membrane fraction）的溶離蛋白。經由分析測試的結果，我們意外的發現利用這種分離的方法可以得到植物本身所生產的核醣核酸複製酵素（RNA-dependent RNA poly-merase）。若我們改以超高速30,000 rpm的轉速將含有核酸複製酵素複合體（replicase complex）的組織膜體部份（membranefraction）沉降下來，再將所沉降下來的沉澱物用detergent予以溶解，最後再以25-55％的甘油梯度離心作進一步的分離。結果發現我們所沉降下來的蛋白質確實含有胡瓜嵌紋病毒核酸複製酵素複合體的活性，再經由測試的結果得知我們所部份純化出的酵素可以將內含性的胡瓜嵌紋病毒核酸（endogenous CMV RNA）做成雙股的病毒核酸。
• 英文摘要： Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into atRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaicvirus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virusinfected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solublized membrane bound fractions. We accidentally foundthat these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity.Similar activity could also be found in other plants tested. Alternately, the membrane bound fractioncould be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation(40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerolgradient fractionation was performed. Activity was tested through the incorporation of [α- (方程式無法摘錄) ]UTP usingendogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractionscontained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were foundto have a double-stranded form through further analysis of the RNase protection assay.
• 中文關鍵字： cucumber mosaic virus; tRNA-like structure; RNA-dependent RNA polymerase
• 英文關鍵字： --