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相關文獻
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頁籤選單縮合
題 名 | Infectivity Assays of in Vitro and In Vivo Transcripts of Papaya Ringspot Potyvirus=木瓜輪點病毒生體內及生體外轉錄體感染力之分析 |
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作 者 | 江主惠; 葉錫東; | 書刊名 | Botanical Bulletin of Academia Sinica |
卷 期 | 38:3 1997.07[民86.07] |
頁 次 | 頁153-163 |
分類號 | 373.9 |
關鍵詞 | 木瓜輪點病毒; 生體內轉錄體; 生體外轉錄體; In vitro transcript; In vivo transcripts; Papaya ringspot potyvirus; |
語 文 | 英文(English) |
中文摘要 | 木瓜輪點病毒(papaya ringspot virus; PRSV)夏威夷株系(HA)有四段cDNA涵蓋整 個病毒RNA基因體,由於彼此之間有重疊,可利用適當的限制酵素及黏結酵素(ligase)將cDNA 逐段接起來置於T3啟動子後面,所構築完成的全長度cDNA,利用生體外轉錄(in vitro transcription)得到的轉錄體,在5'端會多出一個G,在3' poly(A)36後面會多出12個非病毒 核□酸。此轉錄體無論有無冠端(cap)構造均和自然界的病毒RNA具類似分子量。為了證實此 轉錄體確實可對應產生正確病毒蛋白,利用兔子網狀紅血球使RNA在試管條件下進行轉譯作 用,結果發現所轉譯出來的蛋白和自然PRSV RNA轉譯結果相似,以PRSV病毒的抗血清進行免 疫沉澱分析亦可得相同結果。由此可證明此全長度的PRSV構築不但可以轉錄出與自然界PRSV RNA一樣長度的轉錄體,這個轉錄體亦可表現出正常的蛋白。以機械接種方式將含有冠端構造 的PRSV轉錄體接種於系統性寄主木瓜(Carica papaya)與單斑寄主奎藜(Chenopodium quinoa) 上均可產生PRSV的典型病徵。利用PRSV之抗血清進行西方轉漬分析,可測到病毒的鞘蛋白(36 kDa)。在免疫電顯反應中,亦可看到與PRSV血清反應的長絲狀病毒顆粒,由此證明我們確實 已構築一個具感染力的生體外轉錄體。而另一個全長度的構築方式和前者相似,不過其3'端 poly(A)後面尚多出64個非病毒核□酸,雖然此構築在試管的條件下產生的RNA與蛋白均和自 然界PRSV RNA產生的相似,但經過接種木瓜、奎藜均無法感染,顯然此64個非病毒核□酸的 存在會導致病毒活力的喪失。至於生體內轉錄體的構築,係將PRSV全長度cDNA置於35S啟動子 與NOS終止子之間,構築得到含全長度基因體之質體其5'與35S啟動子之間並無其他非病毒核 □酸存在,其3'端poly(A)與NOS終止子之間則多了10個非病毒核□酸及NotI限制酵素序列, 此質體經超高速離心純化後以機械接種或粒子鎗接種均可得到發病之植株。利用PRSV抗血清 進行酵素連結抗體反應與免疫雙向擴散分析均可得到強烈的正反應結果,而無病徵或健株則 無反應。以電子顯微鏡觀察病葉,可看到成群的絲狀病毒顆粒聚集。另一個PRSV生體內轉錄 體的構築其35S啟動子與PRSV 5'之間尚有33個無病毒核□酸而3'端poly(A)與NOS終止子之間 則有64個非病毒核□酸存在,此質體經大量接種奎藜及木瓜葉片卻不能造成感染,顯示35S 質體之感染活性與5'及3'端之非病毒核□酸有關。本研究所構築之木瓜輪點病毒生體外及生 體內具感染力轉錄體均為目前已知植物病毒中最長者。 |
英文摘要 | A full-length cDNA with nucleotide sequence representing the genomic RNA of a Hawaii strain of papaya ringspot potyvirus (PRSV HA) was constructed downstream from a bacteriophage T3 promoter in an in vitro transcription vector. The plasmid was able to generate an in vitro transcript corresponding to PRSV RNA (10326 nt) with one extra guanosine residue at the 5' terminus and 12 nonviral nucleotides at the 3' end following a poly(A)36 tract. In vitro translation products and immunoprecipitation analysis with the antiserum to PRSV verified correctness of the gene expression of the transcript. When the capped transcript was mechanically introduced to the systemic host papaya and the local lesion host Chenopodium quinoa, typical symptoms of PRSV HA appeared at almost the same time as on those host plants inoculated with native PRSV RNA. Western blotting and serologically specific electron microscopy with PRSV antiserum confirmed the infection. The uncapped in vitro transcript and the transcript with longer nonviral nucleotides (64 nt) at the 3' end were not infectious. The full-length cDNA was also constructed with a cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (NOS) terminator in an in vivo expression vector. Purified plasmids were applied directly onto host plants either mechanically or by bombardment with a particle delivery system to analyze their infectivity. The plasmid without extra nucleotides between the 35S promoter and the 5' end of PRSV sequence and with 10 nonviral residues and a NotI site at its 3' end was infectious, as evident from symptom development, and ELISA. immunodiffusion, and serologically specific electron microscopy analyses with PRSV antiserum. The construct with 33 nonviral nucleotides at the 5' end of the PRSV sequence and more than 64 nucleotides at the 3' end was not infectious. The infectious in vitro and in vivo transcripts derived from the full-length cDNA to PRSV RNA are the longest so far recorded for a plant virus. |
本系統中英文摘要資訊取自各篇刊載內容。