https://scholars.lib.ntu.edu.tw/handle/123456789/96217
標題: | 竹嵌紋病毒之分子生物學─子計畫八:竹嵌紋病毒缺失RNA之研究(3/3) | 作者: | 張雅君 | 關鍵字: | 竹嵌紋病;竹嵌紋病毒;缺失性RNA;Bamboo Mosaic;BaMV;Defective RNA | 公開日期: | 2000 | 出版社: | 臺北市:國立臺灣大學植物病理與微生物學系暨研究所 | 摘要: | 為了探討竹嵌紋病毒(Bamboo mosaic virus,簡稱BaMV)的缺失性RNA 之性質,去 年度我們從含有缺失性RNA 之BaMV 病毒顆粒中,選殖出35 個含有0.7-1.3 kb DNA 片段的cDNA 株,並且完成9 個cDNA 株的核酸定序與序列分析,証實皆為BaMV 缺 失性RNA。為分析那一株缺失性RNA 具有生物活性(即能被BaMV RNA 所增殖), 將BaMV 與9 株缺失性RNA 之生體外轉錄體同時接種至菸草原生質體,經24 小時光 照培養後,抽取RNA,以非放射性的BaMV 5'端cDNA 探針進行北方雜配反應,結果 發現至少有6 株缺失性RNA 具有被增殖的能力,且皆不具干擾BaMV 複製的能力。同 屬於potexvirus 的三葉草黃化嵌紋病毒(Clover yellow mosaic virus,簡稱ClYMV)之缺失 性RNA,其基因體皆具有由複製酵素基因5’端與鞘蛋白基因3’端合併形成之融合轉譯 架,若破壞其鞘蛋白基因原有之轉譯架,則該缺失性RNA 會喪失增殖能力。相反地, 目前我們所選殖到具有增殖能力的六株BaMV 缺失性RNA,只有D52 具有此特殊之融 合轉譯架;故鞘蛋白基因轉譯架之維持,對於BaMV 缺失性RNA 的增殖並非必要。為 了測試複製酵素基因轉譯架之存在是否會影響BaMV 缺失性RNA 的增殖能力,因此我 們根據菸草原生質體接種實驗結果,選擇增殖能力最佳的D20 作為野生株,利用人為 突變的方法構築出兩種突變株D20-pml 和D20-spe;其中D20-pml 的複製酵素基因的起 始碼被破壞,而D20-spe 只能轉譯出五個氨基酸。經菸草原生質體接種實驗證實,上述 兩種突變株皆不具生物活性,此結果顯示複製酵素基因轉譯架之存在對BaMV 缺失性 RNA 的增殖極為重要。由上述實驗結果可知,BaMV 和ClYMV 的缺失性RNA 的性質 不同,因此轉譯架對BaMV 缺失性RNA 之意義與影響,仍有待進一步的研究。 In order to study the defective RNAs (D RNAs) of Bamboo mosaic virus (BaMV), 35 cDNA clones were obtained from D RNA-containing BaMV virions, nine of them were sequenced and proved to be BaMV D RNAs. A protoplast infection assay was used to evaluate the biological activity of cloned D RNAs. By co-inoculating the RNA transcripts of BaMV and D RNAs into tobacco protoplasts, the accumulation of D RNAs was analyzed after - 2 - 24 hours. The results indicated that at least six clones of BaMV D RNAs were infectious but none could interfere with the replication of BaMV. The D RNAs of Clover yellow mosaic virus (ClYMV) were reported to contain a fusion open reading frame (ORF) of replicase and coat protein genes, which was essential for their infectivity. On the contrary, D52 was the only infectious clone of BaMV D RNAs containing the in-frame fusion ORF. Therefore, the maintenance of the reading frame of coat protein gene in the fusion ORF was not required for the amplification of BaMV D RNAs in vivo. To test whether the ORF of replicase gene is important for the biological activity of BaMV D RNAs, two mutants were constructed from D20, which had the highest level of accumulation than other clones in protoplast assay. D20-pml was a mutant whose original start codon was destroyed. D20-spe was a frame-shift mutant with only five amino acid residues translated. Both mutants could not maintain the original ORF and were nonviable when assayed in tobacco protoplasts. The results suggested that the maintenance of the replicase ORF is crucial for the accumulation of BaMV D RNAs in vivo. From the above results, the characters of BaMV D RNAs are different from those of ClYMV D RNAs. The significance and influence of ORF on BaMV D RNAs needs further study. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/29754 | 其他識別: | 892311B002023B11 | Rights: | 國立臺灣大學植物病理與微生物學系暨研究所 |
顯示於: | 植物病理與微生物學系 |
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