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研究生:李淑娟
研究生(外文):Lee, Shu-Chuan
論文名稱:影響竹嵌紋病毒缺失性RNA增殖之因子
論文名稱(外文):Factors Affecting the Accumulation of Bamboo Mosaic Potexvirus-associated Defective RNAs
指導教授:張雅君張雅君引用關係
指導教授(外文):Chang, Ya-Chun
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:46
中文關鍵詞:竹嵌紋病毒缺失性RNA增殖
外文關鍵詞:Bamboo Mosaic PotexvirusDefective RNAAccumulation
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竹嵌紋病毒(bamboo mosaic virus,簡稱BaMV)為Potexvirus群之正意單股RNA病毒,全長序列為6.4 kb,具有5個轉譯架構。BaMV在自然界中所產生的缺失性RNA (defective RNA),其基因體由BaMV 5’端序列及3’端序列組合而成,皆具有一個轉譯架構。D20為BaMV缺失性RNA選殖株之一,具有良好之增殖能力,以聚合酵素連鎖反應(polymerase chain reaction)構築D20之突變株。以菸草(Nicotiana benthamiana)原生質體測定D20及其突變株之生物活性,結果顯示轉譯架構的存在決定BaMV缺失性RNA被BaMV增殖的能力,轉譯架構的缺失同時也使D20失去增殖能力,其增殖的能力與轉譯架構的存在有絕對相關性。D20之不同轉譯架構長度突變株實驗結果顯示,此一轉譯架構的長度必須大於99個氨基酸,轉譯長度的增加則有助於加強其增殖能力。利用來自BaMV鞘蛋白基因或是以完全無關之外來基因EGFP取代D20原有之轉譯架構,雖然仍保有相似長度之轉譯架構,但卻無法維持BaMV缺失性RNA的增殖能力,顯示除了轉譯架構的長度外,轉譯架構中的序列部份亦有其重要性。
移除不同長度及不同區域之D20序列發現,缺失性RNA至少需保有BaMV 5’端nt 1-551與3’端nt 936-1118之序列片段才能增殖;當移除序列nt 388-551時會使D20增殖能力喪失,因此其中應含有與增殖能力相關的cis-acting sequence。而序列nt 551-936雖不是D20增殖之必要因子,但可以增加其增殖效率。綜合上述之實驗結果,BaMV缺失性RNA之轉譯架構不能被其他基因取代,轉譯架構長度與其轉譯架構中的序列皆會影響缺失性RNA的增殖能力。
Bamboo mosaic virus (BaMV) belongs to Potexvirus group and has a single-stranded, positive-sense RNA molecule. Genome of BaMV is 6.4 kb in length and contains five open reading frames (ORFs). Naturally occurring defective RNAs (D RNAs) of BaMV are composed of the 5’ and 3’ termini of BaMV genome and contain an ORF. D20, one of BaMV D RNAs, had the highest level of accumulation than others in protoplast assay. D20-derived mutants were constructed by means of polymerase chain reaction-directed mutagenesis. Protoplast infection assays were used to evaluate the biological activity of D20-derived mutants. The results suggested that the maintenance of the ORF is crucial for the accumulation of BaMV D RNAs in vivo. In addition, the length of the ORF influenced the accumulation of D RNAs, and must be no less than 99 amino acids in order to maintain their biological activities. When the sequences of the ORF was replaced by BaMV coat protein gene or foreign EGFP gene, the mutants were nonviable although their ORF lengths similar to D20. The above results indicated that not only the lengths of the ORF but also the sequences within ORF are important for the accumulation of BaMV D RNA.
According to the experimental results of D20 deletion mutants, the minimum sequences required for biological activities of BaMV D RNAs are from regions nt 1-551 and nt 936-1118. D20 mutants with nt 388-551 deleted lost their viabilities. This result suggested that these sequences may contain cis-acting sequence of replication. Although nt 551-936 is not essential for replication, it could increase the level of accumulation of D RNAs. Taken together, the ORF sequence of D RNAs can not be replaced by other genes, and both the lengths and the sequences of the ORF influence the accumulation of BaMV D RNAs in vivo.
中文摘要 1
英文摘要 2
壹、前人研究 3
一、 缺失性與缺失干擾性RNA之特性 3
二、 Potexvirus屬病毒缺失性RNA之研究 4
三、 轉譯架構對缺失干擾性RNA 增殖能力之影響 5
四、 竹嵌紋病毒簡介 6
貳、材料與方法 9
一、實驗材料 9
二、利用聚合酵素連鎖反應構築D20突變株 9
(一) 構築D20之不同轉譯架構長度突變株 9
(二) 構築D20之轉譯架構序列取代突變株 10
(三) 構築D20之不同長度序列移除突變株 10
(四) 構築D20之不同區域片段序列移除突變株 11
(五) PCR反應與限制酵素酵解作用 11
(六) 黏合反應 12
三、轉型試驗及突變株之篩選 12
(一) 勝任細胞(competent cells)之製備 12
(二) 轉型試驗(transformation) 12
(三) 正確突變株之篩選 13
(四) 突變株之核酸定序及序列分析 13
四、 生體外轉錄體接種源之製備 14
(一) 限制酵素直線化質體(plasmid linearization) 14
(二) 生體外轉錄反應(in vitro transcription) 14
五、 菸草原生質體之製備、接種、RNA萃取及電泳分析 15
(一) 供試植物之栽培 15
(二) 原生質體之製備 15
(三) 原生質體之接種 16
(四) 原生質體全RNA (total RNA)之萃取與電泳分析 16
六、 北方氏轉印法及雜配反應 17
(一) BaMV 5’端專一性核酸探針之製備 17
(二) 北方氏轉印法 17
(三) 北方氏雜配反應 18
參、結果 19
一、 不同轉譯架構長度對D20增殖能力之影響 19
二、 轉譯架構之取代對D20增殖能力之影響 20
三、 不同長度序列移除對於D20增殖能力之影響 21
四、 不同區域序列移除對D20增殖能力之影響 22
肆、討論 24
伍、圖表 29
陸、參考文獻 41
柒、附錄 45
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