Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36036
標題: 胡瓜嵌紋病毒衛星核酸複製訊號之探討
Analyses of Replication Signals of Satellite RNAs Associated with Cucumber Mosaic Virus
作者: 張怡莉
YiLi-Chang
關鍵字: Cucumber Mosaic Virus;胡瓜嵌紋病毒;Satellite RNAs;Replication Signals;衛星核酸;複製訊號
出版社: 生物科技學研究所
摘要: 
胡瓜嵌紋病毒 (cucumber mosaic virus, CMV) 為單股三基因體的RNA植物病毒,有些分離株攜帶有衛星核酸(satellite RNA, satRNA),需要依賴輔助病毒進行複製、包被及傳播,但與其輔助病毒無核酸序列同源性。本研究以胡瓜嵌紋病毒NT9株系 (CMV-NT9) 及其衛星核酸M-satRNA為材料,利用PCR的方式造成端點序列突變,來探討衛星核酸複製所需之訊息是由序列或者是結構所決定,以進一步了解衛星核酸之複製機制與輔助病毒複製酵素之交互作用。實驗方法主要為利用帶有30個隨機核苷酸序列的引子進行PCR來取代原本衛星核酸的5’端及3’端序列,突變株經生體外轉錄得到的轉錄體,與輔助病毒共同接種至菸草 (Nicotiana tabacum 、N.benthamiana),再抽取雙股RNA來偵測突變株於植物體中之複製能力,並利用逆轉錄-聚合酵素鏈鎖反應 (RT-PCR) 及核酸定序來確認子代之衛星核酸序列。在實驗進行之前,鑑於隨機序列接種可能出現野生型,因此先將使用的M48 satRNA作修飾,把位於satRNA 164位置上的核苷酸由T修改成A,使其多出一個NdeⅠ切位,方便於判斷獲得的野生型殖株是由於序列隨機度足夠還是接種污染所造成。另外,突變的位置是在端點,無法用端點引子進行RT-PCR,因此以poly (A) polymerase在雙股RNA的3’端加上poly(A) tail以oligo (dT) 進行RT-PCR以得到子代序列。實驗後發現,在雙股RNA正股的3’端可能有未知結構,妨礙poly(A) tail的加入,因此改利用多體的特性,以中間序列引子進行RT-PCR,即得到端點的序列,解序結果發現一突變株,在5’端的序列為5’-GTTTTGTTT-3’ ,與M48 satRNA相比,在5’端多了一個T,在3’ 端少了一個C,但與其他野生型satRNA端點序列差異並不顯著。部分序列多體連接處不完整,經分析後發現缺失序列接點附近具有高度相似性序列,因此研判多體的產生為與同源性序列有關。分析經原生質體接種確定不具複製能力的突變株,及以mfold預測其二級結構,不論其序列和結構皆與原本的衛星核酸差異甚大。另外本實驗比較在自然界存在的胡瓜嵌紋衛星核酸,將mfold預測的最低能量結構直線化,利用叢集分類進行多重序列排列,並與序列分類做比較。結果發現以結構分類與序列分類的的衛星核酸差異甚大,而有些結構在衛星核酸中屬於高度保留。總而言之,在本實驗的環境下,帶有野生型端點序列的衛星核酸最具有競爭力,而分析後發現序列與結構同樣在衛星核酸複製上扮演著重要角色。

Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus. Some strains of CMV harbor satellite RNAs (satRNAs) that are dependent on CMV for their replication, encapsidation, and transmission. The specific aim of this study is to investigate the 5'- and 3'-terminal signals on satRNAs required for high efficiency replication and to distinguish the effects of nucleotide sequences and structures. The 5'- and/or 3'-terminal 30 nucleotides were replaced by random sequences with synthetic primers in polymerase chain reactions (PCR). The transcripts of mutants were used to inoculate the plants with the helper virus, CMV-NT9. The viabilities of the mutants were analyzed by double-stranded RNA (dsRNA) analyses. The nucleotide sequences of the mutant satRNAs progenies were investigated using RT-PCR followed by DNA sequencing. To differentiate between inoculated transcripts and wild-type satRNA contaminations, a selection marker was introduced into the satRNAs by mutating the Thymine164 into an Adenine to create an NdeI restriction site. Since the 5'- and/or 3'-terminal sequences were randomized, poly(A) polmerase was used to add poly(A) tail at 3'ends of dsRNAs, and oligo(dT) primers were used to amplify the progenies. In addition, the multimeric replication forms of satRNAs were also used as templates for RT-PCR with inverse internal primer pair to amplify the terminal sequences. A viable mutant was recovered from the progenies of 324 inoculation tests. Nucleotide sequencing revealed that the mutant contains an addition of T in the 5' terminal T stretch region and a deletion of C in the 3' terminal triple region. The sequence analysis of the multimeric forms revealed that some nucleotides at the junctions between monomers were deleted, and the deletion sites contain similar sequence. The formation of multimers may come from homologous template-switching. Sequence analyses and structural predictions revealed evident differences between viable and non-viable satRNAs. Together, the results indicated that the current wild-type terminal sequences of satRNAs are the most competitive patterns under our experimental conditions. Both structure and sequence may play important roles in the replication of satRNAs.
URI: http://hdl.handle.net/11455/36036
Appears in Collections:生物科技學研究所

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