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研究生:洪怡芳
研究生(外文):Yi-Fang Hung
論文名稱:突變序列對胡瓜嵌紋病毒衛星核酸複製之探討
論文名稱(外文):Mutational studies on the replication of satellite RNAs associated with cucumber mosaic cucumovirus
指導教授:胡仲祺
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:日文
中文關鍵詞:衛星核酸
外文關鍵詞:satellite RNA
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中文摘要
胡瓜嵌紋病毒 (cucumber mosaic virus, CMV) 為Cucumovirus屬的三基因體植物RNA病毒,有些分離株攜帶有衛星核酸(satellite RNA),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。因此,本研究以胡瓜嵌紋病毒NT9株系及衛星核酸C-satRNA為材料,利用序列刪減突變探討衛星核酸高效率複製所需之最少核酸序列訊息,並利用繼代接種及核酸序列分析進一步探討衛星核酸基因體變異及其修復機制,以進一步了解衛星核酸與輔助病毒之交互作用,期作為未來發展為基因載體系統之依據。本研究利用Bal 31酵素進行序列刪減,分別構築在BamHI及NcoI限制酵素切位之序列刪減突變株,以生體外轉錄得到的轉錄體,與輔助病毒(CMV-NT9)共同接種至菸草(Nicotiana tabacum cv. Van Hicks、Nicotiana benthamiana),再抽取雙股RNA來偵測各序列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。在接種N. tabacum後所得具有複製能力的第一子代中,經序列分析可發現,在BamHI切位附近約可容忍3至20個核苷酸刪減,而在NcoI切位附近刪減31個核苷酸,依然保有其複製能力。由具有複製能力的第一子代中發現,約有30% ~70%回復成野生型,且部份子代在第47至55個核苷酸之間仍保有刪減序列。在第二子代及第三子代的序列分析結果中約有30% ~100%回復成野生型,且部份子代在第47至57個核苷酸之間仍保有刪減序列,並可得3到4個不同的核酸插入序列存在第45至第47個核苷酸序列中。由此推測,在第47至57個核苷酸之間對衛星核酸於N. tabacum高效率複製是不需要的。另外,由接種N.benthamiana所得的結果發現,各突變株序列與其第一子代序列的刪減序列位置十分相近,序列完全與突變接種源一樣的比例佔所有選殖株中的55% ~100%,其中在BamHI切位最長可容忍的刪減序列為87個核苷酸,在NcoI切位最長可容忍的刪減序列為31個核苷酸;遠高於N. tabacum中所獲得之結果。因此寄主植物在衛星核酸之演化及適應性上扮演一重要角色來決定衛星核酸序列,衛星核酸通常可藉由突變、分子間重組或基因體的重新排列使之較能適應於不同寄主。此結果將可進一步探討其輔助病毒之複製機制,並可應用於未來病毒病害之防治與基因載體系統之研發。
Abstract
Cucumber mosaic virus (CMV), the type member of the Cucumovirus, is a plant virus with a tripartite genome. Some CMV strains contain a small RNA that has been demonstrated to be a satellite RNA (satRNA). SatRNAs are dependent on helper virus for replication, encapsidation, and transmission, but share little or no sequence similarity to the helper virus. The specific aim of this study is to elucidate the minimum required sequence signals on satRNAs for high efficiency replication to gain further insight into the biological functions of satRNAs. Deletion mutations of satRNA were created around the BamHI and NcoI sites. The transcripts of mutants were synthesized in an in vitro transcription system and were used to inoculate Nicotiana tabacum and Nicotiana benthamiana with the helper virus, CMV-NT9, which is free of satRNAs. The viability of mutants was analyzed by double-stranded RNA analyses. The nucleotide sequences of the mutant satRNAs progenies were investigated using RT-PCR followed by DNA sequencing. The first-generation progenies that contain mutations near the BamHI site could maintain 3 to 20 nucleotides deletions. The mutants with 31 nucleotides deletions near the NcoI site maintained the replication ability. It is found that 30%~70% of the first-generation progenies have reversed to wild type, while the others still maintain deletion sites between nucleotide positions 47 to 55. For the second and third-generation progenies, 30%~100% have reversed to wild type and 3 to 4 different nucleotide insertions were found between nucleotide positions 45 to 47, indicating that the nucleotides between positions 47 to 57 are not required for efficient replication with in N. tabacum. However, from N. benthamiana inoculation test, the first-generation progenies maintained similar deletion sites of the inoculum, and it is found that 55%~100% of progenies contained exactly the same mutation sites. The longest deletions allowed were 87 nucleotides around BamHI site and 31 nucleotides around NcoI site. These results suggested that the host plants played an important role in the evolution and selection of satRNA quasispecies. By mutation and recombination, satRNA may adapt to different hosts. These results might be applied in the understanding of helper virus replication and development of satRNA-based transient expression vector systems.
目錄
中文摘要………………………………………………………………1
英文摘要………………………………………………………………2
壹、 緒論……………………………………………………….……3
貳、 前言……………………………………………………….……4
一.胡瓜嵌紋病毒簡介……………………………………………… 4
1. 經濟重要性…………………………………………………….. 4
2. 分類地位………………………………………………………...5
二.胡瓜嵌紋病毒基因體組成……………………………………….5
1. 基因體結構、組成及功能………………………………….…..5
(1) 三基因體組成………………………………………….. 5
(2) RNA 1與其基因功能…………………………………..…7
(3) RNA 2與其基因功能…………………………………..…8
(4) RNA 3、次基因體RNA 4與其基因功能…………….....9
2. 衛星核酸………………………………………………………..10
(1) 結構及功能………………………………………………10
(2) 調控輔助病毒影響病徵………………………………..12
(3) 衛星核酸複製相關性…………………………………..13
(4) 輔助病毒影響其複製……………………………..……13
(5) 生體內(in vivo)及生體外(in vitro)複製………….14
(6) 胡瓜嵌紋病毒之基因體變異……………………..……15
(6.1) 突變(mutation)……………………………….….…. 15
(6.2) 分子間重排(reassortment; psedorecombination)…. 16
(6.3) 分子內重組(recombination)………………………. 16
(7) 實驗環境或天然環境下所產生的基因體變異………... 17
參、材料與方法…………………………….……………..………19
一.病毒來源…………………………………………………….….19
二.病毒的純化………………………………………………………19
三.序列刪減突變株的選殖………………………………….….. 19
四.轉形作用與轉形株的篩選…………………………….…….. 22
五.序列刪減突變株的刪減及DNA序列之確定………….….…...22
六.接種試驗…………………………………………………….….24
七.轉錄子複製之偵測………………………………….………….25
八.定序樣品之製備………………………………………..……. 26
九.電腦輔助核苷酸序列分析…………………………..…….… 27
肆、實驗結果…………………………………………………...…28
伍、討論………………………………………………...…….… 34
陸、參考文獻…………………………..……………………..… 39
柒、圖表………………………………………………..………….65
陸、參考文獻
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