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研究生:林偉志
論文名稱:喜姆比蘭嵌紋病毒台灣分離株基因序列譯讀與分析
論文名稱(外文):Molecular sequencing and analysis of the viral genome
指導教授:王惠亮
指導教授(外文):Hui-Liang Wang
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:110
中文關鍵詞:喜姆比蘭嵌紋病毒
外文關鍵詞:Cymbidium mosaic virus
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喜姆比蘭嵌紋病毒( Cymbidium mosaic virus;CyMV ),是感染蘭科植物最重要的病毒,在分類上屬於馬鈴薯X群(Potexvirus genus),是一種長絲狀的病毒,其顆粒大小約為13 X 490 nm,在地理的分佈上遍佈溫帶及熱帶地區的非洲、亞洲、澳洲、歐洲、北美及南美洲。傳統的方法是以寄主植物範圍與病徵表現進行CyMV的鑑定與分類,但其分類過程易受到環境和人為因素干擾,結果常有無法周延的遺憾,因此希望透過分子生物學的層面來探討不同系統間的差異。本實驗的目的在進行譯讀喜姆比蘭嵌紋病毒台灣系統的基因序列,並與已解讀完成之CyMV-K2 (韓國)及CyMV-S2(新加坡)兩個系統作近全長度核苷酸和胺基酸序列比對與分析。經譯讀後得知CyMV-TW近全長度核苷酸序列共定序完成6187個核苷酸,其中RdRp(RNA-dependent RNA polymerase)基因共4254個核苷酸,可轉譯成1417個胺基酸,分子量約為159,956 Da、TGB1(triple-gene-block 1)基因共690個核苷酸,可轉譯成229個胺基酸,分子量約為25,863 Da、TGB2(triple-gene-block 2)基因共339個核苷酸,可轉譯成112個胺基酸,分子量約為12,474 Da、TGB3(triple-gene-block 3)基因共276個核苷酸,可轉譯成91個胺基酸,分子量約為9,682 Da、CP(coat protein)基因共672個核苷酸,可轉譯成223個胺基酸,分子量約為 23,808 Da、3'' 端非轉譯區定序完成55個核苷酸;5'' 端非轉譯區定序完成49個核苷酸。經由核苷酸序列相同度及胺基酸序列相似度比對結果顯示,三種系統間各基因的比較結果都達到90 ﹪以上,顯示三者為同種病毒之不同系統。綜合來說,喜姆比蘭嵌紋病毒各系統間的多重核苷酸或胺基酸序列比對皆展現了高同源性,種內的遺傳歧異度相當低。但是泰國與法國系統的CP基因核苷酸序列,和上述三個系統和馬來西亞系統之比較只有49∼53﹪相同度,因此可能不是CyMV病毒,而且其核苷酸序列無法轉譯成完整胺基酸序列,故推測其報告之序列可能是不正確的。在親緣關係上,CyMV-TW與CyMV-S1系統有較接近的親緣關係。此喜姆比蘭嵌紋病毒各系統之序列比對分析資料期能應用在抗病毒基因轉殖植物之構築,以有效防治田間病害。
Cymbidium mosaic virus ( CyMV ) is a member of Potexvirus genus in the family Potexviridae. It is an important virus infecting orchid plants and occurring worldwide. The conventional methods including host range, symptomatology and serology are not able to identify and classify the CyMV strains. Therefore we tried to use molecular biology method to differentiate CyMV strains. The purpose of this study was to sequence the genomic regions of CyMV Taiwan strain(CyMV-TW), and compare the nucleotide and amino acid sequences with those of two reported CyMV strains, CyMV-K2 ( from Korea ) and CyMV-S1 ( from Singapore ). As the result of sequencing, the nearly full length nucleotides of CyMV-TW were 6187. The nucleotide and amino acid sequences for RdRp(RNA-dependent RNA polymerase) gene were 4254 and 1417, respectively, which encoded a protein of MW of 159,956 Da representing RNA-dependent RNA polymerase; for TGB1(triple-gene-block 1)gene were 690 and 229, respectively, which encoded a protein of MW of 25,863 Da representing movement protein 1; for TGB2(triple-gene-block 2)gene were 339 and 112, respectively, which encoded a protein of MW of 12,474 Da representing movement protein 2; for TGB3(triple-gene-block 3)gene were 276 and 91, respectively, which encoded a protein of MW of 9,682 Da representing movement protein 3; for CP(coat protein)gene were 672 and 223, respectively, which encoded a protein of MW of 23,808 Da representing coat protein. The nucleotide sequences of 5'' and 3'' non-translated region were 55 and 49, respectively. As the results of comparison of nucleotide and amino acid sequences, all of three CyMV strains, CyMV-TW, CyMV-S1 and CyMV-K2, showed over 90 ﹪identity and similarity which proved that they are different strains of the same virus. In conclusion, the comparison of multiple alignments of nucleotide and amino acid sequences among CyMV strains displayed high identities in intraspecies. However, the nucleotide sequences of CP gene of Thailand and France strains had only 49~53﹪identities to other known strains of CyMV. Therefore, they may not be the same virus of CyMV. Furthermore the reported nucleotide sequences of CP gene of Thailand and France strains could not be translated into a complete amino acid sequence. In the phylogenetic analysis, CyMV-TW strain had a closer relationship with CyMV-S1. It is expected that the result of this study can help in the construction of the antivirus transgenic plants for controlling CyMV in the future.
中文摘要…………………………………………………… I
英文摘要.………………………………………………….. III
壹、前言及前人研究……………………………………….. 1
一、蘭花及其發生之病害...…………………………….. 1
二、喜姆比蘭嵌紋病毒之特性….………………………. 3
三、喜姆比蘭嵌紋病毒基因之組成.………………….…. 4
四、植物病毒鑑定與病害防治..…………………………. 5
貳、材料與方法………………………………………….….. 8
一、喜姆比蘭嵌紋病毒之來…………………….….. 8
二、病毒核酸萃取與電泳分析.………..……….... 8
(一)病毒核糖核酸之萃取…………………….. 8
(二)病毒核酸之電泳膠體分析…………..…… 9三、病毒基因核酸序列之分析與比……..…….… 9
(一)核酸引子之設計……… ………………. 10
(二)反轉錄聚合酶連鎖反應(RT-PCR)…… 22
(三)反轉錄聚合酶連鎖反應產物之電泳膠體分.. ……………………………………… 24
(四)喜姆比蘭嵌紋病毒台灣系統基因核酸序列之譯讀分析………………………………… 24
(五)喜姆比蘭嵌紋病毒台灣系統基因核酸序列之比對……………………….……………... 24
參、結果……………………………….………….……… . 26
一、病毒核酸萃取與電泳分析…………………………. . 26
二、病毒基因核酸序列之分析與比對….…………..…… 26
(一)反轉錄聚合酶連鎖反應產物之電泳膠體分析…. 26
(二)喜姆比蘭嵌紋病毒台灣系統基因核酸序列之譯讀分析………………………………………… 30
(三)喜姆比蘭嵌紋病毒台灣系統基因序列與國外已發表同種病毒之比較..…………………………. 50
肆、討論..……..……………………………………….… 98
伍、參考文獻………………………………………….. 103
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