臺灣博碩士論文加值系統

蕪菁嵌紋病毒(turnip mosaic virus；簡稱TuMV)，寄主植物在受到其感染後會產生斑駁、嵌紋、葉子捲曲、矮化等病徵。而TuMV之病毒顆粒為長絲狀，長度約720 nm屬於potyvirus群病毒，可經由機械傳播及蚜蟲以非永續性的方式傳播，但不經由種子傳播。本病毒可以感染很多重要的經濟作物，對於十字花科植物危害最為嚴重，如：白菜、蘿蔔、芥菜最重要的病毒，更造成產量損失。所以，被認為是世界上感染十字花科最重要的病毒之一。根據Provvidenti所提出分類方法，將TuMV區分為C1、C2、C3、C4四種系統，但Green和Deng發現在台灣另有一系統訂為C5系統；至於TuMV-TW係由台灣芥菜田篩選到的另一系統。將六個系統病葉萃出液經PEG沈降及硫酸銫等密度離心後可得純化之病毒顆粒，病毒RNA萃取後經電泳顯示其長度均約10 Kb。而蛋白質電泳顯示六個系統之鞘蛋白質分子量約為37 Kda，此六種抗原在和TuMV-TW抗血清反應後，於西方轉漬反應中TuMV-TW 抗血清均可和此六種抗原反應。進一步以反轉錄聚合連鎖反應選殖六個系統之鞘蛋白質基因，解出其核酸序列，並比較六者之鞘蛋白質基因核酸、胺基酸與3'' 端非轉譯區核酸之相同度顯示TuMV-C2與TuMV-C3二個系統關係較相近，而TuMV-C5 與TuMV-TW二個系統關係較相近，且彼此間差異性不大。再比較這六個系統與國外發表之CPP（中國大陸），CAPP（加拿大），CQS、NIAP（韓國），CP、CPRN、MUT（英國）和JAP（日本）等TuMV系統在鞘蛋白質基因之核酸、胺基酸與3''端非轉譯區核酸相同度上做比較，發現TuMV-C5、TuMV-TW與韓國發表之NIAP系統較為接近。由於此六個TuMV系統在血清學上不易區分，為了發展有效對抗TuMV的控制策略和分子診斷技術，分析此一病毒的遺傳差異性是有其必要性。同樣以反轉錄聚合連鎖反應選殖TuMV-C1和TW系統之鞘蛋白質上游基因，包括：細胞核內含體b蛋白質（NIb）基因，解出其核酸序列後，和國外發表之CAPP（加拿大），NIAP（韓國）和JAP（日本）等TuMV系統在NIb基因之核酸及胺基酸相同度上做比較，發現TuMV-TW系統與日本發表之JAP系統較為接近；細胞核內含體a蛋白質（NIa）基因，解出其核酸序列後，和國外發表之CAPP（加拿大），TMU18654（韓國）和JAP（日本）等TuMV系統在NIa基因之核酸及胺基酸相同度上做比較，發現TuMV-TW系統與韓國發表之TMU18654系統較為接近；與病毒基因組結合之蛋白質（VPg）基因，解出其核酸序列後，和國外發表之CAPP（加拿大）和JAP（日本）TuMV系統在VPg基因之核酸及胺基酸相同度上做比較，發現TuMV-TW系統與日本發表之JAP系統較為接近；以TuMV-C1系統之6K2基因與國外發表之CAPP（加拿大）和JAP（日本）TuMV系統在6K2基因之核酸及胺基酸相同度上做比較，發現TuMV-C1系統與日本發表之JAP系統較為接近；以TuMV-C1系統之CI基因與國外發表之CAPP（加拿大）和JAP（日本）TuMV系統在CI基因之核酸及胺基酸相同度上做比較，發現TuMV-C1系統與日本發表之JAP系統較為接近。

TuMV is a member of the potyvirus group, with a flexuous, rod-shaped particle of 720 nm in length. It is transmitted by mechanical inoculation and several species of aphids in a non-persistent manner but not through seeds. TuMV infects many economically important crops, and is one of the most important viruses in the world affecting Brassica species including Chinse cabbage, radish and smooth-leaf mustard, it includes mottling, mosaic, leaf distortion and stunting in infected plants. Provvidenti discribed four strains, C1、C2、C3、C4 of TuMV based on Chinese cabbage differerntial varieties. Green and Deng reported a fifth strain, C5, in Taiwan. The TuMV-TW strain was isolated from a mustard field in Taiwan. Virons of six TuMV strains were purified from infected leaves of mustard by polyethylene glycol precipitation followed by a Cs2SO4 isopycnic centrifugation. Electrophoresis revealed presence of a single RNA band about 10 Kb for each of six strains of TuMV. Relative molecular weight of coat proteins from six TuMV strains were about as 37 KDa. They reacted with an antiserum prepared to TuMV-TW in Western blot assay. The cDNAs of coat protein (CP) genes of the six TuMV strains were prepared by using reverse transcription polymerase chain reaction (RT-PCR). Analysis of nucleotide sequences and amino sequences of CP genes and 3'' non-coding regions were showed that the CP genes of TuMV-C2 and C3, and those of TuMV-C5 and TW had close relationships. The CP genes and 3'' non-coding regions of the six TuMV strains were also compared with other reported TuMV strains of CPP (from China), CAPP (from Canada), CQS、 NIAP (from Korea), CP、CPRN、 MUT (from UK) and JAP (from Japan). Results showed that the CP genes of TuMV-C5 and TW had close relationships with NIAP strain (from Korea). The six TuMV strains were serologically indistinguishable. The cDNAs of coat protein (CP) upstream genes including nuclear inclusion b protein ( NIb ), nuclear inclusion a protein ( NIa ), genome-linked viral protein ( VPg ), 6K2 protein ( p6K2 ) and cytoplasmic inclusion protein ( CI ) of TuMV strains, C1 and TW, were also prepared by RT-PCR. Nucleotide sequences and amino sequences of NIb genes of TuMV strains, C1 and TW were compared with other reported TuMV strains of CAPP (from Canada), NIAP (from Korea) and JAP (from Japan). Results showed that the NIb gene of TuMV-TW had a close relationship with that of JAP strain (from Japan). Comparsion of NIa genes of TuMV strains, C1 and TW were compared with other reported TuMV strains of CAPP (from Canada), TMU18654 (from Korea) and JAP (from Japan). Results indicated that the NIa gene of TuMV-TW had a close relationship with that of TMU18654 strain (from Korea). Comparsion of VPg genes showed that TuMV-TW had a close relationships with JAP strain (from Japan), whereas the p6K2 gene and CI gene of TuMV-C1 strain had close relationships with that of JAP strain (from Japan).

中文摘要……………………………………………………………… Ⅰ
英文摘要…………………………………………………………… Ⅲ
壹、前言……………………………………………………………… 1
貳、材料與方法……………………………………………………… 10
一、病毒純化與病毒核酸之萃取……………………………… 10
(一)、病毒來源…………………………………………… 10
(二)、病毒純化…………………………………………… 10
(三)、病毒核酸萃取及電泳分析………………………… 12
二、病毒鞘蛋白分子量測定及西方轉漬反應………………… 12
(一)、病毒鞘蛋白分子量測定…………………………… 12
(二)、西方轉漬反應分析 ( Western blotting )…………… 13
三、鞘蛋白質及其上游基因之定序與分析…………………… 14
(一)、反轉錄聚合酵素連鎖反應（RT－PCR）………… 14
(二)、RT－PCR產物之電泳膠體分析…………………… 18
(三)、回收cDNA片段…………………………………… 18
(四)、鞘蛋白質及其上游基因核酸序列之譯讀………… 19
(五)、核酸序列之分析…………………………………… 19
參、結果……………………………………………………………… 21
一、病毒純化與病毒核酸之萃取……………………………… 21
(一)、病毒純化…………………………………………… 21
(二)、病毒核酸萃取及電泳分析………………………… 22
二、病毒鞘蛋白分子量測定及西方轉漬反應………………… 22
(一)、病毒鞘蛋白分子量測定…………………………… 22
(二)、西方轉漬反應分析 ( Western blotting ) ………… 26
三、鞘蛋白及其上游基因之定序與分析…………………… 26
(一)、反轉錄聚合酵素連鎖反應（RT－PCR）………… 26
(二)、鞘蛋白質及其上游基因核酸序列之譯讀………… 29
(三)、不同系統鞘蛋白質核酸序列之比較……………… 36
(四)、六個TuMV系統與國外報告TuMV系統鞘蛋白質基
因序列之比較…………………………………… 43
(五)、TuMV-C1和TW系統與國外報告TuMV系統鞘蛋白
質上游基因序列之比較………………………… 51
肆、討論……………………………………………………………… 83
伍、參考文獻………………………………………………………… 90
陸、附錄……………………………………………………………… 95

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