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    Please use this identifier to cite or link to this item: http://ir.nknu.edu.tw/ir/handle/987654321/15954


    題名: 蕪菁嵌紋病毒TW和C1系統生體外具感染力轉錄體之構築
    Construction of in vitro infectious transcripts of Turnip mosaic virus TW and C1 strains
    Authors: 曾君豪
    Chun-Hao Tseng
    貢獻者: 王惠亮
    Hui-Liang Wang
    Keywords: 蕪菁嵌紋病毒;轉錄體
    TuMV;transcripts
    Date: 2002-06-18
    Issue Date: 2011-10-04 15:10:40 (UTC+8)
    Abstract: 蕪菁嵌紋病毒(Turnip mosaic virus;TuMV),屬於Potyviridae科Potyvirus屬,是感染十字花科最重要的病毒,在地理的分佈上十分的廣泛,遍佈溫帶及熱帶地區的非洲、亞洲、澳洲、歐洲、北美及南美洲。目前蕪菁嵌紋病毒的分類是根據Provvidenti所提出的方法,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。本實驗室前人吳孟芳已完成TuMV-TW和C1的鞘蛋白基因解序,而曹佳莉則完成了TuMV-TW和C1其餘序列的譯讀,因此TuMV-TW和C1的全長序列均已經解序完成。本實驗以MMLV反轉錄?分別合成全長的TuMV-TW和C1第一股cDNA,接著分別設計TuMV-TW和C1的核酸引子,5''端引子的設計包含有T7啟動子和病毒體5''端部分的序列;3''端引子的設計則是含有poly-T以及限制?辨識的序列。再以這一組設計的引子來進行病毒體全長cDNA的PCR反應,所構築出的cDNA就會含有T7啟動子辨識序列,所以接下來就可以利用T7 RNA polymerase來進行生體外的轉錄,轉錄出來的病毒轉錄體經過純化和確認過後,接種至MT1品種的芥菜上,經過14和24天後,摘取葉子進行酵素結合抗體檢定法,以確認病毒轉錄體是否具有感染成功。接種含有cap構造TuMV-TW轉錄體的植株經過大約24天後其外觀上有嵌紋狀的病徵產生,而接種未含有cap構造之TuMV-TW轉錄體的植株則沒有病徵的出現;在TuMV-C1方面,則是無論接種有沒有含有cap構造之TuMV-C1轉錄體,其植株均無病徵出現。在吸光值檢測上,經由比對發現含有cap構造之TuMV-TW轉錄體無論14天或24天的吸光值為未接種植株吸光值的兩倍以上,而不含有cap構造之TuMV-TW轉錄體和TuMV-C1的轉錄體之吸光值和未接種植株吸光值比較則沒有顯著之差異;在另一方面,萃取含有cap構造之TuMV-TW轉錄體接種植株之RNA,進行RT-PCR後可以觀察到有10 kb的片段存在,同樣的片段在TuMV-TW病毒體感染的植株之RNA經RT-PCR也可以觀察到,因此可以評估所合成的TuMV-TW生體外具感染性病毒轉錄體的確具有感染性。
    Turnip mosaic virus(TuMV), a member of Potyvirus genus in Potyviridae family, is one of the most important viruses infecting cruciferous crops, and spreads worldwide including Africa, Asia, Australia, Europe, and North and South America. TuMVs are classified into four strains - C1, C2, C3, and C4 according to various symptoms developed on different Chinese cabbages varieties. A fifth strain, C5, and another special strain, TW, were reported in Taiwan. TuMV-TW and C1 were sequenced in our laboratory in 2001. In this study, modified Moloney Murine Leukemia Virus reverse transcriptase was used to synthesize the full-length first strand cDNAs of TuMV-TW and C1. The 5'' terminal primer was designed to include a T7 promoter sequence and partial viral 5'' NTR sequence. The 3'' terminal primer was designed to include a 14-T tail and a restriction enzyme cutting site. Both primers were used to synthesize full-length double-stranded viral cDNA by PCR. With the completion of the full-length dsDNA, TuMV-TW and C1 transcripts were obtained by in vitro transcription with T7 RNA polymerase. Purificated transcripts were inoculated onto mustard MT1 and confirmed by ELISA 14 and 24 days after inoculation. Mottlings were observed on mustard inoculated leaves with capped TuMV-TW transcripts at day 24. No symptom was observed for TuMV-TW transcripts without cap and TuMV-C1 transcripts with or without cap. ELISA test showed positive reaction on plant 14 and 24 days after inoculation with TuMV-TW transcripts with cap. A 10kb RT-PCR product was confirmed from plants infected with TuMV-TW transcripts with cap.
    Appears in Collections:[生物科技學系] 博碩士論文
    [生科系] 王惠亮

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