洪挺軒Hung, Ting-Hsuan臺灣大學:植物病理與微生物學研究所劉亭君Liu, Ting-ChungTing-ChungLiu2010-05-112018-06-292010-05-112018-06-292008U0001-2307200818310200http://ntur.lib.ntu.edu.tw//handle/246246/181922中文摘要瓜輪點病是木瓜的重要病害之一。台灣於 1975 年發現此病害後，即迅速傳播至全省木瓜產地，嚴重影響台灣的木瓜產業。此病由馬鈴薯Y病毒屬（Potyvirus）的木瓜輪點病毒（Papaya ringspot virus, PRSV）所引起。依據在木瓜葉片上所引起的病徵不同，PRSV可分成嚴重嵌紋系統（severe mottling, SM strain）、畸形系統（deformation, DF strain）以及嚴重嵌紋壞疽系統（severe mottling with necrosis, SMN strain）。過去台灣木瓜栽植區所觀察到的病徵以 SM 系統為主，但近年來台灣田間木瓜輪點病逐漸被 DF 與 SMN 系統取代，而且以DF系統更是普遍。此三系統經由全長定序後發現各基因核苷酸序列相似度大多在 95～100％ 間，只有 P1 基因低於 95％，而5’ UTR（untranslated region）僅 91.8％～92.9％。為了瞭解此三系統病徵差異與核酸序列上的關係，本論文即先以目前田間最優勢的DF系統為材料構築出 PRSV 的全長 cDNA 感染性選殖株(infectious clones)，再用胞外轉錄(in vitro transcription)方式合成純系、完整的病毒基因體RNA，接種到木瓜寄主使其產生DF系統的病徵，一方面藉以確定基因體與病徵之間的關係，另一方面也為將來PRSV基因重組試驗奠立基礎，期望找出病徵決定基因。本論文將全長10326個核苷酸的PRSV基因體分成5個大小不同、頭尾重疊（overlapping）的區域，進行反轉錄聚合酶鏈鎖反應(RT-PCR)，增幅的cDNA片段配合pGEM-T質體做選殖，獲得5個選殖株。隨後再分別進行聚合酶鏈鎖反應(PCR)增幅，各片段以限制酶剪接合成全長，最後得到5’端帶有 T3 promoter 的全長度 cDNA質體株 (pT3-NP)。以 XbaI 將 pT3-NP 切割成線狀模版後，進行胞外轉錄，可得到約 2500~3000 ng/μl 之 病毒RNA。以機械接種後約三週可用RT-PCR檢測到PRSV已成功感染部分木瓜植株，感染率為為 25～33％，增幅的片段定序結果確認這些木瓜植株體內的PRSV來自感染性選殖株無誤。觀察發現木瓜感染後已經開始出現葉片畸形、扭曲，葉面縮小的病徵，但尚未出現嚴重的葉片嵌紋。由RT-PCR的持續追蹤也證實受感染的植株體內PRSV有增加的趨勢，表示感染性選殖株的構築已獲得初步成功，唯病徵的進展仍需持續追蹤。Abstract apaya ringspot is one of the most destructive diseases of papaya. This disease invaded Taiwan in 1975, and immediately spread all over the papaya-cultivated areas in Taiwan. This disease is the greatest impact for the papaya industry. It is caused by Papaya ringspot virus（PRSV）,which is the member of the genus Potyvirus. Based on the differences of foliar symptoms, PRSV can be categorized into three main strains such as the severe mottling（SM）, deformation（DF）and severe mottling with necrosis（SMN）strains. The SM strain was ever absolutely predominant in Taiwan, but the DF and SMN have recently become newly rising dominant strains. The alignment of nucleotide sequences reported that three strains are very close with around 97% homology. Their individual genes within the genome also show high homology (95 ~100％) except of the P1 gene (94.5~94.8％) and 5’ UTR（91.8％ ~92.9％）. To facilitate the study of the relation between the symptoms and the nucleotide sequences, this thesis was dedicated to construct the full-length infectious clones to synthesize of PRSV genomic RNA through in vitro transcription for artificial inoculation tests. The DF strain was first chosen as the template for amplification and cloning of several genomic fragments with reverse-transcription polymerase chain reaction (RT-PCR) and the TA-cloning kit (pGEM-T vector). These fragments were further amplified with polymerase chain reaction (PCR), digested with restriction enzymes and assembled into a complete cDNA clone (pT3-NP) of PRSV genome with T4-DNA ligase, The cDNA clone was cut with the XbaI restriction enzyme into a linear form and in vitro transcription was performed. The RNA products were collected (2500~3000 ng/μl) and used in the inoculation tests to confirm their infectivity. PRSV was monitored in several infected papaya plants with RT-PCR assays and the infectious percentage was 25～33％. Sequencing of the RT-PCR products demonstrated these viruses derived from the pT3-NP clone. The leaf-deformation symptoms were observed in the infected papayas 3 weeks after inoculation. The continuous monitoring of PRSV with RT-PCR showed that PRSV was replicating in the hosts, which confirm that the transcripts from pT3-NP are infectious. However, the apparent and typical symptoms of PRSV-DF train such as mottling, green islands and fern-leaf have not been observed so far.目錄委審定書………………………………………………………………………........i謝…………………………………………………………………………………....ii文摘要……………………………………………………………………………...iii文摘要……………………………………………………………………………...iv、 前言………………………………………………………………………………1、 前人研究…………………………………………………………………………4、 材料與方法……………………………………………………………………..13、 試驗植物來源……………………………………………………………..13、 PRSV系統來源與保存…………………………………………………...13、 構築PRSV DF系統之感染性選殖株…………………………….………14. 病毒核酸萃取…………………………………………….…….14. PRSV核苷酸序列之限制酶圖譜分析與專一性引子對之設計………………………………………………………………..14. Two-step RT-PCR增幅反應……………………………………15. PCR產物的純化………………………………………………..15. PCR產物之選殖………………………………………………..16. 限制酶酵解……………………….…………………………….17、 胞外轉錄……………………………………...…………………………...17、 接種試驗…………………………………………………………………..17、 檢測造成感染之系統……………………………………………………..18、 結果……………………………………………………………………………..19、 反轉錄聚合酶連鎖反應進行PRSV 核酸片段增幅與選殖……………………………………………………………………………19、 木瓜輪點病毒基因全長cDNA選殖株之合成………………………....…19. PRSV 全長 cDNA clone 的建立…………………………….…19. 胞外轉錄in vitro transcription……………………………….....20、 胞外轉錄的RNA之感染力試驗……………..………………………..…20、 新的 PRSV-DF 系統專一性引子對設計………………………………..21、 以PRSV系統專一性引子對搭配RT-PCR檢測受感染的植株……………………………………………………………………….….21、 檢測造成感染之系統………………………………………………….….21、 討論……………………………………………………………………….…….22、 參考文獻…………………………………………………………………….….28、 表…………………………………………………………………… ……….…36、 圖…………………………………………………………………………….….37、 附錄……………………………………………………………………………..53application/pdf2884524 bytesapplication/pdfen-US木瓜木瓜輪點病毒感染性選殖株胞外轉錄papayaPapaya ringspot virusinfectious clonein vitro transcripthttp://ntur.lib.ntu.edu.tw/bitstream/246246/181922/1/ntu-97-R95633002-1.pdf