臺灣博碩士論文加值系統

紅龍果(Hylocereus spp.)原產熱帶美洲雨林，屬仙人掌科三角柱屬多年生攀緣性肉質植物，喜高溫多濕之氣候，目前全台栽培面積近1000公頃，產區主要集中在中南部，為國內重要的新興水果。本研究於2005年利用仙人掌病毒X (Cactus virus X, CVX)抗血清，對台大園藝分場種植的紅龍果以間接酵素連結抗體免疫吸附反應(indirect-ELISA)進行檢測，發現園區紅龍果CVX感染率高達68%。進一步由罹病紅龍果植株抽取病毒顆粒，接種到指示植物白藜（Chenopodium quinoa）和紅藜（C. amaranticolor），發現其病徵與先前在台灣所研究的CVX分離株(CVX-Hu)不相同，懷疑此病毒可能為一個新的CVX系統（strain）。因此將病毒顆粒接種至紅藜做三次單斑分離後獲得一分離株稱為NTU，遂進行此病毒株的生物與分子特性研究。由指示植物接種實驗結果可知，NTU除了白藜和紅藜外尚可感染千日紅(Gomphrena globosa)、羽狀雞冠花（Celosia argentea）、洋麻(Hibiscus cannabinus)、菸草(Nicotiana tabacum var. White burley)與蕃茄(Lycopersicon esculentum）。以西方墨點法分析NTU的鞘蛋白，其大小為25 kDa，與CVX-Hu分子量接近。以紅龍果果實切面，進行組織轉印免疫分析法，顯示病毒於整顆果實皆有分佈。利用CVX-Hu序列，設計專一性引子對，成功增幅NTU病毒全長序列並選殖。當進行核酸解序之後，再分別設計NTU的專一性引子對，以RACE和RT-PCR分別增幅5’與3’的非轉譯區片段，並選殖定序，以確定NTU兩端的序列。當完成NTU全長度核酸序列的解序工作後，將NTU的各個基因轉譯成胺基酸序列，與已發表的CVX-Hu胺基酸序列作比對，發現兩者的RdRP與CP相同度分別達85%和86%，故認定為一新發現之CVX分離株，並取名為CVX-NTU。將CVX-NTU病毒分離株各個ORF與其它已知序列的同屬病毒作胺基酸序列比對及類緣分析，結果顯示感染仙人掌科的potexvirus都屬同一個分群中，顯示彼此的類緣關係較密切。另一方面為了解CVX-Hu與CVX-NTU兩種不同分離株在紅龍果分佈的情形，利用ELISA及one tube multiplex RT-PCR對陽明山觀光果園種植的紅龍果進行檢測調查，結果顯示CVX-NTU感染較CVX-Hu普遍，前者高達95%，而後者佔40%且都是複合感染。除此之外，意外發現檢測樣品中有台灣尚未發現過的病毒Zygocactus virus X (ZVX)，且感染率達50%，顯示台灣紅龍果廣受病毒侵染，並且多數為多種病毒複合感染。此研究的另一部份是將CVX-NTU建構成一個具有感染力的病毒選殖株，藉由帶有T7啟動子的病毒全長序列選殖株，產生RNA轉錄體，然後接種於白藜原生質體和植株中，並以北方氏雜合反應分析其感染力，以篩選出具有感染力的選殖株，此部份篩選實驗尚在進行中。未來希望能將CVX發展成可供多方面應用的病毒載體，使其應用層次更多元化。

Pitaya (Hylocereus spp.) is a perennial herbaceous plant in the family Cactaceae originated from the rainforest in tropic America. In Taiwan, there are about 1000-ha pitaya plantation mainly in the middle and southwestern areas. In 2005, we surveyed the pitayas grown in the experimental farm at National Taiwan University by indirect enzyme-linked immunosorbent assay (ELISA) using the polyclonal antiserum specific against Cactus virus X (CVX). The result indicated that up to 68% of tested samples were infected by CVX. Virus particles were purified from infected pitaya and then inoculated to Chenopodium quinoa and C. amaranticolor. The symptoms of the infected plants were different from those caused by CVX-Hu, a previous studied virus isolate in Taiwan. This result suggested there may be a new strain of CVX. To further characterize the biological and molecular features, a virus isolate named NTU was obtained after three successive single lesion isolations in C. amaranticolor. Host range and size of capsid protein of NTU isolate were determined. Virus distribution in pitaya fruit was also studied by tissue blot immunoassay. Specific primers designed according to CVX-Hu sequence were used to amplify the full-length genome of NTU isolate by RT-PCR. To determine the sequences of 5’ and 3’ ends of NTU isolate, RACE and RT-PCR were performed, respectively. When the complete genomic sequence of NTU isolate was obtained and compared with that of CVX-Hu, the amino acid sequence identities of RdRp and CP genes are 85% and 86%, respectively. Therefore, NTU isolate is considered as a new CVX isolate and thus designated as CVX-NTU. Multiple sequence alignment and phylogenetic analysis of CVX-NTU and other completely sequenced potexviruses demonstrated that CVX-NTU has a close relationship with potexviruses infecting plants in the family Cactaceae. To determine the infection frequency of CVX-NTU and CVX-Hu in pitaya, samples collected from Yangmingshan orchard were detected by ELISA and one tube multiplex RT-PCR. Up to 95% of tested samples were infected with CVX-NTU and 35% contained CVX-Hu. In addition, an previously unreported virus, Zygocactus virus X (ZVX), was detected in 50% of collected samples. The results clearly showed pitaya plants are prevalently infected by potexviruses in Taiwan. Another part of the thesis is to construct full-length cDNA clones of CVX. RNA transcripts were synthesized in vitro under the control of T7 promoter at the CVX cDNA clones. Biological activities of the clones were tested by protoplast and plant inoculation assays in C. quinoa. The screening experiment of infectious cDNA clones of CVX is still going on. Hopefully, a CVX-based vector can be developed and applied to many research areas in the future.

中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………3
壹、前言.………………………………………………………………………………5
貳、前人研究…………………………………………………………………………7
叄、材料與方法………………………………………………………………………11
一、病毒的來源與分離……………………………………………………………11
二、生物特性分析…………………………………………………………………11
（一）寄主範圍測試……………………………………………………………11
（二）病毒顆粒純化……………………………………………………………11
（三）病毒鞘蛋白分析…………………………………………………………12
（四）植物組織轉印法…………………………………………………………13
三、分子特性分析………………………………………………………………14
（一）病毒全長核酸序列之增幅和選殖……………………………………14
（二）病毒基因體5’及3’UTR之選殖定序…………………………………17
（三）序列比對及分子類緣關係分析（phylogenetic analysis）……………18
四、紅龍果受仙人掌病毒感染之田間調查……………………………………19
（一）間接酵素聯結免疫反應(indirect-ELISA)………………………………19
（二）單管反轉錄聚合酶連鎖反應(one tube multiplex RT-PCR) ………………19
五、具感染力病毒株之篩選……………………………………………………20
（一）生體外轉錄病毒RNA…………………………………………………20
（二）白藜原生質體製備與接種………………………………………………21
（三）北方氏雜合分析……………………………………………………23
肆、結果………………………………………………………………………………25
伍、討論………………………………………………………………………………33
陸、參考文獻…………………………………………………………………………38
柒、表…………………………………………………………………………………43
捌、圖…………………………………………………………………………………48
附錄…………………………………………………………………………………74

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