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  • 學位論文

紅龍果X病毒之特性分析、感染性選殖株構築與抗血清製備

Characterization, infectious clone construction and antiserum preparation of Pitaya virus X

指導教授 : 張雅君

摘要


紅龍果(pitaya)屬於仙人掌科(Cactaceae)三角柱屬(Hylocereus spp.)的攀緣性肉質植物,喜歡生長在高溫多雨的潮溼氣候,近年來在台灣成為新興且重要的熱帶果樹。根據研究報告顯示,Potexvirus屬的仙人掌病毒X (Cactus virus X, CVX)為第一個被報導可以感染紅龍果的病毒,並且廣泛存在於紅龍果植株中。在2006至2008年期間,本實驗室從陽明山紅龍果園分離並鑑定出三種potexvirus,除了CVX外,尚有在台灣未曾報導過的蟹爪蘭病毒X (Zygocactus virus X, ZVX),以及一種新發現的病毒,暫時命名為紅龍果病毒X (Pitaya virus X, PiVX)。但是之後發現我們所分離保存的PiVX病毒株P37有CVX的污染,因此利用紅藜(Chenopodium amaranticolor)將原本的病毒株重新進行三次單斑分離,並應用已建立的multiplex RT-PCR確定所獲得為單一病毒株,同樣稱其為PiVX-P37。此病毒分離株接種紅藜五到七天後會產生帶有黃暈的褪綠斑,之後轉為壞疽斑。 PiVX-P37的寄主測試結果顯示,雖然此病毒僅能感染紅藜、番杏(Tetragonia expansa)與羽狀雞冠花(Celosia argentea)的接種葉,但是可以系統性感染白藜(C. quinoa)及紅龍果(H. undatus)。由穿透式電顯觀察發現PiVX病毒顆粒為絲狀,長約450-500 nm,寬約12-13 nm。利用蛋白質電泳分析此病毒的外鞘蛋白,大小約24 kDa。為了進一步了解PiVX-P37的特性,我們將此病毒的全長cDNA選殖至具有CaMV 35S啟動子的載體,以建構p35S-PiVX選殖株。然後將質體DNA純化後直接接種至紅藜與白藜,以測試全長cDNA選殖株的感染力。所測試的七個全長cDNA選殖株中有五個具有生物活性,但感染力並不相同。將其中三個具感染力的選殖株全長序列與先前本實驗室獲得的PiVX-P37全長序列進行比對分析,序列相同度皆高於99%。PiVX-P37全長序列不含poly(A)共有6677個核苷酸,且基因體組成與potexvirus相同。將PiVX-P37與其他potexvirus進行序列比對,結果發現與其最相近的為Schlumbergera virus X (SVX),兩者的RdRp與CP核苷酸序列相同度分別為70%及69%,而胺基酸序列相同度分別為77%及81%。根據第八版ICTV 報告對Potexvirus屬病毒種的規定,PiVX-P37確定為一個可以感染紅龍果的新種。此外,我們也發現感染力較弱的p35S-PiVX-1選殖株具有和其他選殖株不同的序列。為研發有效的檢測工具,我們表現並純化PiVX-P37的重組外鞘蛋白作為抗原,完成抗血清之製備。以immunoblot與indirect-ELISA測試,證實PiVX抗血清可專一性地與PiVX-P37作用,並且對病毒的檢測具有高靈敏度。本研究不僅純化出感染紅龍果的新種病毒-PiVX,同時成功建構出具感染力的cDNA選殖株,提供一個感染仙人掌科植物的 potexvirus的良好研究材料,未來並可應用於病毒載體的開發。而所製備的PiVX抗血清,除了應用在學術研究上,也可以用來檢測田間紅龍果PiVX的存在情形。

關鍵字

紅龍果X病毒 紅龍果

並列摘要


Pitaya (Hylocereus spp.), a climbing succulent plant with delicious fruits in the family Cactaceae, is newly cultivated in Taiwan and becoming an important tropical fruit crop. According to the literatures, Cactus virus X (CVX), a widespread potexvirus, is the first reported virus infecting pitaya plants. From the previous field survey in the Yanmingshan orchard from 2006 to 2008, we isolated and identified three potexviruses from pitaya plants. These viruses included CVX, Zygocactus virus X (ZVX), first reported in Taiwan, and Pitaya virus X (PiVX), a new species of potexvirus. Before too long we discovered that the P37 isolate of PiVX was contaminated with CVX. As a result, the original PiVX-P37 sample was used for single lesion isolations on Chenopodium amaranticolor again. Finally, a pure virus isolate was obtained and confirmed by multiplex RT-PCR, and also named as PiVX-P37. This virus isolate produced chlorotic lesions with yellow halo which turned into necrotic lesions on the inoculated leaves of C. amaranticolor at 5-7 days post inoculation. In host range test, PiVX-P37 could locally infect C. amaranticolor, Tetragonia expansa and Celosia argentea, and systemically infected C. quinoa and Hylocereus undatus. The virions of PiVX are in filamentous shape with 450-500 x 12-13 nm by transmission electron microscopy, and its capsid protein is about 24-kDa by SDS-polyacrylamide gel electrophoresis analysis. To further study the properties of PiVX-P37, the full-length cDNA clone with a 35S promoter was constructed and its biological activity was tested by inoculating plasmid DNA to C. amaranticolor and C. quinoa. Five out of seven full-length cDNA clones were infectious on both tested plants, but their infectivities were different. Three of them were completely sequenced, and the sequence identities of these clones are over 99% compared to PiVX-P37. The full-length genome of PiVX-P37 contains 6677 nucleotides without poly(A) tail and five open reading frames. The genome organization of PiVX-P37 is similar to those of other potexviruses. According to sequence comparison of potexviruses, PiVX-P37 is most homologous to Schlumbergera virus X (SVX). The nucleotide sequence identities of RdRp and CP gene between PiVX-P37 and SVX are 70% and 69%, and the amino acid sequence identities are 77% and 81%, respectively. According to the species demarcating criteria of the genus Potexvirus in the 8th ICTV report, there is no doubt that PiVX should be a new species of pitaya-infecting potexvirus. We further noticed the p35S-PiVX-1 clone with weaker infectivity containing sequences different from those of other infectious clones. To develop an effective detection tool, we expressed and purified the recombinant CP of PiVX as the antigen to generate the antiserum. This PiVX antiserum could specifically and sensitively react to PiVX in immunoblot and indirect-ELISA assays. In this study, a new pitaya-infecting potexvirus, PiVX, was purified, and the infectious cDNA clones were successfully constructed. These results provided a new material for studying the cactus-infecting potexviruses and for developing virus-based vector in the future. The anti-PiVX antiserum will be useful in research as well as in disease survey of pitaya.

並列關鍵字

Pitaya virus X pitaya

參考文獻


Liou, M. R., Hong, C. L., and Liou, R. F. 2004. Charaterization of a Cactus virus X infecting Hylocereus undatus and its detection by DAS-ELISA. Plant Patholo. Bull. 13:27-34. (In chinese)
Huang, W. Z., Chung, F. C., Chen, Y. L., Huang, C. H., and Chang, Y. C. 2005. Development and comparision of three detection methods for calla lily-infecting Dasheen mosaic virus. Plant Pathol. Bull. 14:239-250. (In chinese)
Liao, J. Y., Chang, C. A., Yan, C. R., and Deng, T. C. 2003. Detection and incidence of Cactus virus X on pitaya in Taiwan. Plant Pathol. Bull. 12:225-234. (In chinese)
Anderson, E. F., Barthlott, W., and Brown, R. 2001. The Cactus Family Timber Press, Inc., Protland Oregon, USA.
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