台灣新興 Crinivirus 屬瓜類褪綠黃化病毒分子特性與診斷技術之研究

Molecular characterization and diagnosis of a new emerging Cucurbit chlorotic yellows virus in Taiwan

李如婷(Ju-Ting Li)

指導教授 ： 陳宗祺

亞洲大學/健康學院/生物科技學系碩士班/碩士(2009年)

摘要

Closteroviridae 科為正極性單股RNA長絲狀病毒，其下分為三個屬 (genera)，分別為 Closterovirus、Ampelovirus及Crinivirus。Crinivirus 與其他二病毒屬最大的不同在於其具有由兩條正極性單股 RNA 所組成之基因體，分別命名為 RNA1 及 RNA2，且其病毒顆粒較短，介於 650-900 nm × 12 nm 之間，是由粉蝨以半永續性的方式傳播。Crinivirus 危害全球許多重要的經濟作物，造成植株的葉片呈現黃色斑點、退綠黃化、甚至白化的病徵，導致收成量降低而造成嚴重的經濟損失。在 2009 年四月間，於台灣雲林縣崙背鄉的洋香瓜田中，發現洋香瓜上出現典型的 Crinivirus 病徵。採集洋香瓜的罹病組織並以針對 Closterovirus 及 Crinivirus 所設計之簡併性引子對進行巢式反轉錄-聚合酶鏈鎖反應 (nested reverse transcription-polymerase chain reaction, nested RT-PCR)，可增幅出 0.5 kb 的 DNA 片段，經定序及比對分析得知與日本所發現的新興病毒 Cucurbit chlorotic yellows virus (CCYV) 有 100 % 的相同度。本研究以粉蝨傳毒方式將新興病毒感染於菸草 (Nicotiana benthamiana) 中，以獲得一 crinivirus 分離株，命名為 Crinivirus-TW。將 Crinivirus-TW 的 RNA2 完全解序，可得其全長度有 8,041 個核苷酸，可對應產生八個蛋白，分別為 P5、Hsp70h、P6、P60、P9、CP、CPm 和 P26。RNA2的核苷酸序列與日本原始的CCYV分離株序列具有 99% 的相同度，因此 Crinivirus-TW 應為 CCYV 的分離株，並更名為 CCYV-TW。將所有已知 Crinivirus 病毒種的 Hsp70h、P60、CP、CPm 及 P26 等基因進行親緣關係比對，結果得知 CCYV-TW 與 Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) 及 Cucurbit yellow stunting disorder virus (CYSDV) 最為親近。利用 pET 細菌表現系統表現 CCYV-TW 的 CP 用以生產單株抗體 (monoclonal antibody)，所得之單株抗體可用於西方墨點法 (western blotting) 以進行田間之病毒檢測，並可應用於組織點漬免疫分析法 (tissue blot immunoassay, TBIA) 以檢視病毒於韌皮部細胞累積之情形。此外，設計自 Hsp70h 序列的引子對 Crini-hsp70h-f/Crini-hsp70-r 與設計自植物粒線體中的 NADH dehydrogenase (nad5) 基因之引子對 mt-F2/mtR1 共同使用所開發的 multiplex one-step RT-PCR，為相當有效率的檢測方法，可應用於田間 CCYV 之檢測。以此方法進行田間調查，2010 年 2-6 月間，自台灣中部地區共採集 253 個罹病洋香瓜樣本，檢測結果發現，病毒之檢出率從 3.8% 上升至 100%。本調查結果顯示 CCYV 已成為台灣瓜類作物生產上的一大重要限制因子，值得政府單位密切注意。

關鍵字

長線型病毒科 ； 瓜類黃化褪綠病毒 ； 單株抗體

並列摘要

Viruses of the family Closteroviridae have large positive-sense single-stranded RNA genomes encapsidated in long flexuous virions and are divided into three genera, Closterovirus, Ampelovirus and Crinivirus. Different from the other two genera, Crinivirus has bipartite particles of 650-900 × 12 nm comprising two genome segments, denoted RNA1 and RNA2, and is transmitted by whitefly. Criniviruses infect several important crops worldwide, causing symptoms of interveinal chlorosis, yellowing and brittleness of leaves accompanied by severe yield losses. In April 2009, typical crinivirus-induced symptoms were observed on melon (Cucumis melo L.) plants in Yunlin County, Taiwan. The degenerate primers designed for Closterovirus and Crinivirus were used in nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify a 0.5 kb DNA fragment from the symptomatic melon tissues. Subsequently, the amplified DNA fragment was determined to share 100% identity with the reported sequence of Cucurbit chlorotic yellows virus (CCYV), a new Crinivirus species found in Japan. In this study, an isolate of crinivirus denoted as Crinivirus-TW was obtained from a plant of Nicotiana benthamiana inoculated by whitefly transmission. Furthermore, the complete nucleotide (nt) sequence of Crinivirus-TW RNA2 was determined as 8,041 nt in length containing eight open reading frames (ORFs), P5, Hsp70h, P6, P60, P9, CP, CPm and P26. Sequence analyses revealed that the RNA2 sequence of Crinivirus-TW shares 99% nt identity with that of the original Japan isolate of CCYV. Thus, Crinivirus-TW was identified as an isolate of CCYV, renamed CCYV-TW. Phylogenetic analyses of the Hsp70h, P60, CP, CPm and P26 among Crinivirus species revealed that CCYV-TW is closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). The CP of CCYV-TW expressed by the bacterial pET expression system was used as an immunogen for production of monoclonal antibodies (MAbs). The MAbs prepared were successfully used to detect CCYV in diseased plants by western blotting and to examine the accumulation of virus in phloem cells by tissue blot immunoassay (TBIA). Moreover, primer pair Crini-hsp70h-f/Crini-hsp70-r, designed from the Hsp70h gene, coupled with primer pair mt-F2/mtR1, designed from the plant mitochondrial NADH dehydrogenase (nad5) gene, used in multiplex one-step RT-PCR was developed as an effective detection method and applied in field survey. A number of 253 diseased melon samples collected from central Taiwan during February to June in 2010 were tested by multiplex one-step RT-PCR. An increased incidence of 3.8% to 100% was noticed. Our results indicated that CCYV has become an important threat for the production of cucurbits in Taiwan, and it should be majorly concerned by government.

並列關鍵字

Closteroviridae ； Cucurbit chlorotic yellows virus ； Monoclonal antibodie

參考文獻

10. Chang, Y. M., Hsaio, C. H., Yang, W. Z., Hseu, S. H., Chao, Y. J., and Huang, C. H. 1987. The occurrence and distribution of five cucurbit virus on melon and watermelon in Taiwan. J. Agricul. Res. China. 36:389-397.

1. Agranovsky, A. A. 1996. Principles of molecular organization, expression and evolution of Closteroviruses: over the barriers. Adv. Virus Res. 47:119-158.

2. Agranovsky, A. A., Lesemann, D. E., Maiss, E., Hull, R., and Atabekov, J. G. 1995. "Rattlesnake" structure of a filamentous plant RNA virus built of two capsid proteins. Proc. Natl. Acad. Sci. USA. 92:2470-2473.

3. Alzhanova, D. V., Hagiwara, Y., Peremyslov, V. V., and Dolja, V. V. 2000. Genetic analysis of the cell-to-cell movement of beet yellows closterovirus. Virology 268:192-200.

4. Alzhanova, D.V., Napuli, A.J., Creamer, R., and Dolja, V.V. 2001. Cell-to-cell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog. EMBO J. 20, 6997-7007.