蘿蔔嵌紋病毒台灣分離株之特性分析

Characterization of the Taiwan Isolate of ＂Radish mosaic virus＂

王淨竹(C. C. Wang) ； 張怡珊(Y. S. Chang) ； 陳煜焜(Y. K. Chen)

《植物病理學會刊》 23卷2期 (2014/06) Pp. 139-158

https://doi.org/10.6649/PPB.201406_23(2).0005

摘要

苗栗大湖地區採集得疑受病毒感染而在葉片上有嚴重嵌紋病徵之蘿蔔病株，電子顯微鏡鏡檢可觀察到直徑約30 nm的球形病毒顆粒。以病組織粗汁液機械接種於奎藜（Chenopodium quinoa）進行純系分離，並以機械接種方式將單離的病毒株回接至健康蘿蔔苗，能在蘿蔔引起葉片嵌紋、變形及畸脈贅生（enation）的病徵，証實該病毒對蘿蔔的病原性。以豆嵌病毒科（Comoviridae）的簡併式引子對進行半巢式反轉錄聚合酶鏈鎖反應（semi-nested RT-PCR）檢測，證實該病毒為蘿蔔嵌紋病毒的台灣分離株（Radish mosaic virus Taiwan isolate, RaMV-TW）。寄主範圍測試顯示RaMVTW能在8種十字花科作物上產生輪紋和嵌紋的病徵。純化之病毒樣本內含大量直徑約30 nm的球形病毒顆粒。蛋白質電泳（SDS-PAGE）分析顯示病毒具有兩種外鞘蛋白次單位（coat protein subunit），其分子量分別約為41 kDa及25 kDa。RaMV-TW基因體核酸RNA1含6041個核苷酸，具有單一開放讀架（ORF），轉譯出之複合大蛋白，能被protease裂解成5個病毒功能蛋白，由5'端依序分別為Co-factor、helicase、VPg、Protease 和RdRp。RNA2含4012個核苷酸，也具有單一開放讀架（ORF），但卻有兩個ATG起始碼（start codons），根據不同的ATG起始碼能轉譯出包含4個蛋白轉譯架的複合大蛋白，由5'端依序分別為CR/MP、LCP、及SCP。將RaMV-TW的RNA 1（Acce. No. HM032712）及RNA 2（Acce. No. HM032711）分別與已登錄的三個RaMV分離株及6個Comovirus屬病毒之全長基因體RNA1和RNA2核苷酸及蛋白胺基酸序列進行比對分析，結果顯示在三個RaMV分離株中，RaMV-TW和RaMV-CA與RaMV-J的親緣關係最為相近。

關鍵字

十字花科 ； 豆嵌病毒屬 ； 十字花科蔬菜 ； 蘿蔔嵌紋病毒

並列摘要

Radish plants (Raphanus sativus) with severe mosaic on leaves were observed in the fields of Dar- Hu, Miaoli. Spherical virus particles (ca. 30 nm in diameter) could be observed in the preparations of infected crude sap of radish by electron microscopy. Virus isolates obtained by carrying out single lesion isolation on Chenopodium quinoa successively and was back-inoculated to original host to confirm its pathogenicity. The isolated virus induced systemic symptoms of mosaic and enation on the leaves of backinoculated radish plants. Semi-nested RT-PCR tests using Comoviridae degenerate primers amplified a cDNA fragment of expected size which highly homologous to that of Radish mosaic virus (RaMV). The virus thus designated as Radish mosaic virus Taiwan isolate (RaMV-TW). Host range tests revealed that RaMV-TW caused symptoms of mosaic, rugose, enation and ringspot on various cruciferous crops. Purified virus preparations contained spherical virus particles with a diameter of about 30 nm. SDS-PAGE analysis indicated that the virus contains two coat protein subunits, a large coat protein (LCP) and a small coat protein (SCP), with relative molecular mass of about 41 kDa and 25 kDa, respectively. Full length sequences of RNA1 and RNA 2 of RaMV-TW have been determined and deposited in GenBank with accession numbers of HM032712 and HM032711, respectively. The RNA 1 contains 6041 nucleotides and encodes a polyprotein which is further cleaved into 5 functional proteins including protease cofactor (Cofactor), helicase (Hel) , viral genome-linked protein (VPg), protease (Pro), and RNA-dependent RNA polymerase (RdRp). The RNA2 consists of 4012 nucleotides and contains an open reading frame (ORF) with two ATG start codons. The polyprotein derived from RNA 2 can also be further cleaved into 4 functional proteins including cofactor required for the replication of RNA 2 (CR) , movement protein (MP), large coat protein (LCP), and small coat protein (SCP). Based on the nucleotide sequences revealed, RaMV-TW is more closely related to strains RaMV-CA and RaMV-J than to RaMV-1.

並列關鍵字

Brasicaceae ； Comovirus ； Cruciferae ； cruciferous vegetables ； Radish mosaic virus