蘿蔔嵌紋病毒台灣分離株 (RaMV-TW) 之分子特性分析

王淨竹

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蘿蔔嵌紋病毒台灣分離株 (RaMV-TW) 之分子特性分析

Molecular Characterization of the Radish mosaic virus Taiwan isolate (RaMV-TW)

王淨竹 , Masters　　Advisor：陳煜焜　　

繁體中文

十字花科作物；蘿蔔嵌紋病毒；全長度核酸基因體； Comovirus ； Cruciferous vegetables ； Radish mosaic virus ； RaMV ； Full length sequences ； Comovirus

Abstract │ Reference (88) │ Times Cited (2) │ Altmetrics

Abstract 〈TOP〉

台灣地區栽培的十字花科作物種類繁多，為重要的蔬菜之一，但普遍受病毒病為害。2007年於苗栗大湖地區採集得疑受病毒感染之蘿蔔植株，病株葉片上有嚴重之嵌紋(mosaic)病徵，以穿透式電子顯微鏡鏡檢罹病組織粗汁液，可觀察到直徑大小約30 nm的球形病毒顆粒。以罹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純系的病毒回接至健康蘿蔔苗，發現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。使用Maliogka等人(2004)所發表適用於Comoviridae的簡併式引子對，以半巢式反轉錄聚合酶鏈鎖反應(semi-nested RT-PCR)進行檢測，證實該病毒為蘿蔔嵌紋病毒(Radish mosaic virus, RaMV) ，遂以蘿蔔嵌紋病毒台灣分離株(Radish mosaic virus Taiwan isolate, RaMV-TW)為其代稱。寄主範圍測試顯示RaMV-TW能在台灣地區常見的8種十字花科作物上產生輪紋和嵌紋的病徵，感染嚴重者則會呈現葉片皺縮變形的徵狀。罹病葉片粗汁液經過淨化、PEG濃縮、以及40%蔗糖緩衝液為墊層(sucrose cushion)的超高速離心後，所得的部份純化之懸浮液內含大量直徑約30 nm的球形病毒顆粒。利用SDS-PAGE蛋白質電泳分析，顯示所純化的病毒其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。選取目前已發表的RaMV分離株，包括: RaMV-J (AB295643和AB295644 )、RaMV-CA (AB456531和AB456532)及RaMV1 (EU450837 和EU450838)之全長度核酸基因體RNA 1及RNA 2，利用CLUSTALW 程式進行比對後，針對序列較保守的區域設計各基因的專一性引子對進行RaMV-TW全長度基因體核酸序列 RNA1及RNA2之選殖與序列解讀。RaMV-TW基因體核酸RNA1已完成6041個核苷酸的解序，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已完成4012個核苷酸的解序，一樣具有單一開放讀架(ORF)，但RNA2含有兩個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基因體核酸全長序列 RNA1 (Acce. No. HM032712)及RNA2 (Acce. No. HM032711)分別與目前已在NCBI上登錄的三個RaMV分離株及6個Comovirus屬病毒(genus Comovirus)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。

Parallel Abstract 〈TOP〉

Cruciferous vegetables are one of the important crops in Taiwan and generally suffer from virus infections in the field. Radish plants (Raphanus sativus) with severe mosaic symptoms on leaves were observed in the fields of Dar-Hu, Miaoli in 2007. Spherical virus-like particles (ca. 30 nm in diameter) were observed in the preparations of infected crude saps by electron microscopic examination . Pure virus isolate obtained from the single lesion induced by mechanical inoculation on Chenopodium quinoa successively and was back-inoculated to original host to confirm its pathogenicity. The isolated virus induced systemic mosaic and enation symptoms on the leaves of back-inoculated radish plants. Semi-nested RT-PCR tests using Comoviridae degenerate primers ( Maliogka et al., 2004) amplified a cDNA fragment of expected size which highly homologous to the nucleotide sequence of Radish mosaic virus (RaMV). The isolated 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. Virus particles were partially purified through clarification, PEG concentration, and sedimented by ultracentrifugation in a 40% sucrose cushion.  The partially purified virus preparations contained numerous spherical virus particles with a diameter of about 30 nm. SDS-PAGE analysis on the partially purified sample indicating that the virus contains two coat protein subunits, i.e. large coat protein (LCP) and small coat protein (SCP), with relative molecular mass of 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 nt and encodes a polyprotein which is further cleaved into 5 funtional proteins including protease cofactor (Co-factor), helicase (Hel) , viral genome-linked protein (VPg), Protease (Pro), and RNA-dependent RNA polymerase (RdRp) at the order from N-terminus. The RNA2 consists of 4012 nt 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) at the order from N-terminus. Based on the comparison of nucleotide sequences, RaMV-TW is identified as a new isolate of RaMV and highly homologous to other strains of RaMV. Among these, RaMV-TW is more closely related to strains RaMV-CA and RaMV-J than to RaMV-1.

Reference ( 88 ) 〈TOP〉

台灣省政府農林廳。1995。台灣農業年報。380頁。
連結：
陳滄海、吳秀芳、陳翠蓉、陳惠玲。2000。台灣地區十字花科作物病毒病害之發生與血清學診斷。植病會刊 9: 39-46。
連結：
陳滄海。2003。台灣地區甜菜西方黃化病毒(Beet western yellows virus)之發生、鑑定及蚜媒傳播特性與生態。植病會刊 12: 43-56。
連結：
劉興隆。1993。中部地區設施蔬菜之病害調查。台中區農業改良場研究彙報 41: 1-9。
連結：
鍾文全、黃振文。1993。十字花科蔬菜黑斑病菌的存活研究。植保會刊 35: 39-48。
連結：

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