Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/89350
標題: 蕪菁輪斑病毒之特性、傳播與檢測技術之研究
Study on the Characters, Transmission and Detection of Turnip ringspot virus
作者: Ming-You Wu
吳明祐
關鍵字: 青江白菜
蕪菁輪斑病毒
黃條葉蚤
Chinese mustard
Turnip ringspot virus
Phyllotreta striolata
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摘要: 青江白菜 [Brassica greens or Chinese mustard, Brassica campestris L. ssp. chinensis (L.) Mad.]田間發現葉片具嵌紋病徵之病株,將病組織粗汁液機械接種於奎蔾 (Chenopodium quinoa)進行單斑分離。所得之純系病毒株回接於原寄主及數種十字花科作物可證實其病原性。穿透式電子顯微鏡鏡檢顯示病組織中含直徑約30 - 32 nm之球形病毒顆粒。SDS-PAGE分析純化病毒懸浮液發現病毒具有相對分子量分別為41和22 kDa之兩種外鞘蛋白次單位等類似於豆嵌病毒屬 (Comovirus)之病毒特性。以半巢式反轉錄聚合酶連鎖反應 (semi-nested RT-PCR)配合檢測Comovirus之簡併式引子對,可增幅出約800 bp之cDNA片段。經序列解序後發現與蕪菁輪斑病毒 (Turnip ringspot virus, TuRSV)的RNA 1有83 - 90%的核苷酸序列相同度,推測所分離的病毒應是TuRSV,並將之暫名為蕪菁輪斑病毒台灣分離株 (TuRSV-TW)。供試病毒之基因體全長度RNA核苷酸序列已解析。RNA 1 (accession no. GU968732)全長6076個核苷酸,包含1個開放轉譯架構 (open reading frame, ORF),可轉譯出210.8 kDa之複合蛋白。複合蛋白藉病毒之protease裂解成5種功能性蛋白:自5'端依序為protease cofactor (Co-Pro, 34.8 kDa)、RNA helicase (Hel, 67.9 kDa)、virus protein genome-linked (VPg, 3.2 kDa)、protease (Pro, 23.6 kDa)和RNA-dependent RNA polymerase (RdRP, 81.2 kDa)。RNA 2 (accession no. GU968731)全長3960個核苷酸,包含1個開放轉譯架構及2個起始密碼子AUG,可分別轉譯出122.3 kDa與112.9 kDa之兩個複合蛋白。前者可再裂解成cofactor necessary for RNA 2 replication / movement protein (CR / MP, 52.6 kDa)等功能性蛋白及大外鞘蛋白 (large coat protein, LCP; 41.2 kDa)與小外鞘蛋白 (small coat protein, SCP; 28.5 kDa)等兩個結構性蛋白;後者則裂解成移動蛋白 (movement protein, MP)、LCP與SCP。將LCP 與SCP之連續胺基酸序列分別和豆嵌病毒屬病毒 (comoviruses)進行序列比對,結果發現與其他TuRSV分離株有90.6 - 97.3%之胺基酸序列相同度,但與其他豆嵌病毒屬病毒者則只具29.2 - 74.0%之胺基酸序列相同度,證實TuRSV-TW為TuRSV的新分離株,並為在台灣之首次發現。比對TuRSV-TW 及其他豆嵌病毒屬病毒之基因體全長核苷酸序列、LCP胺基酸序列與LCP + SCP之胺基酸序列進行親緣關係分析,結果均顯示TuRSV-TW和蘿蔔嵌紋病毒 (Radish mosaic virus, RaMV)之親緣關係最為接近。由於TuRSV和RaMV皆以十字花科作物為主要寄主,加以RaMV可藉由黃條葉蚤 (striped flea beetle, Phyllotreta striolata)來傳播,因而進行TuRSV經由黃條葉蚤傳播之初步試驗。傳播試驗結果顯示TuRSV可經由黃條葉蚤傳播,且媒介昆蟲的獲毒取食時間 (acquisition feeding time)短至數秒鐘即可獲毒,獲毒後的持毒時間 (retention period)雖可達至少兩週以上,但僅能保持傳播病毒的能力最多達6天。黃條葉蚤傳播病毒的相關機制仍需要進一步探討。病株與蟲體之TuRSV的檢測以反轉錄聚合酶連鎖反應 (RT-PCR)與西方轉漬分析 (Western blotting)進行之,證實可應用RT-PCR於檢測單隻蟲體帶毒之情形。
Chinese mustard [Brassica campestris L. ssp. chinensis (L.) Mad.] with mosaic symptoms on leaves were found in fields. A virus culture has been obtained from three successive single-lesion isolation by mechanically inoculated on Chenopodium quinoa and the virus has been back-inoculated onto original host and some other crucifer crops to confirm its pathogenicity. The virus particles have spherical profile with a diameter of about 30 - 32 nm and consist of two coat protein subunits of 41 and 22 kDa which similar to those properties of a Comovirus. An expected cDNA fragment about 800 bp was amplified by using Comovirus degenerate primers with semi-nested reverse-transcription polymerase chain reaction (semi-nested RT-PCR) and shared 83 - 90% nucleotide sequence identities with that of other isolates of Turnip ringspot virus (TuRSV). The virus isolate was therefore designated as the Taiwan isolate of TuRSV (TuRSV-TW). The full-length sequences of its genomic RNAs 1 and 2 have been sequenced and have been deposited in NCBI Genbank database with accession numbers of GU968732 and GU968731, respectively. The RNA 1 of TuRSV-TW consists of 6076 nucleotides and encodes a single open reading frame (ORF) and expresses as a polyprotein of 210.8 kDa which was further cleaved into 5 functional proteins by viral protease. Those functional proteins include protease cofactor (Co-Pro, 34.8 kDa), RNA helicase (Hel, 67.9 kDa), virus protein genome-linked (VPg, 3.2 kDa), protease (Pro, 23.6 kDa) and RNA-dependent RNA polymerase (RdRP, 81.2 kDa). The RNA 2 of 3960 nucleotides also containes a single ORF but with 2 AUG start codons. Two polyproteins of 122.3 kDa and 112.9 kDa, respectively, can be expressed from each AUG codon. The polyprotein of RNA 2 is either cleaved by viral protease to produce cofactor necessary for RNA 2 replication (CR), large coat protein (LCP) and small coat protein (SCP), or to produce movement protein (MP), LCP and SCP. Functional proteins, including CR / MP (52.6 kDa), LCP (41.2 kDa) and SCP (28.5 kDa) cab be derived from RNA 2 polyprotein. Based on the amino acid sequences of combined coat proteins (LCP + SCP), high similarities of 90.6 - 97.3% can be found between TuRSV isolates while lower similarities of 29.2 - 74.0% were observed when compared that to other comoviruses. The virus studied was identified as a new and the first isolate of TuRSV reported in Taiwan. Phylogenetic analyses based on the full-length of nucleotide sequences of RNA 1 and RNA 2, the LCP and the combined coat protein amino acid sequences between other TuRSV isolates and comoviruses showed that TuRSV-TW is most closely related to Radish mosaic virus (RaMV), also a crucifer-infecting comovirus. As RaMV had been reported to be transmitted by striped flea beetle (Phyllotreta striolata), suggesting TuRSV can be transmitted by striped flea beetle as well. Striped flea beetles were collected from symptomless cruciferous crops on the field and fed on healthy or TuRSV-TW infected Brassica campestris L. ssp. chinensis (L.) Mad. for virus-transmitted tests. Transmission experiemts have shown that the striped flea beetles did transmit TuRSV, the acquisition feeding time was as short as seconds. The striped flea beetles retained TuRSV after acquisition for at least 14 days; however, the beetles kept transmitting virus for 6 days at most. TuRSV could be detected from the infected plants and from those beetles fed on it by RT-PCR and Western blotting. The RT-PCR has been used to detect TuRSV from one single viruliferous beetle successfully.
URI: http://hdl.handle.net/11455/89350
文章公開時間: 10000-01-01
Appears in Collections:植物病理學系

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