Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98167
標題: 感染唐棉的馬鈴薯Y病毒屬新種病毒之鑑定及特性分析
Identification and Characterization of a Novel Potyvirus Infecting Balloon Plant (Gomphocarpus spp.)
作者: 張庭愷
Ting-Kai Chang
關鍵字: 唐棉
唐棉嵌紋病毒
馬鈴薯Y病毒屬新種病毒
病毒鑑定
多元抗血清
Balloon plant
Gomphocarpus mosaic virus
New species of Potyvirus
Virus identification
Polyclonal antiserum
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摘要: 唐棉 (Gomphocarpus spp., syn. Asclepias spp.) 為夾竹桃科 (family Apocynaceae) 蘿藦亞科 (subfamily Asclepiadoideae) 多年生草本植物,又被稱為氣球果 (balloon plant)。2015年11月間,由台南區農業改良場送驗唐棉病株樣本。病株葉片呈嵌紋、葉面凹凸、葉片窄小、葉緣波浪狀等疑似病毒感染之病徵。以電子顯微鏡鏡檢罹病組織粗汁液,可觀察到大小約750-850 x 12 nm之長絲狀病毒顆粒;病組織超薄切片亦可觀察到細胞內有Potyvirus感染所引起之風車狀 (pinwheel) 及板層狀 (laminated) 內含體 (inclusion bodies)。將唐棉病葉粗汁液機械接種於奎藜 (Chenopodium quinoa) 進行三次單斑分離後,得到一病毒分離株CM532。將病毒株CM532以機械接種法分別回接至健康大粒種唐棉 (Gomphocarpus physocarpus syn. Asclepias phycocarpa) 和小粒種唐棉 (G. fruticosus syn. A. fructicosa) 實生苗,可產生與原始罹病唐棉植株相似的病徵。將病毒株CM532機械接種至9科24種的指示植物進行寄主範圍測試,結果顯示夾竹桃科 (Apocenaceae)、藜科 (Chenopodiaceae) 與茄科 (Solanaceae) 中共有14種供試植物會被感染。其中9種供試植物之病徵表現屬於局部單斑寄主;5種屬於系統性寄主。純化之病毒顆粒懸浮液以SDS-PAGE (SDS-polyacrylamide gel electrophoresis) 分析後,可觀察到大小約30 kDa之鞘蛋白條帶。將純化之鞘蛋白進行紐西蘭白兔之免疫注射以製備病毒抗血清,於免疫雙向擴散反應 (double diffusion test) 中,該抗血清對純化之病毒懸浮液產生白色沉澱帶且抗血清之力價達1/2。以間接式酵素連結免疫吸附分析法 (indirect-ELISA) 與西方轉漬反應 (western blotting),該抗血清與接種CM532之唐棉病葉粗汁液產生正反應,且於轉漬膜上位置約30 kDa處有反應帶產生。萃取唐棉病葉組織之總量RNA,搭配Potyvirus簡併式引子對 (PNIbF1/Oligo-dT) 進行反轉錄聚合酶鏈鎖反應 (RT-PCR),可增幅出一條約1.7 kb涵蓋Potyvirus NIb基因3'半部、全長鞘蛋白基因及3'非轉譯區 (3'-UTR) 的cDNA片段。經選殖、解序及BLAST比對後,該cDNA片段與隔山消嵌紋病毒 (keunjorong mosaic virus, KjMV)(accession number JF838187) 之核苷酸序列相同度達71.9%。以KjMV基因序列為藍本設計引子對,增幅並整合各相關片段後可得病毒株CM532之基因體總長度為9,998個核苷酸 (不含尾端PolyA),具有典型Potyvirus的基因架構,包括5'-UTR (174 nt)、一個大蛋白開放轉譯框架 [an open reading frame (ORF) of polyprotein] (9588 nt)、以及3'-UTR (236 nt)。複合大蛋白 (polyprotein) 可酶切出P1 (1311 nt )、HC-Pro (1365 nt)、P3 (1038 nt)、6K1 (156 nt)、CI (1902 nt)、6K2 (159 nt)、NIa-Vpg (567 nt)、NIa-Pro (738 nt)、NIb (1551 nt)、CP (801 nt) 等10個功能性蛋白。另在P3蛋白區域內有一獨立的ORF,可轉譯一段由89個胺基酸所組成的PIPO蛋白。已將完整基因體序列上傳至DNA Data Bank of Japan (DDBJ),並取得一組基因序列資料庫的登記碼 (accession number) 為LC228573。將CM532之複合大蛋白開放轉譯框架 (polyprotein ORF) 之核苷酸和胺基酸序列與目前NCBI GenBank登記的29種potyviruses進行序列比對,顯示CM532 polyprotein ORF之核苷酸與胺基酸序列相同度分別介於52.8-68.4% 與39.1-75.8% 之間。依國際病毒分類委員會 (ICTV) 界定新種potyvirus之規範,本研究之病毒株CM532是Potyvirus 屬的一個新種 (new species) 病毒,暫定名為唐棉嵌紋病毒(gomphocarpus mosaic virus, GoMV),為引起唐棉嵌紋病的病因。
Gomphocarpus physocarpus, commonly known as balloon plant or swan plant, exhibiting virus-like symptoms of mosaic, mottle and crinkling were observed and collected in southwestern part of Taiwan in 2015. Electron microscopic examination revealed the presence of filamentous virus-like particles about 750-850 x 12 nm in crude sap and typical potyviral pinwheel and laminated inclusion bodies in ultrathin sections of infected tissues. A virus culture, CM532, was isolated from a symptom bearing G. physocarpus. It was established in Chenopodium quinoa through three consecutive single lesion isolations. In addition to its original host (i.e. G. physocarpus), CM532 also infects another species of balloon plant (i.e. G. fruticosus) systemically. Both showed the similar symptoms to that of original diseased sample after mechanical inoculation. Among 24 tested plant species belonging to 9 families, CM532 caused local infections mainly on plants of genera Chenopodium and Nicotiana as well as Beta vulgaris but systemic infections on G. physocarpus, G. fruticosus, Catharanthus roseus, Asclepias curassavica, and Nicotiana benthamiana. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed that the CM532 contains a single viral coat protein subunit in molecular weight about 30 kDa. A specific antiserum against the CM532 was produced by immunizing New Zealand white rabbit with purified virus particles. The specific antiserum is reacted positively to CM532 in all tests of SDS-agar gel double diffusion, western blotting, and indirect enzyme-linked immunosorbent assay. Total RNA was extracted from CM532-infected leaves. The potyvirus degenerate primer pair, PNIbF1 and Oligo-dT were used to amplify the 3'region [including partial nuclear inclusion body protein (NIb), entire coat protein (CP) gene and 3' untranslated region (3'-UTR)] of the potyvirus genome. A cDNA fragment of about 1.7 kb was amplified. A database search using BLASTn in GenBank revealed that the cDNA fragment shared 71.9% nucleotide sequence identity to that of keunjorong mosaic virus (KjMV, accession number JF838187).The cDNA of the remaining CM532 genome was amplified by the primers designed based on KjMV. After the assembly of cDNA, the complete genome of CM532 comprises 9,998 nucleotides in length excluding the 3'-terminal poly(A) tail. It contains two open reading frames encoding a polyprotein of 3196 amino acids and a PIPO protein of 89 amino acids. The full-length sequence of the CM532 genome has been deposited in DDBJ as the accession number LC228573. The polyprotein gene shares 52.8-68.4% nucleotide sequence identity and 39.1-75.8% amino acid sequence identity with 29 selected potyviruses. The data presented in this study indicate that the virus CM532, tentatively named as gomphocarpus mosaic virus (GoMV), is a distinct new species of the genus Potyvirus and is the causal agnet of gomphocarpus mosaic disease.
URI: http://hdl.handle.net/11455/98167
文章公開時間: 10000-01-01
Appears in Collections:植物醫學暨安全農業碩士學位學程

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