幾種Tospovirus屬病毒間NSs蛋白的血清學關係比較

Comparison of the serological relationships among the NSs proteins of different tospoviruses

陳羽騏(Yu-Chi Chen)

指導教授 ： 張清安

朝陽科技大學/理工學院/應用化學系生化科技碩士班/碩士(2013年)

摘要

Tospoviruses 乃一群可以經由薊馬傳播之植物病毒，受害之作物病徵通常極為明顯，對生長與品質造成嚴重影響。根據文獻報導目前有三種可能危害蘭花之 tospoviruses 存在；包括台灣已經發生之番椒黃化病毒（Capsicum chlorosis virus, CaCV）、蕃茄斑萎病毒（Tomato spotted wilt virus, TSWV）及尚未但可能入侵台灣之鳳仙花壞疽斑點病毒（Impetiens necrotic spot virus, INSV）。此三種病毒的檢測與鑑別技術便攸關此類病毒病害之發生與傳播是否能被精準的掌握與控制。傳統 tospoviruses 的鑑定是以其 S RNA 互補股所對應之核鞘蛋白（nucleocapsid protein，NP）為檢測之標的與分類之依據。本研究基於相關文獻指出 tospovirus S RNA 之正意股所對應之非結構性蛋白（Non-structural protein, NSs）會累積在感病細胞內形成內含體（inclusion），故擬嘗試探討以 NSs 蛋白作為此等病毒檢測或鑑定標的之可行性。同時針對此等病毒間其 NSs 蛋白之血清類緣關係進行比對，供檢測應用之參考。研究策略乃將感染本省蝴蝶蘭最普遍之 CaCV 及發生於荷蘭蝴蝶蘭上之 INSV 及 TSWV 所對應之 NSs 蛋白以細菌表達外源蛋白之技術，將其大量誘導純化並製備成多元抗體，進行血清學研究。另外將另一種普遍發生於台灣瓜類作物且與 CaCV 同屬 NP 蛋白第四血清型之西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV）之 NSs 蛋白加以純化供血清類緣關係比較。根據上述四種 tospoviruses 之已知 S RNA 基因體序列，設計可分別增幅各病毒 NSs 基因之四組專一性引子對，將四種病毒之 NSs 基因加以增幅，並分別選殖於 pGEM-T Easy（Promega﹐USA）或pCRII-TOPO （Quiagen, USA）載體上，經分別解序並分析後證實利用NSs 基因序列分析所獲得之 tospovirus 親緣關係與演化趨勢結論與文獻上針對 NP 基因所做之相同分析結論幾近相符。分析發現同種病毒之 NSs 基因在核苷酸及胺基酸序列相同度（identity）均落於 94

關鍵字

血清學 ； 非結構性蛋白 ； 番茄斑點萎凋病毒屬

並列摘要

Tospoviruses is a group of plant viruses characterized in their distinct thrips-borne transmissibility. There have been more than 30 different tospoviruses reported globally and most of them induce very severe symptoms on their host plant that result evident loss in yield and quality. Among the three tospoviruses known to infect Phalaenopsis, Capsicum chlorosis virus (CaCV) was currently endemic in Taiwan. The other two including Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) have not yet been found in Taiwan to infect Phalaenopsis. Therefore the latter two tospoviruses are considered as quarantine pathogens for Taiwan’s Phalaenopsis industry. Therefore, the accuracy of the techniques applied to the detection and diagnosis is a major concern on the surveillance and control of the these virus diseases of orchids. Traditionally, the nuclear capsid protein (NP) was used as the major target of detection and taxonomic criteria for tospoviruses. While recently there was literature suggesting the non-structure protein (NSs) encoded in the S-RNA of tospoviruses could be used an alternative target. This study was therefore aiming at clarification of the feasibility of using NSs as a target for quick differentiation of those Phalaenopsis-infecting tospoviruses. In addition, serological relationship of NSs proteins among tospoviruses was also studied due to its importance in application of NSs for virus detection. Our study took the approach of using bacteria expressed protein system to obtain purified NSs protein encoded by the three Phalaenopsis infecting tospoviruses for their antibody production. We also included Watermelon silver mottle virus (WSMoV), the type species of the 4th NP serogroup in tospoviruses, to study the serological variation among NSs proteins of tospoviruses. The study started by the design of primer pairs specific respectively to these tospoviruses to amplify their NSs genes, which were then cloned in pGEM-T Easy (Promega, USA) or pCRII-TOPO (Quiagen, USA) plasmids and sequenced. Analyses of these NSs gene sequences found that those viruses in the same NP serogroup were also closely related among their NSs proteins. The nucleotide and amino acid sequence identities within the same NP serogroup were 70-79

並列關鍵字

serological ； Non-structural protein ； Tospovirus

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