Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31579
標題: 木瓜輪點病毒在木瓜與奎藜上致病性相關基因之探討
Analyses of Pathogenicity-Related Genes of Papaya Ringspot Virus on Plants of Carica papaya and Chenopodium quinoa
作者: 李俊義
Lee, Chun-yee
關鍵字: Papaya Ringspot Virus
木瓜輪點病毒
HC-Pro
出版社: 植物病理學系
摘要: 木瓜輪點病毒(Papaya ringspot virus, PRSV)屬於馬鈴薯Y病毒屬(the genus Potyvirus),寄主範圍狹窄,僅能感染番木瓜科(Caricaceae)、葫蘆科(Cucurbitaceae)及藜科(Chenopodiaceae)植物。木瓜輪點病毒夏威夷株(PRSV HA)在木瓜上引起系統性感染,罹病木瓜在葉片上產生嵌紋、皺縮及扭曲變形病徵,以機械接種於奎蔾上,會在奎蔾上發生過敏性反應,產生局部病斑;而木瓜輪點病毒夏威夷株以亞硝酸誘變後產生的弱病毒株系PRSV HA5-1,在木瓜上亦引起系統性感染,但不產生明顯的病徵,以機械接種在奎蔾上,在感染後奎蔾上並不會產生局部病斑。於之前研究中,將PRSV HA及PRSV HA 5-1之cDNA重組置於T3啟動子後面,構築成重組全長度cDNA,利用生體外轉錄(in vitro transcription)所得到之一系列之生體外轉錄體,接種於木瓜上,依病徵發展程度不同,可分為type I、type II、type III與type IV四種不同病徵型態。為更進一步瞭解與病原性相關之病毒基因序列nt 950-3261中,胺基酸之突變對於病毒感染木瓜產生之病徵型態,及病毒在奎藜上致病性改變之影響,構築了pT3-Aat/Msc、pT3-Msc/Mlu、p51-Aat/Msc、p51-Msc/Mlu、pT3-Nhe/Msc、pT3-NM76mm、p51- Aat /Mlu、p51-Nhe /Mlu等八個重組之構築體,經以限制酵素圖譜與DNA解序比對確認無誤後,利用生體外轉錄,得到重組之生體外轉錄體,接種於木瓜上測試其感染力,同時觀察其發病後之病徵發展。結果顯示,木瓜輪點病毒P1蛋白與HC-Pro蛋白之突變可造成病毒感染木瓜與奎藜後病徵型態改變。若將PRSV HA之P1蛋白C端(nt 950-1653)與HC-Pro蛋白C端(nt 2858-3261)以PRSV HA5-1之片段取代,會造成病毒感染木瓜後,由原本嚴重型病徵轉變為無明顯病徵,顯示PRSV HA之P1蛋白C端以及HC-Pro蛋白C端胺基酸突變,兩處基因之突變即可抑制病毒在木瓜上病徵之發展,使病毒感染之木瓜不產生病徵。含有來自PRSV HA 5-1之HC-Pro蛋白C端與一小部分P3蛋白N端(nt 2858-3261)之重組病毒,接種於其單斑寄主奎藜上,發現皆不會引起奎藜產生局部病斑,顯示木瓜輪點病毒HC-Pro蛋白是奎藜過敏性反應之關鍵基因。本實驗中顯示HC-Pro蛋白除了在奎藜上對病徵表現為決定性之關鍵基因外,對於病毒感染木瓜後之病徵表現亦有重要之影響。
Papaya ringspot virus (PRSV) is a member of the genus Potyvirus, with flexuous particles of 780x12 nm. The host range of PRSV is limited to Chenopodiaceae, Cucurbitaceae and Caricaceae. PRSV HA is a Hawaii strain and infects papaya systemically, with mosaic, leaf narrowing, stunting, and distortion. HA also infects the plants of Chenopodium quinoa and induces local lesions on them. PRSV HA 5-1 is a nitrous acid-induced mutant that infects papaya without conspicuous symptoms. PRSV HA 5-1 can also infect plants of C. quinoa, but does not induce local lesions on the leaves of C. quinoa. A series of full-length chimeric constructs were generated by exchanging the restriction fragments of pHA-T3G and pHA5-1 in the previous study. The symptoms induced by these viruses were divided into four types: type I of severe mosaic and leaf distortion, type II of mild mottling and slight distortion, type III of mild mottling and vein yellowing, and type IV of no conspicuous symptoms. In this study, other eight recombinant clones were constructed to further analyze the pathogenicity-related mutations in the region of nt 950-3261 of PRSV HA 5-1. It was suggested that the mutations in the P1 and HC-Pro protein resulted in the attenuation of the viruses. Recombinant viruses that contained the regions of PRSV HA 5-1 nt 950-1653 and nt 2858-3261 including the C-terminal half of the P1 protein and the C terminal region of the HC-Pro protein caused no symptoms on the plants of papaya. The results indicated that the mutations in the C-terminal region of the P1 protein (nt 950-1653) together with the mutations in the HC-Pro protein (nt 2858-3261) of PRSV HA alter the symptom type from severe (type I) to mild (type IV). The recombinant viruses that infected the plants of C. quinoa without local lesions contained the same mutations in nt 2858-3261 (the C-terminal portion of the HC-Pro gene and a bit of the N-terminal portion of the P3 gene). The data in this study indicated that this region of HC-Pro protein of PRSV had the major effect on virus-induced hypersensitive reaction on C. quinoa. Furthermore, the results in this study indicated the HC-Pro gene of PRSV was the major determinant factor for symptom expression on C. quinoa and was important for the attenuation of symptoms on papaya plants.
URI: http://hdl.handle.net/11455/31579
Appears in Collections:植物病理學系

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