Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31574
標題: 木瓜輪點病毒西瓜型生體外具感染力載體之構築及感染木瓜寄主專一性基因之分析
Construction of in vitro infectious clone of a type W strain of Papaya ringspot virus and analysis of host determinant for papaya
作者: 陳冠君
Chen, Kaun-Chun
關鍵字: Papaya ringspot virus (PRSV)
木瓜輪點病毒
host determinant
寄主專一性基因
出版社: 植物病理學系
摘要: 木瓜輪點病毒 (Papaya ringspot virus, PRSV) 為馬鈴薯Y病毒屬 (the genus Potyvirus) 之一員,依寄主範圍之不同區分為木瓜型 (PRSV type P virus, PRSV P) 及西瓜型 (PRSV type W virus, PRSV W) 兩型,PRSV木瓜型可感染番木瓜科 (Caricaceae)、葫蘆科 (Cucurbitaceae) 及藜科 (Chenopodiaceae);而PRSV西瓜型只能感染葫蘆科及藜科,不能感染番木瓜科。木瓜輪點病不同系統間的序列差異主要由地理分佈所決定,而與能否感染木瓜能力較不相關,因此對於木瓜型能夠感染木瓜的決定基因仍不明瞭。木瓜型的分離株P-HA和P-YK的全長度基因體核酸已由本實驗室解序完成,且其具感染力的全長度cDNA亦已構築完成,本實驗之目的即為研究感染木瓜之決定基因所在位置。首先利用西瓜型嘉義分離株 (W-CI) 三個互相重疊的cDNA片段,構築全長度具感染力的生體外轉錄體pT3WCIF1,並利用噬菌體T3 RNA聚合酶轉錄出具感染力的RNA。經感染力測試,此RNA可在奎藜 (Chenopodium quinoa) 造成單斑,於單斑分離後,接種至木瓜、刺角瓜及矮南瓜上,其病徵形態與野生型的W-CI相同,且都不能感染木瓜。此外,利用木瓜型永康分離株 (P-YK) 與W-CI之具感染力載體進行重組互換,構築四個重組病毒載體pT3P(W P3-CP)、pT3P(W CI-CP)、pT3P(W NIb-CP)及pT3P(W CP),依序置換木瓜型的核苷酸3263-10326、4496-10326、7700-10326及9055-10326成為西瓜型的相對基因體核酸。經生體外轉錄作用後,將RNA接至奎藜上以產生重組病毒P(W P3-CP)、P(W CI-CP)、P(W NIb-CP)及P(W CP),再接種至木瓜、刺角瓜及矮南瓜植株後,所有的重組病毒在刺角瓜上造成的病徵相似,且發現只有P(W NIb-CP) 及P(W CP) 可感染木瓜且在矮南瓜上造成的病徵較像P-YK,而P(W P3-CP) 與P(W CI-CP) 無法感染木瓜且在矮南瓜上造成的病徵較像W-CI。所有的測試病毒都可被酵素連結抗體免疫吸附反應 (enzyme-linked immunosorbent assay, ELISA) 偵測到,並用反轉錄-聚合酶鏈鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) 確認無誤。由以上我們的結果顯示決定能感染木瓜之基因可能在P-YK核苷酸片段4496-7700,且由於P(W P3-CP) 與P(W CI-CP) 在矮南瓜上造成的病徵不同於P(W NIb-CP) 及P(W CP),所以,此片段亦決定在矮南瓜上的病徵表現。此片段包括一部分的CI蛋白及完整的6K及NIa蛋白。
Papaya ringspot virus (PRSV) is a member of the genus Potyvirus. According to the difference in host range, most of PRSV isolates belong to two groups, type P (PRSV P) and type W (PRSV W) viruses. The host range of PRSV W is limited in plants of Chenopodiaceae and Cucuribitaceae, while PRSV P can infect plants of Caricaceae in addition. In this study, the in vitro infectious clone of pT3WCIF1 driven by a bacteriophage T3 promoter was constructed from three overlapping cDNA clones corresponding to different regions of the viral genome of a Chiayi isolate of PRSV W(PRSV W-CI). The in vitro transcripts of pT3WCIF1 were generated by T3 RNA polymerase and introduced to the local lesion host Chenopodium quiona and the virus obtained was denoted W-CIF1. When the virus was transferred to zucchini squash (Cucubita pepo L. var. Zucchini) and horn melon (Cucumis metuliferus) from local lesions of C. quinoa, the symptoms induced on these plants were similar to those induced by the native W-CI. Both of the native virus W-CI and the W-CIF1 were unable to infect papaya (Carica Papaya L.) plants. In addition, the chimeric infectious cDNAs clone of pT3P(W P3-CP), pT3P(W CI-CP), pT3P(W NIb-CP), and pT3P(W CP) were constructed by replacing the nt 3263-10326, nt 4496-10326, nt 7700-10326, and nt 9055-10326 fragments of the previously constructed in vitro infectious clone pT3PYKF1of the P type virus PRSV P-YK with the corresponding segments of pT3WCIF1, respectively, and the recombinant clones were introduced into seedlings of papaya, horn melon, and zucchini squash. All the recombinant viruses caused indistinguishable severe symptoms on horn melon. The viruses of P(W NIb-CP) and P(W CP), derived from pT3P(W NIb-CP) and pT3P(W CP), respectively, were able to infect papaya and zucchini squash, causing severe symptoms similar to those induced by virus P-YKF1 derived from pT3PYKF1. However, the viruses of P(W P3-CP) and P(W CI-CP), derived from pT3P(W P3-CP) and pT3P(W CI-CP), respectively, did not infect papaya, but caused severe symptoms on zucchini squash similar to those induced by W-CIF1. Infections by the recombinants and the native viruses were confirmed by direct double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and verified by reverse transcription-polymerase chain reaction (RT-PCR) coupled with restriction enzyme digestion. Base on the results, it appears that the region of nt 4496-7700 of PRSV P-YK, with the part of the CI gene and the complete 6K and nuclear inclusion a (NIa) genes, may contain the determinant(s) responsible for infecting papaya. Furthermore, symptoms on zucchini squash infected by the viruses P(W P3-CP) and P(W CI-CP) were different from the viruses P(W NIb-CP) and P(W CP), the region of nt 4496-7700 may also affect the virulence of PRSV on squash.
URI: http://hdl.handle.net/11455/31574
Appears in Collections:植物病理學系

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