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Genomic Analysis of The Chiayi Isolate of Papaya Ringspot Type W Virus
PRSV type W virus
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，依其寄主範圍可區分為木瓜型（PRSV Type P virus, PRSV P）及西瓜型（PRSV Type W virus, PRSV W）兩型。PRSV西瓜型病毒原名西瓜嵌紋病毒-1（watermelon mosaic virus-1, WMV-1），可感染藜科（Chenopodiaceae）及葫蘆科（Cucurbitaceae）植物；而PRSV木瓜型病毒除可感染上述兩科植物外，尚可感染番木瓜科（Caricaceae）植物。PRSV西瓜型及木瓜型病毒因寄主範圍不同，為瞭解其基因體上的差異，本研究針對PRSV西瓜型病毒做解序的工作，再與已解序出的PRSV木瓜型病毒做比較，分析其歧異情形。本實驗將自嘉義分離來之PRSV西瓜型分離株（PRSV W-CI）繁殖於刺角瓜（Cucumis metuliferus Naud.），再純化出病毒，並抽取其RNA做為模板分子，構築cDNA基因庫。再依據已知病毒基因體兩端的序列做成特定的探針，從基因庫中選出三個涵蓋病毒基因體5''端前半段的cDNA片段，再由這些片段作成對應病毒基因體中段的探針，進一步從基因庫中選出五個涵蓋病毒基因體3''端的部份的選殖體。此八個選殖體對應97.5%的W-CI基因體，以此八個選殖體解出之序列加上先前已知之3''端序列，W-CI病毒全長的核酸序列即被解出解出。解序的PRSV W-CI 全長有10,332個核酸，轉譯出一條含3,346個氨基酸的複合蛋白。將之與先前已發表的PRSV木瓜型病毒夏威夷分離株PRSV P-HA及台南永康分離出的PRSV P-YK之基因體核酸比對序列的相似性，發現W-CI和P-YK有95.25%的核酸相同度（nucleotide identity），與P-HA有83.47%的核酸相同度；而複合蛋白氨基酸相同度（amino acid identity）分別有96.77%及90.64%。個別分析病毒不同部位之核酸相同度，發現W-CI與P-YK核酸相同度最高的為3''端非轉譯區（98.09%），其次為HC-Pro基因（97.08%），最低的為P1基因（93.24%）。W-CI與P-HA核甘酸相同度最高的為3''端非轉譯區（91.39%），P1基因則為71.66%，最低的為5''端非轉譯區（65.88%）。再個別分析病毒不同基因之氨基酸相同度，W-CI與P-HA及P-YK都在P1蛋白部份有最低的相同度，各為91.96%及66.00%，而最高各在CIP 蛋白（98.11%）及HC-Pro蛋白（98.91%）。這些結果顯示W-CI與P-YK的基因體有較高於P-HA基因體的相似性。即PRSV基因體間的親緣關係與地域分佈有較大的關連，而與病原性如會不會感染木瓜較不相關。由於W-CI基因體全部核酸序列已解序完畢，本研究經過電腦比對已預測出W-CI與P-HA及P-YK基因體RNA對應出的互補股DNA（cDNA）上的限制酵素切位，如此便可利用PRSV P及PRSV W病毒上適當的限制酵素切位在cDNA的層次上進行精細的置換工作，以探討木瓜型病毒決定感染木瓜的基因體關鍵部位。|
Genomic Analysis of the Chiayi Isolate of Papaya Ringspot Type W Virus ABSTRACT Papaya ringspot virus (PRSV) is a member of the genus Potyvirus. According to the difference in host range, most of PRSV isolates have been divided into two groups, type P (PRSV P) and type W (PRSV W) viruses. The host range of PRSV W is limited to Chenopodiaceae and Cucurbitaceae, and PRSV P can infect Caricaceae in addition. In this study, the PRSV W Chiayi isolate (PRSV W-CI) was maintained in Cucumis metuliferus Naud. and used for virus purification and RNA extraction. The viral RNA was used as the template for the synthesis of cDNA for construction of a cDNA library. Two specific probes corresponding to the 5'' and the 3'' regions of W-CI RNA were used for screening the cDNA library by plaque hybridization. Three clones obtained were corresponding to the 5'' half of the W-CI genome. A specific probe corresponding to the central region of W-CI RNA was then obtained from these three clones. Five clones corresponding to the 3'' half region of the W-CI RNA were thus obtained. These eight cDNA clones represented 97.5% of the genome of W-CI. The genomic sequence of W-CI was determined by these eight clones and combined with the previous work of the 3''-terminal sequence. The complete sequence of W-CI contained 10,332 nucleotides in length and encoded a polyprotein of 3,346 amino acid residues. The genome of W-CI was found with 6 nucleotides, 2 amino acids, in addition to those of type P viruses of PRSV, P-HA (from Hawaii) and P-YK (from Taiwan), which genomes have been completely determined previously. Comparison of the full-length genome of W-CI with those of P-HA and P-YK revealed that they share over all 83.47% and 95.26% nucleotide identities, respectively; and 90.64% and 96.77% amino acid identities in polyproteins, respectively. Comparison of the nucleotide identity of individual regions between W-CI and P-YK showed that the 3'' NTR is the most conserved region (98.09%), followed by the HC-Pro gene (97.08%), while the P1 gene is the most variable gene (93.24%). Comparison of the nucleotide identity of individual regions between W-CI and P-HA showed that the 3'' NTR is the most conserved region (91.39%), the P1 gene is of 71.66%, and the 5'' NTR is most variable (65.88%). At the amino acid level, the HC-Pro gene with amino acid identity of 98.91% was found to be the most conserved gene between W-CI and P-YK, and the CIP gene with amino acid identity of 98.11% the most conserved gene between W-CI and P-HA. It was also revealed that the P1 protein is the most variable protein between W-CI and P-HA (66.00% amino acid identity), as well as between W-CI and P-YK (91.96% amino acid identity). These sequence analyses showed that W-CI shares a higher degree of homology with the P type Taiwan isolate P-YK than the P type Hawaii isolate P-HA. The variability of the genome of P and W type viruses were found not related to the host specificity, such as infecting papaya or not, but more reflecting the difference in the geographical distribution. The same restriction enzyme sites in the full-length cDNA of PRSV W-CI, P-YK, and P-HA were predicted by the computer program. The in vitro and in vivo infectious full-length cDNA clones of the two P type viruses P-HA and P-YK will be used to recombine with cDNA clones of W-CI using the unique restriction sites revealed, to generate the P and W type hybrid viruses. The infectivity tests of these hybrid viruses will help determine the genomic region(s) responsible for the host specificity.
|Appears in Collections:||植物病理學系|
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