Nucleotide Sequence Analysis of the Coat Protein Genes of Two Isolates of Sweet potato feathery mottle virus from Central Taiwan

甘藷羽狀斑駁病毒台灣分離株鞘蛋白基因序列分析

王麗媛(Li-Yuan Wang) ； 陳冠君(Kuan-Chun Chen) ； 陳宗祺(Tsung-Chi Chen) ； 葉錫東(Shyi-Dong Yeh)

《植物病理學會刊》 16卷4期 (2007/12) Pp. 203-213

https://doi.org/10.6649/PPB.200712_16(4).0004

摘要

於嘉義地區，自呈現斑駁及嵌紋的甘藷葉片接種於奎藜(Chenopodium quinoa)後，經單斑分離可得兩種不同大小型態之局部黃斑，分別命名為CY1及CY2分離株。由感染CY1或CY2之奎藜萃取得總量核糖核酸(total RNA)，利用對應Potyvirus屬病毒基因體核糖核酸3'端之簡併式引子對Pot1/PotⅡ，以反轉錄聚合酶連鎖反應增幅出約1.2及1.3 kb兩個核酸片段。此核酸片段經選殖及核酸序列分析，分別為長1205及135l個核苷酸(nucleotide, nt)序列。與登錄於美國國家生物科技資訊中心(NCBI)之馬鈴薯Y病毒屬(Poryvirus)核酸序列比對分析，得知為甘藷羽狀斑駁病毒(Sweet potato feathery mottle virus, SPFMV)。由此設計專一性引子，將鞘蛋白基因3'端非中轉譯區(3' untranslated region, 3'-UTR)及Poly (A)尾端解序完成；分別得全長含1249(CY1)及1383(CY2)個核苷酸。由5'端起此序列分別為85及213核苷酸之細胞核內含體b (nuclear inclusion b, NIb) 3'端基因部分序列、SPFMV-CY1全長度鞘蛋白(coat protein, CP)基因為939個核苷酸，SPFMV-CY2為945個核苷酸，而3'端非轉譯區(3'-UTR)均為225個核苷酸及poly A尾端。其中所含之全長度鞘蛋白分別為313及315個胺基酸所組成，兩者之間蛋白酶切位應落於符合potyviruses特質Q/S之間，且由鞘蛋白N端起第9-11個胺基酸有位置具有代表potyvirus蚜蟲傳播能力之DAG序列。比較CY1、CY2兩個分離株鞘蛋白基因核酸序列有80.3%的相同度，86.3%的相似度。與登錄於NCBI之SPFMV 73個系統鞘蛋白基因比對結果顯示，CY1與CY2分別屬於基因群C及O；於演化關係上，CY1與6、Ita、MD1/1分離株親緣最近，CY2與5、O、Arua10a、TZ4及115/1S分離株親緣最近。又分析此兩個分離株與24個potyviruses的演化關係，結果顯示與其他能感染甘藷的病毒SPVY、SPVG及SPLV親緣關係最近，顯示SPFMV與違些病毒具相同演化歷程，其次為TEV、PVA及TuMV。

關鍵字

甘藷羽狀斑駁病毒 ； 核酸選殖 ； 序列分析

並列摘要

Two potyvirus-like isolates, CY1 and CY2, were collected from sweet potato displaying leaf symptoms of mosaic or vein mottling at Chia-Yi area. Taiwan, by single-lesion isolation on Chenpodium quinoa plants. Using the degenerate primers for potyviruses, a 1.2 kb and a 1.3 kb DNA fragments were amplified from CY1-and CY2-infected tissues of C. quinoa, respectively, by reverse-transcription polymerase chain reaction (RT-PCR). After clotting and sequencing, the two cDNA fragments were found to be of 1205 and 1351 bp. and corresponding to a part of the 3' end of nuclear inclusion (NIb) region and the 5' end of the coat protein (CP) region of potyviruses. For amplification of the region corresponding to the 3' end of CP region, the 3' untranslated region (3'-UTR), and the poly A tail, RT-PCR was conducted with the oligo (dT) primer and the specific Primers designed from the known sequence. The assembled cDNA sequences of 1249 and 1383 bp, respectively, from CY1 and CY2 were elucidated to reflect the 3'-terminal region of nuclear inclusion b (NIb) protein gene [85 nt (28 aa) / 213 nt (71 aa)], the complete CP gene [939 nts (313 aa) /945 nts (315 aa)], and 3'-uTR (both 225 nt) and a poly (A) tail. Sequence analysis indicated that the two viruses were isolates of Sweet potato feathery mottle virus (SPFMV). The two isolates showed 80.6% nucleotide identity and 86.3% amino acid identity in their CP genes. A putative proteolytic cleavage site Q/S was predicted between NIb protein and CP. A DAG triplet for aphid transmissibility of potyviruses was found at the 9-11 residues from the N-terminus of both CP genes. Phylogenetic analysis of CP sequences revealed that SPFMV-CY1 belonged to the group C and is as closely related to the isolates 115/1S, 5, Arua 10a, O and TZ4. The sequence relationships between the two isolates and potyviruses revealed that SPFMV-CY1, SPFMV-CY2 were closely to the sweet potato infecting potyviruses, SPVY, SPVG and SPLV, reflecting a more recent evolutionary relationship.

並列關鍵字

Sweet potato feathery mottle virus ； nucleic cloning ； sequence analysis