草莓潛隱輪斑病毒感染葵百合之分子生物學證據

Molecular Evidence for the Infection of Strawberry Latent Ringspot Virus in Lily

張清安( C. A Chang) ； 林玫珠( M. J Lin) ； 陳金枝( C. C Chen) ； 鄧汀欽( T. CDeng) ； 廖吉彥( J. Y Liao)

《植物病理學會刊》 10卷2期 (2001/06) Pp. 55-64

https://doi.org/10.6649/PPB.200106_10(2).0001

摘要

草莓潛隱輪斑病毒(Strawberry latent ringspot virus, SLRSV)是一種可以藉由機械性傷口及線蟲傳播的病毒，國內並未有其發生之機紀錄。本研宄室於1999年偶然發現此病毒發生於種球由荷蘭進口之葵百合(cv. Stargazer)植株，由於該植株葉片呈現與感染百合斑紋病毒(Lily mottle virus , LiMV)雷同之系統性嵌紋病徵，因此當時嘗試以對多數potyviruses具廣效性之引子對，進行反轉錄聚合?連鎖反應(RT-PCR)進行確認，卻發現有一樣品並未如預期產生2.0kb大小之核酸片段，反而出現大小僅為0.7kb之核酸產物(P700)。此產物經選殖及定序分析發現其序列與SLRSV RNA2之3'端非轉譯區(non-translated region ,NTR)序列(X75165, Everett et al., 1994)達到90%以上之相同度，因此根據已知SLRSV之RNA2核酸序列設計一對可以增幅出涵蓋完整鞘蛋白基因之引子對，就同一田間樣品再度進行RT-PCR反應，結果增幅出四個DNA 產物，其中一個大小為2.7kb之產物經選殖及解序後獲得個一全長含2763個核甘酸之序列(Tw26)。解析此序列發現其只含有一個轉譯架構，可對應一個含870個胺基酸(aa)之複合蛋白，推測此複合蛋白可於胺基酸247-248及635-636等二個位置上發生裂解，產生三個分別可能為247aa之未知蛋白(UNP)、388 aa之大鞘蛋白(LCP)及235 aa之小鞘蛋白(SCP)，複合蛋白之後有56個核甘酸之NTR。此種基因架構與文獻上所述SLRSV RNA2者符合，唯NTR部份較已知者短，可能仍未完整。故進一步利用已知的P700與Tw26序列重新設計引子對進行選殖，終於獲得涵蓋全長NTR之Tw-ntr3序列。將Tw-ntr3與Tw26序列合併後獲得一全長3224bp之核酸片段(SLR-TW-R2)。進行序列分析後發現，在已登錄的二個SLRSV RNA2序列中SLR-TW-R2與Everett et al.(1994)所報告之X75165序列在核酸層次上達到84%之相同度，而胺基酸層次之相同度甚至高達96%以上。SLR-TW-R2與Kreiah et al.(1994)所報告之X77466序列雖然在核甘酸相同度上略低，約為70-78%，但二者在LCP之胺基酸序列上相同度高達96.4%，此一分子生物證據顯示SLR-TW-R2序列應源自於一種與SLRSV親緣極為接近的病毒或系統，同時亦呼應過去以色列學者所提出SLRSV可以感染百合之生物學發現。

關鍵字

百合 ； 草莓潛隱輪斑病毒 ； 序列分析

並列摘要

Strawberry latent ringspot virus (SLRSV), a tentative species of the Genus Nepovirus in Comobiridae, has never been reported in Taiwan . In 1998, our laboratory detected a 0.7kb PCR product with SLRSV-related sequence from one of the oriental lily plants(cv. Stargazer),which was grown from lily bulbs imported from the Netherland, by polymerase chain reaction .The 0.7kb PCR product(P700) was subsequently sequenced and found that it had 90% of nucleotide identities with the 3’-terminal including part of the non-translated region of SLRSV RNA-2. To further confirm the existence of SLRSV sequence in lily ,we designed a primer pair to amplify the entire coat protein gene region based on the reported RAN-2 sequence of SLRSV and obtained a PCR product(Tw26) corresponding to the estimated size of 2.7kb. Tw26 was later cloned and sequenced and found that it comprised of 2763 base pairs encoding a single polyprotein sequence of 870 amino acid(aa) residues, which could be processed into three mature proteins, i.e. 247 aa unknown protein(UNP)、388 aa large coat protein(LCP) and 235 aa small coat protein(SCP). Downstream the polyprotein , there was a 56 nucleotide non-translated region (NTR), which was shorter than the previously known SLRSV RNA2 sequence. By combining the sequence information of P700 and Tw26, a complete 3224 bp sequence S(SLR-TW-R2) with presumably full length of NTR was obtained after PCR cloning and sequencing. After analyzing in Scan DNASIS program, the nucleotide and amino acid sequences of SLR-TW-R2 were found closely resembling to two known sequences of SLRSV (X75165, X77466) from the Genbank. It had 84% and 96% of identity with the nucleotide and amino acid sequences of the coat protein of X75165, respectively. On the other hand, SLR-TW-R2 showed only 70-78% of identity with the nucleotide sequence of X77466, but they shared over 96.4% of identity in the amino acid sequence of LCP. These results strongly suggest that the sequence of SLR-TW-R2 is originated from a virus identical to SLRSV or at least form a closely related strain .In 1995, the same virus was identified by immunological means in Isratel to infect Stargazer lily plants imported from the Netherlands. To our knowledge, this report provided for the first time the molecular evidence showing the infection of SLRSV in lily plants.

並列關鍵字

Lily ； Strawberry latent ringspot virus ； RNA2 ； sequence analyses