番茄环斑病毒悬钩子分离物复制酶基因的克隆及序列分析

番茄环斑病毒(ToRSV)是一种高风险的检疫性有害生物,可侵染桃、李、葡萄、苹果、樱桃等多种植物。2000—2001年,我国从法国进口的葡萄插条上检出过此病毒。以ToRSV悬钩子分离物(Rasp-2)总RNA为模板,采用RT-PCR方法扩增病毒复制酶基因(RdRp)的cDNA片断并将其克隆到pMD18-T载体上。序列测定结果表明,Rasp-2RdRp基因由2133个核苷酸组成,编码711个氨基酸;Rasp-2半胱氨酸蛋白酶和RdRp之间的蛋白酶切割位点为Q/V。Rasp-2与其他分离物及株系RdRp基因的核苷酸序列同源性为79%～84%,氨基酸序列同源性为89%～94%。聚类分析结果显示,葡萄黄脉株系(GYV)与其他分离物及株系关系最远,Rasp-2与其他分离物及株系亲缘关系较近。证实Rasp-2与另外2个悬钩子分离物Rasp和Rasp-1的核苷酸序列存在明显变异。

Cloning and sequence analysis of the RNA dependent RNA polymerase gene of tomato ringspot virus raspberry isolate

ToRSV is a highly risk plant quarantine pest in China. Its hosts are peach, plum, grape, apple, cherry and so on. From 2000 to 2001, we had found ToRSV from vines imported from France. The RNA dependent RNA polymerase (RdRp) coding region of Tomato ringspot virus from raspberry was sequenced. It comprised 2133 nucleotides and encoded a putative protein of 711 amino acids. The protease cleavage site between the c-protease and RdRp was found to be Q/V. The nucleotide and amino acid sequence of RdRp gene of this isolate shared 79% to 84% and 89 % to 94% similarities to that of other ToRSV isolates or strains, respectively. Phylogenetic analyses of the sequences indicated that the relationship between GYV and other ToRSV isolates or strains was furthest, whereas the Rasp-2 and other ToRSV isolates or strains were most closely related. The study also indicated that there was obvious variation between Rasp-2 and other Raspberry isolates.