河北省马铃薯Y病毒株系分子鉴定及其RT-PCR检测

Molecular identification of Hebei potato virus Y isolate and its detection by RT-PCR

根据马铃薯Y病毒(PotatovirusY,PVY)的外壳蛋白基因序列,设计合成了一对寡核苷酸引物,从感染PVY的马铃薯田间病叶组织中提取出病毒的RNA,进行cDNA合成及PCR扩增,得到一条长度约800bp的特异PCR扩增产物,与理论设计的基因片段大小一致,本试验建立了快速、灵敏、简便的PVY检测的方法。将RT-PCR扩增产物克隆到质粒pGEM-T上,并进行了序列分析。结果表明,克隆的cDNA片段由801个核苷酸组成,编码267个氨基酸组成的理论分子量为29kD蛋白,与PVY外壳蛋白的大小一致,与基因库登记的代表性株系相比,它们之间的核苷酸序列同源性为89 6%～98 8%,氨基酸序列的同源性为93 3%～98 5%,说明PVYCP基因具有高度的保守性(记为PVY-HBEI),与国内报道的PVY-CHU、PVY-ZHJ和PVY-ZHOU株系的氨基酸序列同源性分别为96 6%、98 5%和97 8%,与PVYN和PVYO株系的氨基酸序列同源性分别为93 6%和97 8%,从分子水平上确定PVY-HBEI归属于普通株系(PVYO株系)。

A pair of DNA primers were designed and synthesized based on the nucleotide sequence of the coat protein(CP) gene of potato Y virus (PVY).The PVY RNA was directly extracted from virus-infected potato leaves collected in field and utilized for cDNA synthesizing; then it was amplified by polymerase chain reaction (PCR).A specific PCR fragment about 800bp thus was obtained and was the expected length of PVY CP gene. The experiment provided a rapid, sensitive and relatively inexpensive means of PVY detection. The unique amplified product was then cloned into the pGEM-T vector and sequenced. The result showed that the cloned segment was 801bp and encodes 267 amino acid residues with relative molecular weight of KD which is the length of the PVY CP, Comparison of the nucleotide sequence between the CP gene and those of several PVY isolates showed that the homology of are 89.6%(98.8%, and the homology of the putative amino acid sequence was from 93.3% to 98.5%,respectively. This suggested that the CP gene of PVY isolates is a relatively conserved sequence within the PVY genome (it was called PVY-HBEI). The homology of the amino acid sequence with China isolates PVY-CHU, PVY-ZHJ and PVY-ZHOU were 96.6%, 98.5%和97.8%,respectively.The homology between PVY-HBEI and PVY^N and PVY^O were 93.6% and 97.8%,resepectivly. Thus it was identified as an isolate of PVY^O.