中国马铃薯Y病毒的检测鉴定及CP基因的分子变异

Detection and Molecular Variation of Potato virus Y CP Gene in China

【目的】查明马铃薯Y病毒(Potato virus Y,PVY)病在中国的发生情况,及时、准确地鉴定出PVY并对其分子变异进行分析。【方法】采用ELISA方法对采自中国14个省(直辖市)马铃薯种植区疑似受PVY感染的样品进行了检测,并对其中的部分材料镜检验证,然后根据CP基因序列设计1对简并引物针对随机选择感染PVY的14个省(直辖市)代表样品进行CP基因扩增克隆,将测序得到的序列进行分子变异分析,并使用贝叶斯法(Bayesian inference,BI)重建系统发育关系。【结果】ELISA检测结果表明,691份样品中220个样品与PVY抗体呈阳性反应,其余呈阴性反应;ELISA检测的阳性材料在透射电镜下均可观察到明显的风轮状内含体,14个PVY分离物均成功扩增出预期大小(约800 bp)的特异性片段,CP基因的核苷酸序列与已报道PVY不同株系的核苷酸序列一致性均在88%以上;在14个PVY分离物CP基因中共发现有29个多态性位点,其中6个简约信息位点,23个单一变异位点。系统发育分析结果显示,14个PVY分离物与PVYN:O株系相聚成簇,表明其在系统发育关系上,与PVYN:O株系的亲缘关系最近。【结论】PVY CP基因高度保守,但不同地区分离物也存在一定的分子变异,本研究可为今后了解PVY病毒病流行、变异趋势及其防治提供依据。

【Objective】 The objectives of this study are to investigate the occurrence of Potato virus Y（PVY） disease in China,to develop fast and accurate PVY detection methods and to understand sequence variations in PVY CP gene.【Method】ELISA method was used to detect the infected leaf samples randomly collected from 14 provinces（municipalities） in China during 2011 and 2012.A subsample of the infected leaves was examined using a transmission electron microscope.CP gene of PVY was amplified by reverse-transcription polymerase chain reaction（RT-PCR） using a pair of degenerate primers designed from the conserved regions of published CP gene sequences.Sequence variation was analyzed and phylogenetic tree was re-constructed using Bayesian inference method.【Result】ELISA revealed that 220 out of the 691 samples assayed were infected with PVY.The pin-wheel inclusion bodies were visualized clearly under the transmission electron microscope in the subsamples of ELISA-positive leaves.RT-PCR amplifications of the 14 ELISA-positive subsamples randomly selected from different provinces（municipalities） all generated an expected fragment of ≈800 bp in size.CP genes in the selected 14 subsamples shared more than 88% nucleotide identity with the reported sequences in other PVY strains.Among the 14 CP gene sequences in the subsamples,29 polymorphic sites were detected,including 6 parsimony informative and 23 singleton variable sites.Some of the sequence variations were geography-specific.Bayesian inference of phylogenetic trees revealed that 14 PVY isolates were in the same branch with reference PVYN：O strains,suggesting that the 14 PVY isolates analyzed in this study shared high homology with PVYN：Ostrain.【Conclusion】CP gene in PVY is highly conserved but some geographical variation in sequences exists.The results reported in this manuscript provide useful insights in understanding the evolution of PVY,the epidemiology and control of PVY disease in potato.