中国农业科学 ›› 2013, Vol. 46 ›› Issue (15): 3125-3133.doi: 10.3864/j.issn.0578-1752.2013.15.006

• 植物保护 • 上一篇    下一篇

中国马铃薯Y病毒的检测鉴定及CP基因的分子变异

 高芳銮1, 沈建国2, 史凤阳1, 方治国1, 谢联辉1, 詹家绥1   

  1. 1.福建省植物病毒学重点实验室,福建农林大学植物病毒研究所,福州 350002
    2.福建出入境检验检疫局检验检疫技术中心,福州 350001
  • 收稿日期:2013-01-25 出版日期:2013-08-01 发布日期:2013-05-16
  • 通讯作者: 通信作者谢联辉,Tel:0591-83769704;E-mail:fjxlh@126.com;通信作者詹家绥,Tel:0591-83856973;E-mail:jiasui.zhan@fafu.edu.cn
  • 作者简介:高芳銮,E-mail:raindy@fafu.edu.cn;沈建国,E-mail:shenjg_agri@163.com。高芳銮与沈建国为同等贡献作者
  • 基金资助:

    国家现代农业马铃薯产业技术体系(CARS-10)、福建省自然科学基金项目(2013J01088)、福建省教育厅科技项目(JA12119)、福建出入境检验检疫局科技项目(FK2011-07)

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

 GAO  Fang-Luan-1, SHEN  Jian-Guo-2, SHI  Feng-Yang-1, FANG  Zhi-Guo-1, XIE  Lian-Hui-1, ZHAN  Jia-Sui-1   

  1. 1.Key Laboratory of Plant Virology of Fujian Province, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002
    2.Inspection & Quarantine Technology Center, Fujian Exit-Entry Inspection and Quarantine Bureau, Fuzhou 350001
  • Received:2013-01-25 Online:2013-08-01 Published:2013-05-16

摘要: 【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。

关键词: 马铃薯Y病毒 , ELISA , 外壳蛋白基因 , 分子变异 , 贝叶斯法

Abstract: 【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:O strain.【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.

Key words: Potato virus Y , ELISA , coat protein gene (CP gene) , molecular variation , Bayesian inference method