进境荷兰百合中车前草花叶病毒的分子鉴定及序列分析

Molecular identification and sequence analysis of Plantago asiatica mosaic virus in Lily from Netherlands

对一株进境隔离种植中疑似病毒症状的荷兰百合进行检测、鉴定。采用RT-PCR方法对疑似样品的部分基因序列进行扩增、测序,比对后,建立系统发育树进行亲缘关系分析。结果表明,采用香石竹潜隐病毒属和马铃薯X病毒属(CarlapCar1I/M4T和Potex 5/Potex 1RC)简并引物均可扩增到预期大小的片段(1 257 nts和737 nts)。序列分析显示,扩增片段与车前草花叶病毒(Plantago asiatica mosaic virus,Pl AMV)3'端序列(包含TGBp2、TGBp1、CP,3’-NTS)和部分依赖于RNA的RNA聚合酶基因序列一致性可达79.9%~99.5%和80.5%~99.3%。利用PlAMV的特异性引物(PlAMV-cpup/PlAMV-cpdw)进行检测,结果进一步证实该百合样品含有PlAMV。通过建立系统发育树得出,该病毒分离物与来自荷兰的PlAMV百合分离物优先聚为一簇,表明二者的亲缘关系最近。这是我国口岸首次截获车前草花叶病毒的报道。

The objectives of this study are to investigate a sample of Netherlands lily showing disease symptoms like that of virus infection. This sample was grown in the quarantined glasshouse. Partial genomic fragments was amplified by RT-PCR using two pairs of degenerate primers（Carla-pCar1I/M4T and Potex 5/Potex 1RC） designed for detection of the genera Calavirus and Potexvirus,respectively. Sequence analysis was performed and phylogenetic tree was reconstructed. RT-PCR amplifications of the positive sample both generated expected fragments（1 257 and 737 nts）. Sequence analysis showed that the two fragments shared 79.9%-99.5% and 80.5%-99.3% nucleotide sequence identities with that of the Plantago asiatica mosaic virus（PlAMV） 3’terminal（including TGBp2,TGBp1,CP,3’-NTS genes） and RNA polymerase gene sequences,respectively. The existence of PlAMV in the Netherlands lily was further confirmed by RT-PCR using the coat protein-specific primers（PlAMV-cpup/PlAMV-cpdw） of this virus. Phylogenetic analysis indicated that this isolate was clustered together with viral isolates from Netherlands lily,suggesting a high homology to the Netherlands lily viral isolates.To our knowledge,this is the first report of capturing PIAMV at the port of entry in China.