利用RT-PCR由大叶白蜡检出烟草花叶病毒

Detection of Tabacco mosaic virus from Fraxinus chinensis by RT-PCR

从表现花叶、卷叶症状的大叶白蜡病株上获得一病毒分离物 ,与烟草花叶病毒抗血清呈阳性反应。通过以上试验结果 ,分别提取花叶、卷叶症状白蜡病叶总RNA。根据烟草花叶病毒RNA序列设计引物 ,进行RT -PCR检测 ,扩增出大约 80 0bp的预期特异片段。将PCR产物连接 pMD 18-T载体 ,转化大肠杆菌DH5α,得到了含有目的片段的重组子 ,序列分析表明 ,与GenBank中登录序列号为AJ0 11933.1同源性达 99% ,确定该病毒分离物为烟草花叶病毒 (TabaccomosaicvirusTMV)。

An isolate of Tobacco mosaic virus (TMV) was isolated from Fraxinus chinensis (white ash) leaves with typical mosaic and leafroll symptoms in Beijing. The virus-infected plants reacted with an antiserum prepared against TMV using antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA). A pair of primers were designed based on the sequence of TMV RNA. RT-PCR was applied with total RNA isolated from 0.1g of infected white ash expressing mosaic and leafroll respectively. A fragment of about 800 bp was amplified from the infected samples. The PCR product was cloned into pMD 18-T Vector, and transformed into Escherichia coli DH5α cells. The recombinant plasmid was obtained and sequenced. The nucleotide sequence covered the entire CP gene and contained 815 nucleotides (including a pair of primers). The sequence shared 99% homology with the Vicia faba isolate (GenBank Accession No. AJ011933.1). we concluded that the virus isolated from white ash in Beijing is Tabacco mosaic virus (TMV).