侵染扶桑的烟草花叶病毒分离物鉴定

Identification of tobacco mosaic virus isolate from Hibiscus rosa - sinensis Linn

从表现叶斑驳症状的扶桑病株上获得一病毒分离物,电镜下可见约300 nm×18 nm的杆状粒子,其与烟草花叶病毒抗血清呈明显的阳性反应,dsRNA约为6.4 kbp。根据烟草花叶病毒(tobacco.mosaic virus,TMV)的RNA序列设计引物,进行RT-PCR检测,扩增出约800 bp的预期特异片段。将PCR产物连接pMD18-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,与周雪平等报道的序列(GenBank AJ011933.1)同源性达99％。通过生物学、病毒粒子观察、血清学以及分子生物学实验结果,确定该病毒分离物为TMV。

The rod-shaped virus particles of about 300 nm×18 nm were isolated from Hibiscus rosa - sinensis expressing systemic symptoms, including mottles in leaves. The virus-infected plants reacted with an antiserum prepared against tobacco mosaic virus (TMV) using antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA). The double -stranded RNA pattern revealed one single band of about 6.4kb. A pair of primers was designed based on the sequence of TMV RNA. RT-PCR was performed with total RNAs isolated from 0.1 g of infected Nicotiana tabacum and H. rosasinensis, 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 the primer sequence) . The sequence shared 99% homology with the Vicia faba isolate (GenBank Accession No.AJ011933.1).On the basis of host range, symptoms on indicator plants, serology, particle morphology and sequence of CP gene, we concluded that the virus isolated from H. rosa -sinensis in Beijing is tobacco mosaic virus (TMV).