烟草花叶病毒丁香分离物的分离与鉴定

Isolation and identification of Tobacco mosaic virus infecting Syringa oblate

从表现花叶症状的丁香病株上获得一病毒分离物 ,其在电镜下为约 3 0 0 nm× 1 8nm的杆状粒子 ;电泳分析表明感病组织中 ds RNA大约为 6.4kbp,而其外壳蛋白分子量约为 1 7.6k Da。以上实验结果初步将该病毒分离物鉴定为烟草花叶病毒属 ( Tobamovirus)。根据该属病毒复制酶基因序列设计通用引物 ,进行 RT-PCR检测 ,扩增出约 1 0 0 0 bp的预期特异片段( Gen Bank AY5 6670 3 )。将 PCR产物克隆后测序 ,序列分析表明 ,与从蚕豆中分离的 TMV-B株系序列 ( Gen Bank AJ0 1 1 93 3 .1 )同源性为 99.90 %。根据烟草花叶病毒 ( Tobacco mosaicvirus,TMV)的 RNA CP基因序列设计引物 ,进行 RT-PCR,扩增出约 80 0 bp的预期特异片段 ( Gen Bank AY5 6670 2 ) ,序列分析表明 ,与 TMV-B株系序列 ( Gen Bank AJ0 1 1 93 3 .1 )同源性达99% ,上述实验结果表明 ,该病毒分离物为 TMV。由于该分离物与 TMV-B在指示植物上的症状存在明显差异 ,所以 ,作者把该分离物暂命名为 TMV-S。

Rod-shaped virus particles about 300 nm×18 nm were isolated from Syringa oblate expressing systemic mosaic in leaves. The double-stranded RNA pattern revealed one single band of about 6.4 kbp. The virus contained coat protein of approximately 17.6 kDa. According to these results, the virus isolate was identified as a member of Tobamovirus. A pair of primers were designed based on Tobamovirus RNA RdRp gene, and a fragment of about 1 000 bp(GenBank AY566703)was amplified by RT-PCR. The PCR product was cloned and sequenced. The sequence shared 99.90% homology with TMV-B strain (GenBank Accession No. AJ011933.1). Another pair of primers was designed based on TMV RNA CP gene. A fragment of about 800 bp(GenBank AY566702)was amplified. The recombinant plasmid was obtained and sequenced. The sequence shared 99% homology with TMV-B strain (GenBank Accession No. AJ011933.1). CP gene sequence and amino acids shared 99.37%, 100% homology with TMV-B strain respectively. On the basis of above results, the virus isolated from S. oblate in Beijing is Tobacco mosaic virus (TMV).