北京地区地黄花叶病病原的分子鉴定

Molecular identification of viral pathogens inducing Rehmannia glutinosa mosaic disease in Beijing

为明确北京地区发生的地黄花叶病的病原,在进行生物学接种分离纯化的基础上利用RT-PCR方法对其进行了分子鉴定。通过接种指示植物和进行单斑分离,获得了病毒的纯分离物。经RT-PCR和序列测定及分析,有明显花叶症状的北京地黄样品受黄瓜花叶病毒Cucumber mosaic virus（CMV）和蚕豆萎蔫病毒2Broad bean wilt virus2（BBWV2）复合侵染。为进一步明确BBWV2地黄分离物（BBWV2-Rg）的分类地位,克隆了BBWV2-Rg RNA2多聚蛋白基因,并进行了序列测定和分析,结果表明该多聚蛋白基因由3 195个核苷酸组成,编码1 064个氨基酸。经序列比对分析,BBWV2-Rg编码的外壳蛋白大亚基LCP基因和小亚基SCP基因核苷酸序列与已发表的BBWV2其它株系相应基因核苷酸序列的同源性分别为78.69%~89.30%和76.99%~90.52%,氨基酸序列同源性分别为91.29%~97.51%和87.82%~96.45%。

To identify the viruses that induced Rehmannia glutinosa mosaic disease in Beijing,biological inoculation and RT-PCR used.Virus isolates were purified through single lesions and propagated on indicator plants.Results of RT-PCR and sequencing showed that there were Cucumber mosaic virus（CMV） and Broad bean wilt virus 2（BBWV2） in mosaic R.glutinosa leaves.To further characterize the BBWV2 isolate R.glutinosa（BBWV2-Rg）,the polyprotein gene coded by BBWV2-Rg RNA2 was cloned and sequenced,and it consisted of 3195 nucleotides（nt）,which encoded 1064 amino acid（aa） residues.Sequence alignments showed that BBWV2-Rg LCP and SCP,in nucleotide identity,were from 78.69% to 89.30% and from 76.99% to 90.52% to that of other BBWV2 strains,respectively,and in deduced amino acid,were from 91.29% to 97.51% and from 87.82% to 96.45% separately identical to that of other BBWV2 strains.