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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%。
In order to clarify the pathogen of Diantle mosaic disease occurring in Beijing, the molecular identification was carried out by RT-PCR based on the isolation and purification of biological inoculation. The pure isolates of the virus were obtained by inoculation of the indicated plants and single spot separation. The samples of Rehmannia glutinosa rhizome with obvious mosaic symptoms were infected by cucumber mosaic virus (CMV) and broad bean wilt virus2 (BBWV2) by RT-PCR and sequence analysis. To further clarify the taxonomic status of BBWV2-Rehmannia glutinosa isolate (BBWV2-Rg), the BBWV2-Rg RNA2 polyprotein gene was cloned and sequenced. The results showed that the polyprotein gene consisted of 3 195 nucleotides , Encoding 1 064 amino acids. Sequence alignment analysis revealed that the nucleotide sequences of the LCP gene and small subunit SCP gene encoded by BBWV2-Rg were 78.69% homologous to the corresponding published nucleotide sequences of other strains of BBWV2, respectively ~ 89.30% and 76.99% ~ 90.52%, respectively. The amino acid sequence homologies were 91.29% ~ 97.51% and 87.82% ~ 96.45%, respectively.