侵染半夏的黄瓜花叶病毒RNA3全序列分析及侵染性克隆构建

Full-length sequence analysis of CMV-PGs genomic RNA3 infecting Pinellia ternate and constructing infectious clone

对从我国甘肃省天南星科植物半夏上获得的黄瓜花叶病毒分离物(CMV-PGs)的RNA3进行全长克隆和序列分析及侵染性克隆构建.结果表明:CMV-PGsRNA3序列全长2179nt,与CMV-Fny株系RNA3的同源性为83.1%.选取已报道CMV株系的38个RNA3全长序列,根据CP核苷酸序列进行同源性比较,系统进化树分析表明:CMV-PGs属于亚组ⅠB,但是相对独立.进一步构建CMV-PGsRNA3的侵染性克隆,通过体外转录获得具有侵染活性的RNA转录本,将CMV-PGsRNA3的侵染性转录本与CMV-FnyRNA1和RNA2的侵染性转录本混合接种烟草,成功获得假重组体F1F2P3.

Isolate CMV-PGs was obtained from NA sequence of genomic RNA3 of the isolate Pinellia ternate in Gansu Province. The full-length cDwas determined by RT-PCR and sequene analysis. The results showed that CMV-PGs RNA3 consisted of 2 179 nucleic acids, and the overall sequence identity between CMV-PGs RNA3 and CMV-Fny RNA3 was 83.1%. Phylogenetic analysis of CP suggested that CMV-PGs belonged to subgroup Ⅰ B, but relatively independent from reported strains of this subgroup. In addition, infectious clone of CMV-PGs RNA3 was constructed. Infectious RNA transcript of CMV-PGs RNA3 was generated by external transcription. Along with the RNA transcripts of CMV-Fny, the RNA transcripts were combined to reform wild type CMV-Fny and form the pseudorecombinant between CMV-Fny RNA1, RNA2 and CMV-PGs RNA3 （namely FIF2P3）.