| Begomovirus belongs to the family of Geminiviridae.Phylogenetic analysis showed that Begomoviruses is significantly different from geminivirus.Therefore,the Begomoviruses which infection sweet potato was named "Sweepoviruses".At present,there are 7 species of Begomovirus infecting sweetpotato in China.Sweet potato leaf curl Georgia virus(SPLCGV)is one of major viruses infecting sweetpotato in China.But,at present,we are not very clear about its biological characteristics,genome variation and so on,which lead to the blindness of the monitoring and early warning and prevention.In this study,the coat protein gene of SPLCGV isolates from China were cloned by PCR.Sequence analysis showed that the CP gene of eleven SPLCGV isolates was 765 bp and encoded 254 amino acid residues.The CP of SPLCGV isolates shared 91.4%-100% and95.7%-100% identities at the nucleotide and amino acid level respectively.Furthermore,the CP gene of a SPLCGV isolate was cloned into expression vector p ET-28a(+)for overexpression in prokaryotic cells.The result of SDS-PAGE showed that a 21.7k Da specific fusion was produced after induction with IPTG,the CP gene experssed in Escherichia coli.BL21 at a high level.Based on the nucleotide sequences of 7 species,a sensitive and stable real-time PCR assay was established for detecting Sweepoviruses based on primers and probes from the coat protein gene sequences.The results showed that the this method can be used to detect the presence of sweepoviruses and the slope value of standard curves with plasmid DNA were 0.999 and-3.261,the amplification efficiency of PCR was 102.603%.The real time fluorescent assay has a highesensitivity,about 100 fold,in detection.The real-time PCR assay demonstrated here for rapid and quantitative detection of Sweepoviruses which can provides a technical means for the early warning and epidemiological study of Sweepoviruses. |
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