Cloning and identification of the plasmid replicationgene repC in Mesorhizobium huakuii

Abstract:

Abstract: [Objective] The repC gene is the principal initiation protein gene for plasmid replication. We identified the repC-like sequences from Mesorhizobium huakuii strain HN3015 and its derivatives of plasmid curing. [Methods] Primers of RC1 and RC3 were used to amplify the repC-like sequences by a polymerase chain reaction, the PCR products were obtained and cloned into plasmid vector pMD-18T and then sequenced. Location of the repC sequences on different plasmids in the strains tested was carried out by Southern blotting. The nucleotides homology analysis of the repC gene was carried out using the BLAST. Amino acid sequences were deduced using the ExPASy. Multiple sequences alignments were performed using the ClustalW. Analysis of protein secondary structure was carried out using the PredictProtein. [Results] The repC-like sequences were obtained from the strains tested. The sizes of the PCR products were about 750 bp. The results of Southern blotting showed that the repC-like sequences were only associated with a plasmid in the stains tested. [Conclusions] The repC sequences of the strains tested showed 100% sequence similarity, but were obviously different from that of other rhizobia strains.

Key words:Keywords: Mesorhizobium huakuii; repC; PCR amplification; Identification