华癸中生根瘤菌质粒复制基因repC的克隆与鉴定

摘要：【目的】repC为质粒复制必需的起始蛋白基因。本研究旨在对华癸中生根瘤菌菌株HN3015及其质粒消除突变株进行repC基因的克隆和鉴定。【方法】采用通用引物RC1和RC3进行repC基因的PCR扩增，扩增产物克隆到载体pMD-18T，然后测序。利用Southern 杂交对repC基因定位。利用在线软件分析基因的序列特征，BLAST 工具进行同源性搜索；ExPASy推断其氨基酸的序列；ClustalW进行同源核苷酸和氨基酸序列的多重比较分析；PredictProtein 进行蛋白二级结构分析。【结果】

Cloning and identification of the plasmid replicationgene repC in Mesorhizobium huakuii

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.