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【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;Predict Protein进行蛋白二级结构分析。【结果】供试菌株均扩出了750bp左右的repC序列。Southern杂交结果显示:repC基因在每个供试菌株中只存在于一个质粒上。【结论】生物信息学分析结果表明:供试华癸中生根瘤菌菌株的repC序列相似性高达100%,但与其它根瘤菌的repC基因有明显差异。本研究对深入阐明华癸中生根瘤菌质粒复制机制具有一定参考价值。
【Objective】 repC is the initiator protein necessary for plasmid replication. The aim of this study was to clone and identify the repC gene of Huachui Zhongnorhizobium strain HN3015 and its plasmid-abolishing mutant. 【Method】 The PCR products of repC gene were amplified by universal primers RC1 and RC3. The amplified product was cloned into vector pMD-18T and sequenced. The repC gene was located by Southern hybridization. Homology search was performed using BLAST tools using on-line software to analyze the sequence features of the genes; ExPASy deduced amino acid sequences; ClustalW multiple comparisons of homologous nucleotide and amino acid sequences; and Predict Protein for protein secondary structure analysis. 【Result】 The results showed that 750bp repC sequences were amplified from the tested strains. Southern blot results showed that the repC gene was present on only one plasmid per tested strain. 【Conclusion】 The results of bioinformatics analysis showed that the similarity of the repC sequences of the tested R. solanacearum strains was up to 100%, but there was a significant difference with other rhizobia repC genes. This study has some reference value for further elucidating the mechanism of plasmid replication in Rhizobium japonicus.