华癸中生根瘤菌质粒复制基因repC的克隆与鉴定  

Cloning and identification of the plasmid replication gene repC in Mesorhizobium huakuii

作　　者：胡国元[1] 王善明[2] 李伟伟[1] 李友国[2] 

机构地区：[1]武汉工程大学绿色化工过程省部共建教育部重点实验室,武汉430073 [2]华中农业大学农业微生物学国家重点实验室,武汉430070

出　　处：《微生物学报》2009年第11期1465-1469,共5页Acta Microbiologica Sinica

基　　金：湖北省自然科学基金(2007ABA316);湖北省教育厅科学技术研究计划(D20081509)~~

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;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] 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 RCl 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.

关 键 词：华癸中生根瘤菌 repC PCR扩增 鉴定 

分 类 号：S154.3[农业科学—土壤学]