植物乳杆菌PC518质粒pLP224的发现以及pMV158家族质粒的保守性和多样性分析

Characterization of plasmid pLP224 from Lactobacillus plantarum PC518 and analysis of conservation and diversity of pMV158 plasmid family

【背景】植物乳杆菌含有丰富的天然质粒,分析这些质粒的序列特征有利于分析质粒所携带的遗传信息。【目的】分析从植物乳杆菌PC518分离的新质粒pLP224,聚类分析其所属家族质粒的保守性与多样性。【方法】提取植物乳杆菌PC518的质粒,酶切后构建质粒DNA文库,测序和BLAST鉴定文库中的新序列;通过反向PCR完成质粒全序列测定,注释新质粒;使用进化树软件MEGA X构建质粒的Rep蛋白进化树,并分析结合序列的变化。【结果】从植物乳杆菌PC518分离出一个质粒pLP224,大小为1766bp,其中(G+C)mol%含量为41.39%,与已知质粒的最大序列相似性为86.85%。推定其复制方式为滚环复制,属于pMV158家族成员。17个pMV158家族质粒的Rep蛋白分析表明:pMV158家族质粒的Rep蛋白进化距离越近,其dso位点的结合序列相似性越高,进化距离越远则其序列相似性越低。【结论】pLP224是pMV158家族的新成员。pMV158家族质粒在dso位点的切开序列上保守,在结合序列上多样。其Rep蛋白随结合序列变化而不同。这种差异有利于pMV158家族不同成员在同一宿主的共存,是家族成员持续存在并稳定进化的基础。

[Background]Lactobacillus plantarum has a lot of natural plasmids.Analyzing the sequence characteristics of these plasmids was beneficial to research the genetic information of them.[Objective]To identify a new plasmid isolated from L.plantarum PC518 and analyze conservation and diversity of plasmids belonging to a family.[Methods]Plasmid DNA was isolated from L.plantarum PC518,and two plasmid DNA libraries were constructed after enzyme digestion.New sequences in the libraries were identified by sequencing and BLAST alignment.The whole sequence of the plasmid was completed by reverse PCR.The new plasmid was annotated.The phylogenetic tree of Rep protein was built by MEGA X,and analysis of binding sequence was carried out.[Results]Plasmid pLP224 was a 1766 bp circular molecule with a(G+C)mol%content of 41.39%.The maximum sequence similarity with known plasmids was 86.85%.pLP224 belonged to the pMV158 family of rolling-circle replication.The results of phylogenetic tree based on Rep protein showed that the closer evolution distance was the higher similarity of binding sequence,and the farther evolution distance was the lower similarity of binding sequence.[Conclusion]pLP224 was a new member of the pMV158 family.The nick sequences of dso site belonging to the pMV158 family were conservative,and the binding sequences belonging to the pMV158 family were diverse.The Rep protein varies with the binding sequence.The difference of Rep protein was conducive to the coexistence in a host,which was the basis for the continuous existence and stable evolution of a family.