志贺菌成簇的规律间隔短回文重复序列的检测及同源性分析

Detection and homology analysis of clustered regularly interspaced short palindromic repeats in Shigella

目的:检测志贺菌成簇的规律间隔短回文重复序列(CRISPR),并与GenBank全基因组中志贺菌重复序列和间隔序列对比,分析其结构特征及同源性。方法:利用PCR法扩增获得志贺菌CRISPR,采用生物信息学方法对重复序列及间隔序列进行同源性分析;运用多序列比对分析间隔序列特点及其与侧翼序列间的联系;BLAST分析间隔序列与质粒和噬菌体的同源性;weblogo分析重复序列碱基频率及RNAfold预测其RNA二级结构;重复序列的同源聚类分析并用BLAST分析重复序列与其他细菌的同源性。结果:被测3株临床志贺菌及9株全基因组志贺菌中均含有不同数量CRISPR;同一个CRISPR中,间隔序列有的相似、有的完全不同,某些CRISPR间隔序列的部分序列与CRISPR侧翼序列存在同源性,某些间隔序列可能是信息基因和操纵基因的拼接重组;重复序列保守性不一致,可能与细菌进化存在一定的联系,重复序列碱基的差异影响茎的长度,从而影响二级结构的稳定性及CRISPR的功能。重复序列与个别远缘菌种具有较高的同源性。结论:志贺菌存在多样的CRISPR结构,不同CRISPR的重复序列保守性不同。某些间隔序列部分与CRISPR侧翼序列同源,某些间隔序列是多基因拼接而成。

Objective To detect the clustered regularly interspaced short palindromic repeats（CRISPR）of Shigella,and to analyze the characteristics and homology of their repeats and spacers with complete genome Shigellain GenBank.Methods The CRISPR were obtained by PCR amplification method,and the homology of repeats and spacers was analyzed by bioinformatics;multiple sequence alignment was used to analyze the features of spacers and the relationship between the spacers and flanking sequences;BLAST was used to analyze the homology between the spacers and plasmids and phages;weblogo was used to analyze the frequency of the bases of the repeats and RNAfold was used to predict the secondary structures of the repeats RNA;homology clustering analysis of repeats was performed and BLAST was used to analyze the homology of the repeats with other bacteria.Results All 3studied clinical strains and 9complete genome Shigella contained different number of CRISPR.In the same CRISPR,the spacers were similar or different;the partial sequences of the spacers in some CRISPR and flanking sequences of CRISPR had the same sequences.Some spacers possible came from the splicing and gene recombination between the informational genes and operational genes.There may be relationship between the repeats which were not completely conserved and bacterial evolution.The differences of the bases of the repeats affected the length of stem,thus affected the stability of the secondary structure and the function of CRISPR.The repeats had high similarity with the several individual species of distant relationships.Conclusion The different CRISPR structures exist in Shigella,and the conservation of the repeats in different CRISPR is different.The partial sequences of some spacers have homology with the flanking sequences of CRISPR,and some spacers come from the gene splicing.