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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 regular interval short palindrome repeat (CRISPR) of Shigella clustering and to compare its structure and homology with the sequence and sequence of Shigella in GenBank. Methods: The CRISPR of Shigella was amplified by PCR, and the homology analysis of repeats and spacer sequences was carried out by bioinformatics methods. The sequence alignment and its relationship with the flanking sequences were analyzed by using multiple sequence alignment. The BLAST analysis Sequence homology with plasmids and phages; weblogo analysis of repeat base sequence frequencies and RNAfold prediction of RNA secondary structure; homologous clustering analysis of repeated sequences and analysis of the homology of the repeated sequences with other bacteria by BLAST. Results: The three strains of clinical Shigella and nine strains of Shigella contain different numbers of CRISPRs. In the same CRISPR, there are some similar and some completely different spacer sequences. Some sequences of some CRISPR spacer sequences and The homology of the CRISPR flanking sequences may exist. Some spacer sequences may be splicing recombination between the information genes and the manipulation genes. The inconsistent repeats may be related to the evolution of the bacteria. The differences in the bases of the repeats affect the length of the stem and thus Affect the stability of secondary structure and the function of CRISPR. Repeats and individual distant species have high homology. Conclusion: Shigella has a variety of CRISPR structure, different CRISPR repeat sequence conservation. Some of the spacer sequences are homologous to the CRISPR flanking sequences and some of the spacer sequences are spliced with multiple genes.