多杀性巴氏杆菌CRISPR-Cas系统生物信息学分析

Bioinformatic Analysis of CRISPR-Cas System in Pasteurella multocida

【目的】了解多杀性巴氏杆菌中CRISPR-Cas系统的结构特征及cas基因的分布,为不同CRISPR亚型的应用及多杀巴氏杆菌的抗病毒进化研究提供基础。【方法】通过CRISPRCasfinder在线网站筛选多杀性巴氏杆菌基因组中的CRISPR簇和cas基因;通过生物信息学方法对各亚型系统中repeat和spacer序列保守性、repeat二级结构、cas基因核苷酸序列保守性、CRISPR簇相似性等进行分析。【结果】在选取的19株多杀性巴氏杆菌基因组中均存在Ⅰ-F、Ⅱ-C、Ⅲ-A亚型CRISPR系统。在相同亚型的CRISPR系统中repeat序列有着较保守的回文重复序列,但在不同CRISPR亚型中repeat序列差异较大。Ⅱ-C亚型的tracrRNA可以与crRNA形成保守的结构。在各亚型CRISPR系统中,新获取的spacer序列可以插入到CRISPR簇的5′-端、3′-端或中间位置。通过对各菌株spacer序列分析发现,在菌株内或菌株间的CRISPR序列中均存在着相同的spacer序列,这可能与细菌在遗传进化过程中repeat-spacer发生序列重组或基因水平转移产生的趋同进化有关。【结论】19株多杀性巴氏杆菌中含有Ⅰ-F、Ⅱ-C、Ⅲ-A 3种CRISPR-Cas系统,不同CRISPR亚型中repeat序列差异较大,spacer特征性重复的分布可能与多杀性巴氏杆菌在遗传进化过程中repeat-spacer发生序列重组有关。

【Objective】This study was aimed to understand the structural characteristics of CRISPR-Cas system and the distribution of cas gene in Pasteurella multocida,and provide a basis for the application of different CRISPR subtypes and the antiviral evolution of Pasteurella multocida.【Method】The CRISPR cluster and cas gene were screened in the genome of Pasteurella multocida via CRISPRCasfinder online website.The conservation of repeat and spacer sequence,the secondary structure of repeat,the conservation of nucleotide sequence of cas gene,and the similarity of CRISPR cluster in various subtype system were investigated using bioinformatics analysis.【Result】There were three subtypes(Ⅰ-F,Ⅱ-C andⅢ-A)of CRISPR-Cas system identified in 19 selected Pasteurella multocida strains.The repeat sequences in the same CRISPR subtypes had a relatively conservative palindrome repeat sequence,whereas the palindromic repeats varied in the different CRISPR subtypes.The tracrRNA could bind with crRNA in subtypeⅡ-C system and form conservative structures.In CRISPR system of each subtype,the newly acquired spacer was integrated both at the 5′-end,3′-end or internal locations of CRISPR cluster.Through the analysis of the spacer sequence of each strain,it was found that the same spacer sequence existed in the CRISPR sequence within or between strains,which might relate to the sequence recombination of repeat-spacer in bacteria or convergent evolution of gene level shifts during genetic evolution.【Conclusion】The 19 strains of Pasteurella multocida contained 3 CRISPR-Cas systems ofⅠ-F,Ⅱ-C andⅢ-A.The repeat sequence of different CRISPR subtypes was significantly different.The distribution of spacer characteristic repeats might be related to the sequence recombination of repeat spacer in Pasteurella multocida during genetic evolution.