变异链球菌CRISPR-Cas9系统csn2基因突变株的转录组学分析

Transcriptomic Analysis of csn2 Gene Mutant Strains of Streptococcus mutans CRISPR-Cas9 System

目的对变异链球菌CRISPR-Cas9系统csn2基因突变株的转录组基因表达差异进行分析。方法培养变异链球菌UA159、csn2基因的缺失菌株Δcsn2及回补菌株。提取总RNA,采用高通量测序技术,进行转录组测序,基于差异表达基因GO和KEGG数据库分析的基础上,对其参与的生物学过程进行挖掘,采用qRT-PCR的方法验证转录组测序结果。结果转录组结果显示与UA159相比,Δcsn2中基因表达量变化在1倍以上的基因有176个(P<0.05),其中上调表达基因72个,下调表达基因104个,通过GO功能富集分析及KEGG代谢通路富集分析,发现上调和下调的差异表达基因(DEG)均参与的功能为氨基酸转运和代谢。除此之外,上调的DEG参与的生物过程主要与碳水化合物代谢、能量产生和转化、转录等有关;下调的DEG主要与脂质代谢、DNA的复制、重组和修复、信号转导机制、核苷酸转运和代谢等生物过程有关,还有部分DEG的功能尚不清楚。qRT-PCR验证发现,与UA159及Δcsn2/pDL278-csn2菌株相比,与支链氨基酸形成相关的基因leuA、leuC和leuD在Δcsn2中的表达显著下调。结论通过转录组测序和qRT-PCR验证发现Δcsn2中与支链氨基酸合成与细胞膜通透性相关的基因表达量发生了明显变化。

Objective To explore the differences in transcriptional levels between mutant strains of csn2 gene of CRISPR-Cas9 system of Streptococcus mutans(S.mutans)and wild-type strains.Methods The S.mutans UA159,csn2-gene-deleted strains(Δcsn2)and csn2-gene-covering strains(Δcsn2/pDL278-csn2)of S.mutans were cultivated.Total RNA was extracted,and high-throughput sequencing technology was used for transcriptome sequencing.Based on the GO analysis and the KEGG analysis of the differentially expressed genes,the biological processes involved were thoroughly examined.The qRT-PCR method was used to verify the transcriptome sequencing results.Results The transcriptome results showed that,compared with UA159,there were 176 genes inΔcsn2 whose gene expression changed more than one fold(P<0.05),of which 72 were up-regulated and 104 were down-regulated.The GO enrichment analysis and the KEGG enrichment analysis revealed that both the up-regulated and down-regulated differentially expressed genes(DEG)were involved in amino acid transport and metabolism.In addition,the biological processes that up-regulated DEGs participated in were mainly related to carbohydrate metabolism,energy production and conversion,and transcription;down-regulated DEGs were mainly related to lipid metabolism,DNA replication,recombination and repair,signal transduction mechanisms,nucleotide transport and metabolism.The functions of some DEGs were still unclear.Results of qRT-PCR verified that the expressions of leuA,leuC and leuD(genes related to the formation of branched-chain amino acids)were significantly down-regulated inΔcsn2 when compared with UA159 andΔcsn2/pDL278-csn2.Conclusion Through transcriptome sequencing and qRT-PCR verification,it was found that the expression of genes related to branched-chain amino acid synthesis and cell membrane permeability inΔcsn2 changed significantly.