生物膜形成中间状态下副溶血弧菌的基因转录谱分析

Transcriptomic profile of Vibrio parahaemolyticus in the intermediate state of biofilm formation

【目的】探究生物膜形成中间状态下副溶血弧菌的差异基因表达情况,为今后研究生物膜形成调控机制提供基因信息。【方法】以非生物膜形成条件下为参照,采用Illumina HiSeq测序平台进行转录组测序(RNA sequencing,RNA-seq)研究,分析生物膜形成中间状态下副溶血弧菌的基因表达情况,并采用实时定量PCR(quantitative real-time PCR,qPCR)进行验证。【结果】本研究共获得979个差异显著性表达基因(differentially expressed gene,DEG),其中下调基因379个,上调基因600个。基因本体(gene ontology,GO)分类分析结果显示,共有363个DEGs注释到分子功能、细胞组分和生物学过程3个一级分类和30个二级分类;京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)代谢途径分析结果显示,共有706个DEGs归到37个代谢通路中(Q value<0.05),差异表达基因主要集中在细胞代谢和转运通路上;蛋白相邻类的聚簇(clusters of orthologous groups,COG)分类结果显示,有888个DEGs可归为20个类别,涉及DEGs最多的为氨基酸转运与代谢、一般功能预测基因、能量产生与转换以及未知功能基因。qPCR验证挑选的DEGs变化趋势均与RNA-seq的结果一致。此外,从转录组数据中共筛选出10个c-di-GMP代谢相关基因、1个侧生鞭毛蛋白基因(lafA)、13个极生鞭毛合成相关基因、6个荚膜多糖合成相关基因、6个胞外多糖合成相关基因、5个IV型菌毛合成相关基因、膜融合蛋白(membrane fusion protein,Mfp)基因(cpsQ-mfpABC)、14个III型分泌系统1(T3SS1)相关基因、14个Vp-PAI基因(1个tdh2和13个T3SS2基因)、3个VI型分泌系统1(T6SS1)相关基因、6个T6SS2基因、45个推定调控子基因和15个推定的外膜蛋白基因。【结论】生物膜形成引起副溶血弧菌基因表达谱发生明显变化,差异表达基因中包含生物膜形成相关基因、关键毒力基因和许多推定调控子基因等,这为后续研究生物膜形成调控机制提供重要信息。

[Objective]To investigate the differentially expressed genes(DEGs)of Vibrio parahaemolyticus in the intermediate state of biofilm formation,and thus provide gene information for the future studies about the regulatory mechanisms of biofilm formation.[Methods]Illumina HiSeq and RNA sequencing(RNA-seq)assay were employed to analyze the gene expression of V.parahaemolyticus in the intermediate state of biofilm formation,and the results were then validated by quantitative real-time PCR(qPCR).[Results]A total of 979 DEGs were identified,including 379 down-regulated genes and 600 up-regulated genes.According to the results of gene ontology(GO)annotation,363 DEGs were annotated to three functional categories(biological process,molecular function,and cellular component)and 30 sub-categories.The results of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment showed that 706 DEGs were enriched in 37 pathways(Q value<0.05)and mainly involved in cellular metabolism and transport pathways.The results of clusters of orthologous groups(COG)classification showed that 888 DEGs were assigned to 20 categories,and the DEGs were mainly involved in amino acid transport and metabolism,general function prediction only,energy production and conversion,and function unknown.The expression trends of the DEGs validated by qPCR were consistent with the results of RNA-seq.In addition,the biofilm-associated genes and major virulence genes were identified from the RNA-seq data,including 10 c-di-GMP metabolism-associated genes,1 lateral flagellar gene(lafA),13 polar flagellar genes,6 capsular polysaccharide synthesis genes,6 exopolysaccharide synthesis genes,5 type IV pilus synthesis genes,6 membrane fusion protein(mfp)genes(cpsQ-mfpABC),14 type III secretion system 1(T3SS1)genes,14 Vp-PAI genes(tdh2 and 13 T3SS2 genes),3 type VI secretion system 1(T6SS1)genes,6 T6SS2 genes,45 putative regulator genes,and 15 putative outer membrane protein genes.[Conclusion]A large number of genes demonstrate changed expression levels during the biofilm formation of V.parahaemolyticus,including the biofilm-associated genes,key virulence factor genes,and putative regulator genes.The data presented here provided important gene information for the future studies about the regulation of biofilm formation.