基于RNA-Seq技术对血链球菌spxA_2敲除株的转录组分析  

Use of RNA-seq to identify and characterize Streptococcus sanguinis spxA2

作　　者：徐梦雅 王迪 李子豪 李婷 郑兰艳 

机构地区：[1]中国医科大学基础医学院病原生物学教研室,辽宁沈阳110122

出　　处：《中国病原生物学杂志》2018年第1期45-50,54,共7页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81170211)

摘　　要：目的应用RNA测序(RNA-Seq)技术分析spxA2调控的靶基因,以期揭示spx蛋白的转录调控机制,为血链球菌致病机制的深入研究奠定基础。方法采用高通量测序技术对血链球菌野生株和spxA2突变株进行转录组分析,将测序所产生的数据绘到SK36参考基因组上,获得各个基因的表达信息,应用基因组比对结果进行基因定量。利用edgeR软件分析△spxA2和WT中的基因差异表达情况。随机抽取若干差异表达基因,通过实时定量PCR对基因的差异表达情况进行验证。应用GOTermFinder软件,找出差异表达基因中显著富集GO条目,并推测差异表达基因行使的主要生物学功能;通过Pathway分析进一步了解差异表达基因所参与的代谢通路及其具体的生物学意义。结果将序列数据与公共数据库COG、GO、KEGG、NR、NT和Swiss-Pro进行BLAST比对,89.1%(1203个)独立基因得到功能注释。其中,859个独立基因注释到COG并被划分到25个功能类别,910个独立基因注释到GO数据库,707个(52.4%)独立基因被归为KEGG中的32条代谢途径。对△spxA2组和WT组进行基因表达差异分析,确定spxA2基因敲除株有17个基因表达上调,5个基因表达下调。GO功能显著性富集和Pathway显著性富集分析,表明筛选到的差异表达基因主要富集在氨基酸代谢生物学过程和酶活性细胞组分,共得到2条显著富集的代谢通路,涉及丙氨酸、天冬氨酸和谷氨酸代谢及牛磺酸和牛磺酸代谢。结论RNA-Seq所得序列数据在测序深度、覆盖率以及与血链球菌SK36标准株基因组的比对结果均较理想,转录组测序结果基本能反映血链球菌的整个转录组情况。spxA2是一个全局性转录调控因子,参与多个基因的转录调控,该蛋白的改变可导致血链球菌丙氨酸、天冬氨酸和谷氨酸代谢以及牛磺酸和牛磺酸代谢通路发生变化,可为深入研究血链球菌致病机制及其与代谢途径之间的联系提供重要依�Objective To use RNA-seq and bioinformatic analysis to identify and characterize target genes regulated by Streptococcus sanguinis spxA2 in order to lay the foundation for further elucidation of the role of spxA2 in the pathogene- sis and metabolic pathways of S. sanguinis. Methods The transcriptome profiles of wild-type S. sanguinis and the dxspxA2 strain were characterized with high-throughput sequencing technology using the HiseqTM 2500 platform and bioinformatie analysis. After raw sequencing data were processed, the normalized expression pattern for S. sanguinis transcripts was calculated using reads mapped onto the reference genome. Changes in the levels of expression of the tran- scripts depended largely on FPKM values for wild-type S. sanguinis and the spxA2 mutant. Enrichment analysis of GO terms was performed using a hypergeometrie test in order to identify differentially expressed genes （DEGs） with specific biological functions. S. sanguinis DEGs were mapped onto the KEGG pathway database using KOBAS 2.0 to identify pathways that were significantly regulated when spxA2 was deleted. Results Unigene sequences were matched against public databases such as COG （Cluster of Orthologous Groups of proteins）, GO （Gene Ontology）, KEGG （Kyoto Ency- clopedia of Genes and Genomes）, NR （Non-redundant Protein Sequence Database in GenBank）, NT （Non-redundant Nu- cleotide Sequence Database in GenBank）, and Swiss-Prot （Swiss-Prot Protein Sequence Database）. One thousand two hundred and three of the unigenes （89. 1%） were functionally annotated. Eight hundred and fifty-nine unigenes were matched to 25 functional categories in the COG database, 910 unigenes were matched to the GO database, and 707 unigenes were matched to 32 pathways in the KEGG database. Comparative analysis of the transcriptome of AspxA2 and wild-type S. saT＇zguinis revealed 22 DEGs, including 17 up-regulated and 5 down-regulated genes. GO functional enrich- ment analysis and pathway enrichment analysis indicated that D

关 键 词：血链球菌 spxA2 转录组测序 转录组分析 差异表达基因 

分 类 号：R378.12[医药卫生—病原生物学]