贵州医科大学学报

2021, v.46;No.254(11) 1241-1248+1270

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基于RNA-Seq技术分析不同生物被膜形成能力的临床耐药鲍曼不动杆菌基因表达差异
Analysis of the gene expression difference of clinically resistant Acinetobacter baumannii with different biofilm formation ability though RNA-Seq

刘巍巍;国果;吴兆颖;毛成菊;李忠旬;贾利娜;尚小丽;彭建;吴建伟;
LIU Weiwei;GUO Guo;WU Zhaoying;MAO Chengju;LI Zhongxun;JIA Lina;SHANG Xiaoli;PENG Jian;WU Jianwei;Department of Human Parasitology,School of Basic Medical Sciences,Guizhou Medical University;Key Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University;School of Biology & Engineering,Guizhou Medical University;

摘要(Abstract):

目的了解不同生物被膜形成能力的临床分离耐碳青霉烯类鲍曼不动杆菌菌株(CRAB)基因表达的差异。方法选取8株临床分离CRAB,将生物被膜形成能力强的菌株4、55、78、117号作为强生物被膜(BF)组,将生物被膜形成能力弱的菌株13、177、191、196号作为弱生物被膜(WBF)组,培养两组菌株形成生物被膜,分别提取RNA进行文库构建和转录组测序(RNA-Seq),以鲍曼不动杆菌AB030(NZ_CP009257.1)的基因组作为参考基因组对测序数据进行分析,鉴定差异表达基因(DEGs),并进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析,选取8个DEGs利用实时荧光定量PCR(qRT-PCR)验证转录组测序结果。结果 RNA样品完整无污染,测序数据错误率为0.03%;BF组与WBF组相比,鉴定到171个DEGs,其中106条基因为上调表达,65条基因为下调表达;这些基因主要涉及DNA结合、膜蛋白、菌毛调控、ATP的合成和分解等;GO分析显示较多基因被注释到生物过程部分,KEGG分析显示DEGs富集到多种物质代谢途径;选取的8个DEGs利用qRT-PCR进行验证,结果与转录组测序结果趋势一致。结论不同生物被膜形成能力的临床CRAB的基因表达存在差异,多基因和多条信号通路影响生物被膜的形成。
Objective To understand the transcriptome variation in gene expression of clinically carbapenem-resistant Acinetobacter baumannii with different biofilm formation ability.Methods Eight clinically isolated carbapenem-resistant Acinetobacter baumannii were selected,and the strains 4,55,78,and 117 with strong biofilm formation ability were grouped into a group and named as strong biofilm(BF) Group,the strains 13,177,191,and 196 with weak biofilm formation ability were divided into another group and named the Weak Biofilm(WBF) group.Two groups of strains were cultivated to form biofilm,and ribonucleic acid(RNA) samples were extracted,library construction and transcriptome sequencing were performed.Use the genome of Acinetobacter baumannii AB030(NZ_CP009257.1) as the reference genome to analyze the sequencing data,identify differentially expressed genes(DEGs),and perform Gene Ontology(GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis.To validate the results of RNA-Seq,eight DEGs were selected for Quantitative Real-time PCR(qRT-PCR).Results The RNA sample is intact and pollution-free,and the error rate of sequencing data does not exceed 0.03 %.After transcriptome sequencing,compared with the WBF group,171 DEGs were identified in the BF group,of which 106 genes were up-regulated and 65 genes were down-regulated.These genes mainly involved in DNA binding,membrane protein,fimbriae regulation,ATP synthesis and decomposition,etc.GO analysis showed that more genes were annotated to the biological process part.KEGG analysis showed that DEGs were enriched in a variety of material metabolism pathways,such as pyruvate metabolism,methane metabolism,etc.,as well as signal pathways such as biosynthesis of antibiotics and vancomycin resistance.Eight DEGs were selected and analysised by qRT-PCR,and the results were consistent with the results of RNA-Seq.Conclusion There are differences in the gene expression of clinical CRABs with different biofilm formation ability,and multiple genes and multiple signal pathways affect the formation of biofilms.

关键词(KeyWords): 细菌;耐碳青霉烯类鲍曼不动杆菌;生物被膜形成能力;菌毛;外膜蛋白;信号通路;差异表达基因
bacteria;carbapenem-resistant Acinetobacter baumannii(CRAB);biofilm formation;fimbria;outer membrane protein;pathway;differentially expressed genes(DEGs)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基因(81660347);; 国家科技支撑计划(2011BAC06B12);; 贵州省科技厅科技支撑计划[黔科合支撑(2019)2823]

作者(Authors): 刘巍巍;国果;吴兆颖;毛成菊;李忠旬;贾利娜;尚小丽;彭建;吴建伟;
LIU Weiwei;GUO Guo;WU Zhaoying;MAO Chengju;LI Zhongxun;JIA Lina;SHANG Xiaoli;PENG Jian;WU Jianwei;Department of Human Parasitology,School of Basic Medical Sciences,Guizhou Medical University;Key Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University;School of Biology & Engineering,Guizhou Medical University;

DOI: 10.19367/j.cnki.2096-8388.2021.11.001

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