敲除小RNA gcvB后沙门菌的转录组分析
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摘要
为筛选与沙门菌小RNA gcvB相关的靶基因,采用高通量的转录组测序(RNA-seq)技术对野生型沙门菌和敲除小RNA gcvB沙门菌的mRNA进行对比分析,全面地预测GcvB的靶基因的数量与种类,通过GO和KEGG数据库对筛选基因进行比对注释,进一步分析筛选基因的主要功能及涉及的生物过程。并利用荧光定量PCR对部分差异表达基因进行验证。结果显示:差异表达基因共1 244个,其中基因表达上调678个,基因表达下调566个。GO富集分析显示,差异表达基因主要涉及氧化还原活性、铁离子结合、运动活性等分子功能,细菌鞭毛的细胞成分,细胞呼吸、钴胺素代谢过程和钴胺素生物合成过程等生物过程。KEGG数据库分析显示,差异表达基因主要参与细菌趋化性、卟啉和叶绿素代谢、不同环境中的微生物代谢、双组分系统、甲烷代谢等14个信号通路。对筛选出的10个差异表达基因进行荧光定量PCR验证,结果显示:差异表达基因相对表达量与RNA-seq测序结果基本一致。本研究为进一步探明小RNA gcvB与靶基因相互作用、小RNA的调控机制,以及沙门菌致病机制奠定了基础。
In this article, high-throughput transcriptome sequencing(RNA-seq) technology was used to screen potential regulatory target genes of small RNA(sRNA) gcvB in Salmonella. Comparative analysis of mRNA in wild-type strain and knockout small RNA gcvB strain has been used. The number and type of GcvB target genes were comprehensively predicted, the main functions and the biological processes of the selected genes were compared by the GO and KEGG databases for further analysis. Some differentially expressed genes were verified by real-time PCR. The results showed that there were 1 244 differential expression genes have been screened out, including 678 up-regulated genes and 566 down-regulated genes. GO enrichment analysis showed that the differentially expressed genes mainly involved molecular functions such as redox activity, iron binding, locomotor activity, cellular components of bacterial flagella, cellular respiration, cobalamin metabolism, and cobalamin biosynthesis. KEGG database analysis showed that differential expression genes mainly participated in 14 signaling pathways, including bacterial chemotaxis, porphyrin and chlorophyll metabolism, microbial metabolism in different environments, two-component systems and methane metabolism. Ten differentially expressed genes in WT strain and ΔGcvB strain were selected from transcriptome analysis and carried out the further Real-time PCR test, and the results show that the trend of gene expression is consistent in both transcriptome analysis and in Real-time PCR test. The study lays the foundation for further exploration of the interaction between sRNA GcvB and target genes, the regulation of sRNA and the pathogenic mechanism in Salmonella.
In this article, high-throughput transcriptome sequencing(RNA-seq) technology was used to screen potential regulatory target genes of small RNA(sRNA) gcvB in Salmonella. Comparative analysis of mRNA in wild-type strain and knockout small RNA gcvB strain has been used. The number and type of GcvB target genes were comprehensively predicted, the main functions and the biological processes of the selected genes were compared by the GO and KEGG databases for further analysis. Some differentially expressed genes were verified by real-time PCR. The results showed that there were 1 244 differential expression genes have been screened out, including 678 up-regulated genes and 566 down-regulated genes. GO enrichment analysis showed that the differentially expressed genes mainly involved molecular functions such as redox activity, iron binding, locomotor activity, cellular components of bacterial flagella, cellular respiration, cobalamin metabolism, and cobalamin biosynthesis. KEGG database analysis showed that differential expression genes mainly participated in 14 signaling pathways, including bacterial chemotaxis, porphyrin and chlorophyll metabolism, microbial metabolism in different environments, two-component systems and methane metabolism. Ten differentially expressed genes in WT strain and ΔGcvB strain were selected from transcriptome analysis and carried out the further Real-time PCR test, and the results show that the trend of gene expression is consistent in both transcriptome analysis and in Real-time PCR test. The study lays the foundation for further exploration of the interaction between sRNA GcvB and target genes, the regulation of sRNA and the pathogenic mechanism in Salmonella.
引文
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[2] ALBINO L A A,ROSTAGNO ■,et al.Isolation,characterization,and application of bacteriophages for Salmonella spp.biocontrol in pigs[J].Foodborne Pathog Dis,2014,11(8):602-609.
[3] PARKER A,GOTTESMAN S.Small RNA regulation of TolC,the outer membrane component of bacterial multidrug transporters[J].J Bacteriol,2016,198(7):1101-1113.
[4] JORGENSEN M G,THOMASON M K,HAVELUND J,et al.Dual function of the McaS small RNA in controlling biofilm formation[J].Genes Dev,2013,27(10):1132-1145.
[5] HAO Y,UPDEGROVE T B,LIVINGSTON N N,et al.Protection against deleterious nitrogen compounds:role of σS-dependent small RNAs encoded adjacent to sdiA[J].Nucleic Acids Res,2016,44(14):6935-6948.
[6] BEISEL C L,STORZ G.The base-pairing RNA spot 42 participates in a multioutput feedforward loop to help enact catabolite repression in Escherichia coli[J].Mol Cell,2011,41(3):286-297.
[7] PAPENFORT K,ESPINOSA E,CASADES■,et al.Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella[J].Proc Natl Acad Sci U S A,2015,112(34):E4772-4781.
[8] FONTAINE F,GASIOROWSKI E,GRACIA C,et al.The small RNA SraG participates in PNPase homeostasis[J].RNA,2016,22(10):1560-1573.
[9] KAKOSCHKE T K,KAKOSCHKE S C,ZEUZEM C,et al.The RNA Chaperone Hfq is essential for virulence and modulates the expression of four adhesins in Yersinia enterocolitica[J].Sci Rep,2016,6:29275.
[10] PAPENFORT K,VOGEL J.Small RNA functions in carbon metabolism and virulence of enteric pathogens[J].Front Cell Infect Microbiol,2014,4:91.
[11] GOTTESMAN S.Micros for microbes:non-coding regulatory RNAs in bacteria[J].Trends Genet,2005,21(7):399-404.
[12] SHARMA C M,DARFEUILLE F,PLANTINGA T H,et al.A small RNA regulates multiple ABC transporter mRNAs by targeting C/A-rich elements inside and upstream of ribosome-binding sites[J].Genes Dev,2007,21(21):2804-2817.
[13] MELAMED S,PEER A,FAIGENBAUM-ROMM R,et al.Global mapping of small RNA-target interactions in bacteria[J].Mol Cell,2016,63(5):884-897.
[14] SHARMA C M,PAPENFORT K,PERNITZSCH S R,et al.Pervasive post-transcriptional control of genes involved in amino acid metabolism by the Hfq-dependent GcvB small RNA[J].Mol Microbiol,2011,81(5):1144-1165.
[15] URBANOWSKI M L,STAUFFER L T,STAUF-FER G V.The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli[J].Mol Microbiol,2000,37(4):856-868.
[16] JIN Y,WATT R M,DANCHIN A,et al.Small noncoding RNA GcvB is a novel regulator of acid resistance in Escherichia coli[J].BMC Genomics,2009,10:165.
[17] BARRETO B,ROGERS E,XIA J,et al.The small RNA GcvB promotes mutagenic break repair by opposing the membrane stress response[J].J Bacteriol,2016,198(24):3296-3308.
[18] PAYá G,BAUTISTA V,CAMACHO M,et al.Small RNAs of Haloferax mediterranei:identification and potential involvement in nitrogen metabolism[J].Genes,2018,9(2):83.
[19] LOVE M I,HUBER W,ANDERS S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J].Genome Biol,2014,15(12):550.
[20] ABDI H.The bonferroni and Sidak corrections for multiple comparisons[M]//SALKIND N J.Encyclopedia of Measurement and Statistics.Thousand Oaks,CA:SAGE,2007.
[21] KANEHISA M,ARAKI M,GOTO S,et al.KEGG for linking genomes to life and the environment[J/OL].Nucleic Acids Res,2008,36(Database issue):D480-D484 [2019-02-01].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2238879/.
[22] PULVERMACHER S C,STAUFFER L T,STAUFFER G V.The role of the small regulatory RNA GcvB in GcvB/mRNA posttranscriptional regulation of oppA and dppA in Escherichia coli[J].FEMS Microbiol Lett,2008,281(1):42-50.
[23] SHEPPARD D E,PENROD J T,BOBIK T,et al.Evidence that a B12-adenosyl transferase is encoded within the ethanolamine operon of Salmonella enterica[J].J Bacteriol,2004,186(22):7635-7644.
[24] PARSONS J B,FRANK S,BHELLA D,et al.Synthesis of empty bacterial microcompartments,directed organelle protein incorporation,and evidence of filament-associated organelle movement[J].Mol Cell,2010,38(2):305-315.
[25] BOBIK T A,HAVEMANN G D,BUSCH R J,et al.The propanediol utilization (pdu) operon of Salmonella enterica serovar typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in coenzyme B12-dependent 1,2-propanediol degradation[J].J Bacteriol,1999,181(19):5967-5975.
[26] FAN C G,BOBIK T A.The N-terminal region of the medium subunit (PduD) packages adenosylcobalamin-dependent diol dehydratase (PduCDE) into the Pdu microcompartment[J].J Bacteriol,2011,193(20):5623-5628.
[27] LEMON B,TJIAN R.Orchestrated response:a symphony of transcription factors for gene control[J].Genes Dev,2000,14(20):2551-2569.
[28] JIANG H,HU Y J,YANG M,et al.Enhanced immune response to a dual-promoter anti-caries DNA vaccine orally delivered by attenuated Salmonella typhimurium[J].Immunobiology,2017,222(5):730-737.
[29] SALAM M A,KATZ J,ZHANG P,et al.Immunogenicity of Salmonella vector vaccines expressing SBR of Streptococcus mutans under the control of a T7-nirB (dual) promoter system[J].Vaccine,2006,24(23):5003-5015.
[30] MCPHERSON M J,BARON A J,PAPPIN D J C,et al.Respiratory nitrate reductase of Escherichia coli.Sequence identification of the large subunit gene[J].FEBS Lett,1984,177(2):260-264.
[31] BERGERON J R C,WORRALL L J,DE S,et al.The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body[J].Structure,2015,23(1):161-172.
[32] NILAVONGSE A,BRONDIJK T H C,OVERTON T W,et al.The NapF protein of the Escherichia coli periplasmic nitrate reductase system:demonstration of a cytoplasmic location and interaction with the catalytic subunit,NapA[J].Microbiology,2006,152(11):3227-3237.
[33] GRAHL S,MAILLARD J,SPRONK C A E M,et al.Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide[J].Mol Microbiol,2012,83(6):1254-1267.
[34] DARWIN K H,MILLER V L.Type III secretion chaperone-dependent regulation:activation of virulence genes by SicA and InvF in Salmonella typhimurium[J].EMBO J,2001,20(8):1850-1862.
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