基于RNA-Seq筛选APEC及其PhoP/Q缺失株感染雏鸡肠道免疫相关基因
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摘要
本研究以禽致病性大肠杆菌(APEC)及其PhoP/Q缺失株感染雏鸡小肠为模型,以分析其免疫相关基因的表达变化为目的,采用转录组测序(RNA-Seq)技术对感染APEC及其PhoP/Q缺失株的雏鸡小肠样本RNA进行测序,分析免疫相关基因的表达变化,结果为野生株攻毒组与对照组相比、野生株攻毒组与缺失株攻毒组相比、缺失株攻毒组与对照组相比,分别筛选出131、105、172个差异表达基因(fold change≥2, FDR≤0.05),GO功能分类结果显示分别有87、99、159个基因得到注释,这些基因主要富集到氧化还原过程、脂蛋白转运、血管内皮细胞迁移、免疫反应、凋亡过程负调控、肝素结合、铁离子结合、CCR趋化因子受体结合等功能,得出APEC及其PhoP/Q缺失株感染雏鸡后引起机体肠道免疫相关基因变化的结论,根据GO功能注释筛选出PTPRC、LCP1、YFV等免疫相关基因,为深入研究雏鸡肠道免疫提供依据。
The purpose of this study was to analyze the expression change of immune-related genes based on the chickens small intestinal infected with avian pathogenic Escherichia coli(APEC) and its PhoP/Q deletion strain.The RNAs of small intestine samples of chickens infected with APEC and its PhoP/Q deletion strains were sequenced by transcriptome sequencing(RNA-Seq) technique, and the expression changes of immune-related genes were analyzed. After comparing wild-type strain inoculation group with the control group, wild-type strain inoculation group with deletion strain inoculated group, and deletion strain inoculated group with the control group, we selected 131, 105, 172 differentially expressed genes(Fold Change≥2, FDR≤0.05), respectively. GO function classification results showed that 87, 99, 159 genes were annotated respectively. These genes were mainly enriched in oxidation-reduction process, lipoprotein transport, blood vessel endothelial cell migration, immune response, negative regulation of apoptotic process, heparin binding, iron ion binding, CCR chemokine receptor binding and other functions. It was concluded that APEC and its PhoP/Q deletion strains caused changes in intestinal immune related genes after infection in chicks. According to GO function annotation, PTPRC, LCP1,YFV and other immune-related genes were screened, which might provide basis for further study of intestinal immunity in chicks.
The purpose of this study was to analyze the expression change of immune-related genes based on the chickens small intestinal infected with avian pathogenic Escherichia coli(APEC) and its PhoP/Q deletion strain.The RNAs of small intestine samples of chickens infected with APEC and its PhoP/Q deletion strains were sequenced by transcriptome sequencing(RNA-Seq) technique, and the expression changes of immune-related genes were analyzed. After comparing wild-type strain inoculation group with the control group, wild-type strain inoculation group with deletion strain inoculated group, and deletion strain inoculated group with the control group, we selected 131, 105, 172 differentially expressed genes(Fold Change≥2, FDR≤0.05), respectively. GO function classification results showed that 87, 99, 159 genes were annotated respectively. These genes were mainly enriched in oxidation-reduction process, lipoprotein transport, blood vessel endothelial cell migration, immune response, negative regulation of apoptotic process, heparin binding, iron ion binding, CCR chemokine receptor binding and other functions. It was concluded that APEC and its PhoP/Q deletion strains caused changes in intestinal immune related genes after infection in chicks. According to GO function annotation, PTPRC, LCP1,YFV and other immune-related genes were screened, which might provide basis for further study of intestinal immunity in chicks.
引文
Cheng Y.Y.,Wang X.L.,Wang P.Z.,Li T.,Hu F.Z.,Liu Q.,Yang F.,Wang J.,Xu T.,and Han W.L.,2016,SUSD2 is frequently downregulated and functions as a tumor suppressor in RCC and lung cancer,Tumor.Biol.,37(7):9919-9930
Dalerba P.,Kalisky T.,Sahoo D.,Rajendran P.S.,Rothenberg M.E.,Leyrat A.A.,Sim S.,Okamoto J.,Johnston D.M.,Qian D.L.,Zabala M.,Bueno J.,Neff N.F.,Wang J.B.,Shelton A.A.,Visser B.,Hisamori S.,Shimono Y.,van de Wetering M.,Clevers H.,Clarke M.F.,and Quake S.R.,2011,Single-cell dissection of transcriptional heterogeneity in human colon tumors,Nat.Biotechnol.,29(12):1120-1127
De Clercq S.,Boucherie C.,Vandekerckhove J.,Gettemans J.,and Guillabert A.,2013,L-Plastin nanobodies perturb matrix degradation,podosome formation,stability and lifetime in THP-1 macrophages,PLoS One,8(11):e78108
Dubovsky J.A.,Chappell D.L.,Harrington B.K.,Agrawal K.,Andritsos L.A.,Flynn J.M.,Jones J.A.,Paulaitis M.E.,Bolon B.,Johnson A.J.,Byrd J.C.,and Muthusamy N.,2013,Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrance-associated antigen critical to niche homing,Blood,122(19):3308-3316
Fan W.L.,2015,RNA-seq analysis revealed functional genes associated with resistance to Salmonella enterica in chicken,Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Zhao G.P.,pp.15-27(凡文磊,2015,利用RNA-seq技术挖掘鸡肠炎沙门氏菌抗性相关功能基因,硕士学位论文,中国农业科学院,导师:赵桂苹,pp.15-27)
Gaur U.,Xiong Y.Y.,Luo Q.P.,Yuan F.Y.,Wu H.Y.,Qiao M.,Wimmers K.,Li K.,Mei S.Q.,and Liu G.S.,2014,Breed-specific transcriptome response of spleen from six to eight week old piglet after infection with Streptococcus suis type 2,Mol.Biol.Rep.,41(12):7865-7873
Gobel T.W.,Schneider K.,Schaerer B.,Mejri I.,Puehler F.,Weigend S.,Staeheli P.,and Kaspers B.,2003,IL-18 stimulates the proliferation and IFN-γrelease of CD4+T cells in the chicken:conservation of a Th1-like system in a nonmammalian species,J.Immunol.,171(4):1809-1815
Guan S.P.,and Lu A.G.,2015,Role of chemokine CCL19 in immune cell mediated antitumor effect,Shanghai Jiaotong Daxue Xuebao(Yixueban)(Journal of Shanghai Jiao Tong University Medical Science),35(3):454-459(关少培,陆爱国,2015,趋化因子CCL19在介导免疫细胞抗肿瘤中的作用,上海交通大学学报(医学版),35(3):454-459)
Huang B.Y.,2015,Modulation of virulence by the two-component system PhoP/Q in Avian pathogenic Escherichia coli,Thesis for M.S.,College of Animal Science and Technology,Anhui Agricultural University,Supervisor:Qi K.Z.,pp.1-7(黄博言,2015,禽致病性大肠杆菌PhoP/Q二元调控系统对毒力基因调控的研究,硕士学位论文,安徽农业大学动物科技学院,导师:祁克宗,pp.1-7)
Li F.C.,2014,SNP to stimulation molecular CD274,CD80 regulation research in gastric cancer,Thesis for M.S.,Soochow U-niversity,Supervisor:Chen W.C.,and Wang W.P.,pp.4-18(李伏超,2014,SNP对共刺激分子CD274,CD80在胃癌中表达调控研究,苏州大学,导师:陈卫昌,汪维鹏,pp.4-18)
Matulova M.,Rajova J.,Vlasatikova L.,Volf J.,Stepanova H.,Havlickova H.,Sisak F.,and Rychlik I.,2012,Characterization of Chicken Spleen transcriptome after Infection with Salmonella enterica serovar Enteritidis,PLoS One,7(10):e48101
Porcu M.,Kleppe M.,Gianfelici V.,Geerdens E.,De Keersmaecker K.,Tartaglia M.,FoàR.,Soulier J.,Cauwelier B.,Uyttebroeck A.,Macintyre E.,Vandenberghe P.,Asnafi V.,and Cools J.,2012,Mutation of the receptor tyrosine phosphatase PTPRC(CD45)in T-cell acute lymphoblastic leukemia,Blood,119(19):4476-4479
Qing L.,2006,PhoP,a novel global regulator,directly activates a global regulon contributing to intracellular growth of Yersinia pestis,Dissertation for Ph.D.,Academy of Military Medical Sciences,Supervisor:Yang R.F.,pp.1-27(秦珑,2006,整体调控子phop直接调控鼠疫耶尔森氏菌胞内生存能力的研究,博士学位论文,中国人民解放军军事医学科学院,导师:杨瑞馥,pp.1-27)
Shin D.,Lee J.,Huang H.,and Groisman E.A.,2006,A positive feedback loop promotes transcription surge that jump-starts Salmonella virulence circuit,Science,5805(314):1607-1609
Taku O.,and Honjo T.,2006,The PD-1-PD-L pathway in immunological tolerance,Trends in Immunology,27(4):195-201
Thoraval P.,Afanassieff M.,Bouret D.,Luneau G.,Esnault E.,Goto R.M.,ChausséA.M.,Zoorob R.,Soubieux D.,Miller M.M.,and Dambrine G.,2003,Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity,Immuno Genetics,55:647-651
Trapnell C.,Pachter L.,and Salzberg S.L.,2009,TopHat:discovering splice junctions with RNA-Seq,Bioinformatic,25(9):1105-1111
Trapnell C.,Williams B.A.,Pertea G.,Mortazavi A.,Kwan G.,van Baren M.J.,Salzberg S.L.,Wold B.J.,and Pachter L.,2010,Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation,Nat.Biotechnol.,28(5):511-515
Watson A.P.,Evans R.L.,and Egland K.A.,2013,Multiple functions of Sushi Domain Containing 2(SUSD2)in breast tumorigenesis,Mol.Cancer Res.,11(1):74-85
Yang S.,2014,The roles of PI3K/Akt cell signal pathway and innate immunity restriction factor SAMHD1 in PRRSV infection,Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Tong G.Z.,pp.1-19(杨莘,2014,PI3K/Akt信号通路与天然免疫限制性因子SAMHD1在PRRSV感染过程中的作用研究,博士学位论文,中国农业科学院,导师:童光志,pp.1-19)
Ziegler T.R.,Evans M.E.,Fernandez-Estivariz C.,and Jones D.P.,2003,Trophic and cytoprotective nutrition for intestinal adaptation,mucosal repair,and barrier function,Annu.Rev.Nutri.,23(1):229-261
Dalerba P.,Kalisky T.,Sahoo D.,Rajendran P.S.,Rothenberg M.E.,Leyrat A.A.,Sim S.,Okamoto J.,Johnston D.M.,Qian D.L.,Zabala M.,Bueno J.,Neff N.F.,Wang J.B.,Shelton A.A.,Visser B.,Hisamori S.,Shimono Y.,van de Wetering M.,Clevers H.,Clarke M.F.,and Quake S.R.,2011,Single-cell dissection of transcriptional heterogeneity in human colon tumors,Nat.Biotechnol.,29(12):1120-1127
De Clercq S.,Boucherie C.,Vandekerckhove J.,Gettemans J.,and Guillabert A.,2013,L-Plastin nanobodies perturb matrix degradation,podosome formation,stability and lifetime in THP-1 macrophages,PLoS One,8(11):e78108
Dubovsky J.A.,Chappell D.L.,Harrington B.K.,Agrawal K.,Andritsos L.A.,Flynn J.M.,Jones J.A.,Paulaitis M.E.,Bolon B.,Johnson A.J.,Byrd J.C.,and Muthusamy N.,2013,Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrance-associated antigen critical to niche homing,Blood,122(19):3308-3316
Fan W.L.,2015,RNA-seq analysis revealed functional genes associated with resistance to Salmonella enterica in chicken,Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Zhao G.P.,pp.15-27(凡文磊,2015,利用RNA-seq技术挖掘鸡肠炎沙门氏菌抗性相关功能基因,硕士学位论文,中国农业科学院,导师:赵桂苹,pp.15-27)
Gaur U.,Xiong Y.Y.,Luo Q.P.,Yuan F.Y.,Wu H.Y.,Qiao M.,Wimmers K.,Li K.,Mei S.Q.,and Liu G.S.,2014,Breed-specific transcriptome response of spleen from six to eight week old piglet after infection with Streptococcus suis type 2,Mol.Biol.Rep.,41(12):7865-7873
Gobel T.W.,Schneider K.,Schaerer B.,Mejri I.,Puehler F.,Weigend S.,Staeheli P.,and Kaspers B.,2003,IL-18 stimulates the proliferation and IFN-γrelease of CD4+T cells in the chicken:conservation of a Th1-like system in a nonmammalian species,J.Immunol.,171(4):1809-1815
Guan S.P.,and Lu A.G.,2015,Role of chemokine CCL19 in immune cell mediated antitumor effect,Shanghai Jiaotong Daxue Xuebao(Yixueban)(Journal of Shanghai Jiao Tong University Medical Science),35(3):454-459(关少培,陆爱国,2015,趋化因子CCL19在介导免疫细胞抗肿瘤中的作用,上海交通大学学报(医学版),35(3):454-459)
Huang B.Y.,2015,Modulation of virulence by the two-component system PhoP/Q in Avian pathogenic Escherichia coli,Thesis for M.S.,College of Animal Science and Technology,Anhui Agricultural University,Supervisor:Qi K.Z.,pp.1-7(黄博言,2015,禽致病性大肠杆菌PhoP/Q二元调控系统对毒力基因调控的研究,硕士学位论文,安徽农业大学动物科技学院,导师:祁克宗,pp.1-7)
Li F.C.,2014,SNP to stimulation molecular CD274,CD80 regulation research in gastric cancer,Thesis for M.S.,Soochow U-niversity,Supervisor:Chen W.C.,and Wang W.P.,pp.4-18(李伏超,2014,SNP对共刺激分子CD274,CD80在胃癌中表达调控研究,苏州大学,导师:陈卫昌,汪维鹏,pp.4-18)
Matulova M.,Rajova J.,Vlasatikova L.,Volf J.,Stepanova H.,Havlickova H.,Sisak F.,and Rychlik I.,2012,Characterization of Chicken Spleen transcriptome after Infection with Salmonella enterica serovar Enteritidis,PLoS One,7(10):e48101
Porcu M.,Kleppe M.,Gianfelici V.,Geerdens E.,De Keersmaecker K.,Tartaglia M.,FoàR.,Soulier J.,Cauwelier B.,Uyttebroeck A.,Macintyre E.,Vandenberghe P.,Asnafi V.,and Cools J.,2012,Mutation of the receptor tyrosine phosphatase PTPRC(CD45)in T-cell acute lymphoblastic leukemia,Blood,119(19):4476-4479
Qing L.,2006,PhoP,a novel global regulator,directly activates a global regulon contributing to intracellular growth of Yersinia pestis,Dissertation for Ph.D.,Academy of Military Medical Sciences,Supervisor:Yang R.F.,pp.1-27(秦珑,2006,整体调控子phop直接调控鼠疫耶尔森氏菌胞内生存能力的研究,博士学位论文,中国人民解放军军事医学科学院,导师:杨瑞馥,pp.1-27)
Shin D.,Lee J.,Huang H.,and Groisman E.A.,2006,A positive feedback loop promotes transcription surge that jump-starts Salmonella virulence circuit,Science,5805(314):1607-1609
Taku O.,and Honjo T.,2006,The PD-1-PD-L pathway in immunological tolerance,Trends in Immunology,27(4):195-201
Thoraval P.,Afanassieff M.,Bouret D.,Luneau G.,Esnault E.,Goto R.M.,ChausséA.M.,Zoorob R.,Soubieux D.,Miller M.M.,and Dambrine G.,2003,Role of nonclassical class I genes of the chicken major histocompatibility complex Rfp-Y locus in transplantation immunity,Immuno Genetics,55:647-651
Trapnell C.,Pachter L.,and Salzberg S.L.,2009,TopHat:discovering splice junctions with RNA-Seq,Bioinformatic,25(9):1105-1111
Trapnell C.,Williams B.A.,Pertea G.,Mortazavi A.,Kwan G.,van Baren M.J.,Salzberg S.L.,Wold B.J.,and Pachter L.,2010,Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation,Nat.Biotechnol.,28(5):511-515
Watson A.P.,Evans R.L.,and Egland K.A.,2013,Multiple functions of Sushi Domain Containing 2(SUSD2)in breast tumorigenesis,Mol.Cancer Res.,11(1):74-85
Yang S.,2014,The roles of PI3K/Akt cell signal pathway and innate immunity restriction factor SAMHD1 in PRRSV infection,Dissertation for Ph.D.,Chinese Academy of Agricultural Sciences,Supervisor:Tong G.Z.,pp.1-19(杨莘,2014,PI3K/Akt信号通路与天然免疫限制性因子SAMHD1在PRRSV感染过程中的作用研究,博士学位论文,中国农业科学院,导师:童光志,pp.1-19)
Ziegler T.R.,Evans M.E.,Fernandez-Estivariz C.,and Jones D.P.,2003,Trophic and cytoprotective nutrition for intestinal adaptation,mucosal repair,and barrier function,Annu.Rev.Nutri.,23(1):229-261