F17大肠杆菌在湖羊羔羊个体脾脏中LncRNA表达谱变化
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摘要
【目的】通过筛选对大肠杆菌(E.coli)F17菌毛非腹泻型与腹泻型的绵羊脾脏中差异表达的lncRNA,来探究lncRNA对绵羊抗腹泻的作用。【方法】本研究通过对湖羊羔羊口服E.coli F17菌株获得非腹泻和腹泻型个体,利用羔羊肠道细菌计数、病理组织切片验证攻毒成功性;构建非腹泻组和腹泻组羔羊脾脏的cDNA文库,使用Illumina HiSeq 2500平台进行配对测序;通过Gene Ontology(GO)和KEGG Pathway富集分析对差异表达转录本功能描述和细胞通路分析,利用FPKM法估计lncRNA和mRNA转录物的表达水平,并用高通量测序技术RNA-seq筛选出非腹泻和腹泻个体脾脏中的差异表达lncRNA;然后利用荧光定量PCR技术检测了非腹泻组和腹泻组羔羊脾脏组织中DE lncRNA和DE mRNA的表达水平,来验证筛选的DE lncRNA在非腹泻组过程中发挥作用。【结果】羔羊口服E.coli F17菌株后,出现非腹泻和腹泻两种表型,腹泻组羔羊肠道中的细菌数量显著高于非腹泻组(P<0.05),同时腹泻组羔羊空肠黏膜组织出现不同程度的损伤,色泽暗沉,小肠绒毛部分脱落。笔者利用RNA-seq在非腹泻和腹泻羔羊脾脏中筛选出34个差异表达的(DE)lncRNA,703个的DE mRNA,随机选择一共12个DE lncRNA和DE mRNA,用q-PCR验证它们在非腹泻型和腹泻型羔羊体内的相对表达水平,发现与RNA-seq结果一致。通过Gene Ontology(GO)和KEGG Pathway富集分析,将DE lncRNA与GO数据库进行比对的结果表明一共有34条lncRNA被注释和分类到302个功能亚类中,绵羊蛋白质结合(GO:0005515),细胞核(GO:0005634),poly(A)RNA结合(GO:0044822),细胞质(GO:0005737),组织重塑(GO:0048771),内肽酶活性的调节(GO:0052548)),6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶复合物(GO:0043540),磷脂酰肌醇磷酸化(GO:0046854),果糖-2,6-二磷酸2-磷酸酶活性(GO:0004331),钙依赖性磷脂酶C活性(GO:0050429)等10个功能亚类的lncRNA较多,而其余的功能亚类的lncRNA分布较少。将DE lncRNA与KEGG通路数据库进行比对的结果表明,一共有34条lncRNA被注释和归类到149个KEGG通路中,绵羊甲状腺激素信号通路(路径:ko04919),Spliceosome(路径:ko03040),白细胞跨内皮迁移(路径:ko04670),神经营养因子信号通路(路径:ko04722),溶酶体(路径:ko04142),MAPK信号通路-途径(路径:ko04011),鞘脂信号通路(路径:ko04071),吞噬体(路径:ko04145),氧化磷酸化(路径:ko00190)等9个KEGG通路的lncRNA较多,而其余的KEGG通路的lncRNA分布较少。通过lncRNA-mRNA相互作用网络分析,发现6个共表达基因:MYO1G、TIMM29、CARM1、ADGRB1、SEPT4、DESI2。【结论】探究了对于腹泻产生非腹泻和腹泻型羔羊脾脏中lncRNA的表达谱,发现了非腹泻和腹泻羔羊脾脏中差异表达的lncRNA,有助于找出羔羊如何抵抗腹泻的发生机制,为羔羊抵抗腹泻提供科学的依据。
【Objective】The objective of this study was to investigate the effect of lncRNA on anti-diarrhea in sheep by screening lncRNA differentially expressed in E. coli F17 fimbriae non-diarrhea and diarrhea sheep spleen. 【Method】 In this study, individuals with non-diarrhea and diarrhea were obtained by oral administration of E. coli F17 strain to Lake Lamb. The success of the challenge was verified by using intestinal counts and pathological sections of the lambs. A cDNA library of spleen from lambs in non-diarrhea group and diarrhea group was constructed and sequenced by using Illumina HiSeq 2500 platform.Functional description and cell pathway analysis of differentially expressed transcripts were performed by Gene Ontology(GO)and KEGG Pathway enrichment analysis by using FPKM method. The expression levels of lncRNA and mRNA transcripts were screened by high-throughput sequencing technology RNA-seq for differential expression of lncRNA in spleens of non-diarrhea and diarrhea individuals; then, Quantitative PCR was used to detect spleen tissues in non-diarrhea and diarrhea lambs. The expression levels of differentially expressed(DE) lncRNA and DE mRNA were used to verify the role of screened DE lncRNA in the non-diarrhea group. 【Result】 After oral administration of E. coli F17 strain, there were two phenotypes of non-diarrhea and diarrhea. The number of bacteria in the intestine of the diarrhea group was significantly higher than that in the non-diarrhea group(P<0.05), and the jejunal mucosa of the diarrhea group appeared different degrees of damage, dull color, part of the small intestine villi off. We used RNA-seq to screen 34 DE lncRNAs and 703 DE mRNAs in non-diarrhea and diarrhea lamb spleens. A total of 12 DE lncRNA and DE mRNA were randomly selected and verified by q-PCR. Relative expression levels in the diarrhea and non-diarrhea lambs were found to be consistent with RNA-seq results. The comparison between DE lncRNA and GO database by GO and KEGG pathway enrichment analysis indicated that a total of 34 lncRNAs were annotated and classified into302 functional subclasses. There were more than one functional subclass of lncRNA, such as sheep protein binding(GO:0005515), nuclear(GO: 0005634), poly(A) RNA binding(GO: 0044822), cytoplasm(GO: 0005737), tissue remodeling(GO:0048771), regulation of endopeptidase activity(GO: 0052548)), 6-phosphate fructose-2-kinase/fructose-2,6-bisphosphatase complex(GO: 0043540), phosphatidylinositol phosphorylation(GO: 0848654), fructose-2, 6.2-phosphite 2-phosphatase activity(GO: 0004331) and calcium-dependent phospholipase C activity(GO: 0050429), while the remaining functional subclasses had less lncRNA distribution. The alignment of DE lncRNA with the KEGG pathway database indicated that a total of 34 lncRNAs were annotated and classified into 149 KEGG pathways, the sheep thyroid hormone signaling pathway(path: ko04919),Spliceosome(path: ko03040), white blood cell cross Endothelial migration(path: ko04670), neurotrophin signaling pathway(path:ko04722), lysosome(path: ko04142), MAPK signaling pathway-pathway(path: ko04011), sphingolipid signaling pathway(path:ko04071), phagocytosis the body(path: ko04145), oxidative phosphorylation(path: ko00190) and other 9 KEGG pathways had more lncRNAs, while the remaining KEGG pathways had less lncRNA distribution. Through lncRNA-mRNA interaction network analysis, we found six co-expressed genes: MYO1G, TIMM29, CARM1, ADGRB1, SEPT4, and DESI2. 【Conclusion】 This study explored the expression profile of lncRNA in the spleen of non-diarrhea and diarrhea lambs for diarrhea. It was found that lncRNA differentially expressed in the spleen of non-diarrhea and diarrhea lambs, which helped to find out how lambs resist diarrhea and provided a scientific basis for lambs to resist diarrhea.
【Objective】The objective of this study was to investigate the effect of lncRNA on anti-diarrhea in sheep by screening lncRNA differentially expressed in E. coli F17 fimbriae non-diarrhea and diarrhea sheep spleen. 【Method】 In this study, individuals with non-diarrhea and diarrhea were obtained by oral administration of E. coli F17 strain to Lake Lamb. The success of the challenge was verified by using intestinal counts and pathological sections of the lambs. A cDNA library of spleen from lambs in non-diarrhea group and diarrhea group was constructed and sequenced by using Illumina HiSeq 2500 platform.Functional description and cell pathway analysis of differentially expressed transcripts were performed by Gene Ontology(GO)and KEGG Pathway enrichment analysis by using FPKM method. The expression levels of lncRNA and mRNA transcripts were screened by high-throughput sequencing technology RNA-seq for differential expression of lncRNA in spleens of non-diarrhea and diarrhea individuals; then, Quantitative PCR was used to detect spleen tissues in non-diarrhea and diarrhea lambs. The expression levels of differentially expressed(DE) lncRNA and DE mRNA were used to verify the role of screened DE lncRNA in the non-diarrhea group. 【Result】 After oral administration of E. coli F17 strain, there were two phenotypes of non-diarrhea and diarrhea. The number of bacteria in the intestine of the diarrhea group was significantly higher than that in the non-diarrhea group(P<0.05), and the jejunal mucosa of the diarrhea group appeared different degrees of damage, dull color, part of the small intestine villi off. We used RNA-seq to screen 34 DE lncRNAs and 703 DE mRNAs in non-diarrhea and diarrhea lamb spleens. A total of 12 DE lncRNA and DE mRNA were randomly selected and verified by q-PCR. Relative expression levels in the diarrhea and non-diarrhea lambs were found to be consistent with RNA-seq results. The comparison between DE lncRNA and GO database by GO and KEGG pathway enrichment analysis indicated that a total of 34 lncRNAs were annotated and classified into302 functional subclasses. There were more than one functional subclass of lncRNA, such as sheep protein binding(GO:0005515), nuclear(GO: 0005634), poly(A) RNA binding(GO: 0044822), cytoplasm(GO: 0005737), tissue remodeling(GO:0048771), regulation of endopeptidase activity(GO: 0052548)), 6-phosphate fructose-2-kinase/fructose-2,6-bisphosphatase complex(GO: 0043540), phosphatidylinositol phosphorylation(GO: 0848654), fructose-2, 6.2-phosphite 2-phosphatase activity(GO: 0004331) and calcium-dependent phospholipase C activity(GO: 0050429), while the remaining functional subclasses had less lncRNA distribution. The alignment of DE lncRNA with the KEGG pathway database indicated that a total of 34 lncRNAs were annotated and classified into 149 KEGG pathways, the sheep thyroid hormone signaling pathway(path: ko04919),Spliceosome(path: ko03040), white blood cell cross Endothelial migration(path: ko04670), neurotrophin signaling pathway(path:ko04722), lysosome(path: ko04142), MAPK signaling pathway-pathway(path: ko04011), sphingolipid signaling pathway(path:ko04071), phagocytosis the body(path: ko04145), oxidative phosphorylation(path: ko00190) and other 9 KEGG pathways had more lncRNAs, while the remaining KEGG pathways had less lncRNA distribution. Through lncRNA-mRNA interaction network analysis, we found six co-expressed genes: MYO1G, TIMM29, CARM1, ADGRB1, SEPT4, and DESI2. 【Conclusion】 This study explored the expression profile of lncRNA in the spleen of non-diarrhea and diarrhea lambs for diarrhea. It was found that lncRNA differentially expressed in the spleen of non-diarrhea and diarrhea lambs, which helped to find out how lambs resist diarrhea and provided a scientific basis for lambs to resist diarrhea.
引文
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[2]STIRM S,ORSKOV I,ORSKOV F.K88,an episome-determined protein antigen of Escherichia coli.Nature,1966,209(5022):507-508.
[3]KIM T,JEON Y J,CUI R,LEE J H,PENG Y,KIM S H,TILI E,ALDER H,CROCE C M.Role of MYC-regulated long noncoding RNAs in cell cycle regulation and tumorigenesis.Journal of National Cancer Institute,2015,107(4).doi:10.1093/jnci/dju505.
[4]OUYANG J,ZHU X M,CHEN Y H,WEI H T,CHEN Q H,CHI X J,QI B M,ZHANG L F,ZHAO Y,GAO F,WANG G S,CHEN J L.NRAV,a long noncoding RNA,modulates antiviral responses through suppression of interferon-stimulated gene transcription.Cell Host Microbe,2014,16(5):616-626.
[5]LI Z,RANA T M.Decoding the noncoding:prospective of lncRNA-mediated innate immune regulation.RNA Biology,2014,11(8):979-985.
[6]TURNER M,GALLOWAY A,VIGORITO E.Noncoding RNA and its associated proteins as regulatory elements of the immune system.Nature Immunology,2014,15(6):484-491.
[7]HEWARD J A,LINDSAY M A.Long non-coding RNAs in the regulation of the immune response.Trends Immunology,2014,35(9):408-419.
[8]REN C,DENG M,FAN Y,YANG H,ZHANG G,FENG X,LI F,WANG D,WANG F,ZHANG Y.Genome-wide analysis reveals extensive changes in LncRNAs during skeletal muscle development in Hu Sheep.Genes,2017,8(8):283-313.
[9]ZHANG Y,YANG H,HAN L,LI F,ZHANG T,PANG J,FENG X,REN C,MAO S,WANG F.Long noncoding RNA expression profile changes associated with dietary energy in the sheep testis during sexual maturation.Scientific Reports,2017,7(1):5180.
[10]YUE Y,GUO T,YUAN C,LIU J,GUO J,FENG R,NIU C,SUN X,YANG B.Integrated analysis of the roles of long noncoding RNA and coding RNA expression in sheep(Ovis aries)skin during initiation of secondary hair follicle.PLoS ONE.2016,11(6):e:0156890.
[11]徐兴文.羊大肠杆菌病防治.中国畜禽种业,2017,13(4):112-113.XU X W.Prevention and treatment of sheep colibacillosis.The Chinese Livestock and Poultry Breeding,2017,13(4):112-113.(in Chinese)
[12]张文静.羊大肠杆菌病的防控措施.畜牧兽医科技信息,2017(6):76.ZHANG W J.Prevention and control measures for sheep colibacillosis.Chinese Journal of Animal Husbandry and Veterinary Medicine,2017(6):76.(in Chinese)
[13]KONG L,ZHANG Y,YE ZQ,LIU XQ,ZHAO SQ,WEI L,GAO G.CPC:assess the protein-coding potential of transcripts using sequence features and support vector machine.Nucleic Acids Research,2007,36:345-349.
[14]SUN L,LUO H T,BU D C,ZHAO G G,YU K T,ZHANG C H,LIU Y N,CHEN R S,ZHAO Y.Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts.Nucleic Acids Research,2013,41(17):166.
[15]FINN R D,BATEMAN A,CLEMENTS J,COGGILL P,EBERHARDT R Y,EDDY S R,HEGER A,HETHERINGTON K,HOLM L,MISTRY J,SONNHAMMER E L,TATE J,PUNTA M.The Pfam protein families database.Nucleic Acids Research,2014,42(Database issue):222-230.
[16]LI A,ZHANG J,ZHOU Z.PLEK:A tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme.Bmc Bioinformatics,2014,15(1):311.
[17]TRAPNELL C,WILLIAMS B A,PERTEA G,MORTAZAVI A,KWAN G,VAN BAREN M J,SALZBERG S L,WOLD B J,PACHTER L.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.Nature Biotechnology,2010,28(5):511-515.
[18]ANDERS S,HUBER W.Differential expression analysis for sequence count data.Biology,2010,11:R106.
[19]KANEHISA M,ARAKI M,GOTO S,HATTORI M,HIRAKAWA M,ITOH M,KATAYAMA T,KAWASHIMA S,OKUDA S,TOKIMATSUT,YAMANISHI Y.KEGG for linking genomes to life and the environment.Nucleic Acids Research.2008,36:480-484.
[20]LEWIS S J,HEATON K W.Stool form scale as a useful guide to intestinal transit time.Scandinavian Journal of Gastroenterology,1997,32(9):920-924.
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