感染大肠杆菌F17湖羊羔羊脾脏中差异circRNA分析
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摘要
【背景】羊大肠杆菌病是一种以剧烈腹泻和败血症为特征的急性传染病,是规模化羊场最为常见高发的细菌性疾病之一,尤其是初生羔羊易被产肠毒素大肠杆菌(ETEC)感染,引起羔羊腹泻,又叫羔羊白痢,使养殖场遭受严重的经济损失。而传统的抗生素治疗方案存在诸多缺陷。【目的】本研究通过让湖羊羔羊口服大肠杆菌F17菌株获得不腹泻和腹泻的羔羊个体,筛选出服用大肠杆菌F17菌毛后不腹泻与腹泻型个体中差异表达的circRNA,进而探究circRNA在绵羊抗腹泻中的作用,从而发现与抗大肠杆菌病性状相关的候选基因。从circRNA层面上,加深对绵羊拮抗大肠杆菌F17菌株的认识,确定绵羊拮抗大肠杆菌F17菌株的功能基因。【方法】用CIRI软件从头预测circRNA,利用RNA-seq技术,首次筛选出感染大肠杆菌F17菌株后不腹泻与腹泻型羔羊个体脾脏中差异表达的(DE)circRNA,对差异表达转录本进行GO富集分析,结合GO注释结果对其功能进行描述。统计每个GO条目中所包括的差异转录本个数,并用Fisher's exact test计算每个GO条目中差异转录本富集的显著性。然后随机选择6个DE circRNA,利用q-PCR分别验证这6个DE circRNA在不腹泻和腹泻组羔羊脾脏内的相对表达水平,进而利用Miranda软件来预测与miRNA结合的circRNA以及miRNA的靶基因,根据miRNA靶基因的功能注释来阐明此部分circRNA的功能,分析circRNA-miRNA-mRNA相互作用,最后用q-PCR验证circRNA在不腹泻组和腹泻组羔羊体内的相对表达水平。【结果】绘制参考序列后,鉴定出已知的7 730个circRNA,DE circRNA与GO数据库进行比对,发现一共有60条circRNA被注释和分类到297个功能亚类中。利用RNA-seq在不腹泻和腹泻羔羊脾脏中筛选出60个差异表达的(DE) circRNA,其中31个上调和29个下调,用q-PCR验证随机选择的6个DE circRNA在不腹泻组和腹泻组羔羊体内的相对表达水平,发现与RNA-seq结果一致。利用Miranda分析circRNA-miRNA-mRNA相互作用,发现6个circRNA、5个miRNA和8个mRNA之间存在一定的靶标关系,用q-PCR验证mRNA在不腹泻组和腹泻组羔羊体内的相对表达水平,发现与RNA-seq结果一致。【结论】探究了对于不腹泻和腹泻羔羊脾脏中circRNA的表达谱,进一步了解其在绵羊抗病发生过程中的调控作用。发现了不腹泻和腹泻羔羊脾脏中差异表达的circRNA,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。
【Background】Sheep colibacillosis is an acute infectious disease by E. coli that is characterized by severe diarrhea and sepsis, which is one of the most common bacterial diseases in large-scale sheep farms. Especially the newborn lamb is infected by enterotoxin-producing Escherichia coli(ETEC), causing lamb diarrhea, also known as lamb white diarrhea. Sheep colibacillosis let the farm to suffer serious economic losses, but the traditional antibiotic treatment program has many defects. 【Objective】In this study, we obtained oral diarrhea and diarrhea lambs by oral administration of E. coli F17 strain, and screened out circRNAs that were differentially expressed in individuals with diarrhea and diarrhea after taking E. coli F17 pili, and then explored circRNA for sheep in anti-diarrhea to find candidate genes associated with resistance to E. coli disease traits. From the circRNA level, the understanding of sheep antagonizing E. coli F17 fimbriae was deepened, and the functional genes of sheep antagonizing E. coli F17 fimbriae were determined.【Method】We used CIRI software to predict circRNA from scratch, and firstly screened differentially expressed(DE)circRNA in spleen of Escherichia coli F17 fimbriae without diarrhea and diarrhea by using RNA-seq technology. Then we performed GO on differentially expressed transcripts, and its function was described in combination with GO annotation results. The number of differential transcripts included in each GO entry was counted and the significance of differential transcript enrichment in each GO entry was calculated by using Fisher's exact test. Then, 6 DE circRNAs were randomly selected, and the relative expression levels of the 6 DE circRNAs in the spleen of lambs without diarrhea and diarrhea were verified by q-PCR, and the miRNAs and miRNA target genes were predicted by Miranda software. The function of this part of circRNA was elucidated based on the functional annotation of the miRNA target gene, and the circRNA-miRNA-mRNA interaction was analyzed. Finally, the relative expression level of mRNA in the non-diarrhea group and the diarrhea group lamb was verified by q-PCR.【Result】After mapping the reference sequence, we identified 7730 circRNAs, and DE circRNA was compared with the GO database. A total of 60 circRNAs were annotated and classified into 297 functional subclasses. Using RNA-seq to screen 60 DE circRNAs in spleens of diarrhea-free and diarrhea lambs,31 of which were up-regulated, and 29 of which were down-regulated. Relative expression levels of randomly selected 6 DE circRNAs in non-diarrhea and diarrhea lambs by q-PCR and found to be consistent with RNA-seq results. Using Miranda to analyze circRNA-miRNA-mRNA interactions, we found a certain target relationship between 6 circRNAs, 5 miRNAs and 8 mRNAs. q-PCR was used to verify the relative expression of mRNA in lambs without diarrhea and diarrhea, and the results were found to be consistent with RNA-seq results.【Conclusion】 This study explored the expression profile of circRNA in the spleen of lambs without diarrhea and diarrhea, and further understood its regulation in the process of disease resistance in sheep. The discovery of circRNA,which was differentially expressed in the spleen of lambs without diarrhea and diarrhea, could help to find out how the lambs resist the mechanism of diarrhea, and to provide a scientific basis for improving the lamb's ability to resist diarrhea.
【Background】Sheep colibacillosis is an acute infectious disease by E. coli that is characterized by severe diarrhea and sepsis, which is one of the most common bacterial diseases in large-scale sheep farms. Especially the newborn lamb is infected by enterotoxin-producing Escherichia coli(ETEC), causing lamb diarrhea, also known as lamb white diarrhea. Sheep colibacillosis let the farm to suffer serious economic losses, but the traditional antibiotic treatment program has many defects. 【Objective】In this study, we obtained oral diarrhea and diarrhea lambs by oral administration of E. coli F17 strain, and screened out circRNAs that were differentially expressed in individuals with diarrhea and diarrhea after taking E. coli F17 pili, and then explored circRNA for sheep in anti-diarrhea to find candidate genes associated with resistance to E. coli disease traits. From the circRNA level, the understanding of sheep antagonizing E. coli F17 fimbriae was deepened, and the functional genes of sheep antagonizing E. coli F17 fimbriae were determined.【Method】We used CIRI software to predict circRNA from scratch, and firstly screened differentially expressed(DE)circRNA in spleen of Escherichia coli F17 fimbriae without diarrhea and diarrhea by using RNA-seq technology. Then we performed GO on differentially expressed transcripts, and its function was described in combination with GO annotation results. The number of differential transcripts included in each GO entry was counted and the significance of differential transcript enrichment in each GO entry was calculated by using Fisher's exact test. Then, 6 DE circRNAs were randomly selected, and the relative expression levels of the 6 DE circRNAs in the spleen of lambs without diarrhea and diarrhea were verified by q-PCR, and the miRNAs and miRNA target genes were predicted by Miranda software. The function of this part of circRNA was elucidated based on the functional annotation of the miRNA target gene, and the circRNA-miRNA-mRNA interaction was analyzed. Finally, the relative expression level of mRNA in the non-diarrhea group and the diarrhea group lamb was verified by q-PCR.【Result】After mapping the reference sequence, we identified 7730 circRNAs, and DE circRNA was compared with the GO database. A total of 60 circRNAs were annotated and classified into 297 functional subclasses. Using RNA-seq to screen 60 DE circRNAs in spleens of diarrhea-free and diarrhea lambs,31 of which were up-regulated, and 29 of which were down-regulated. Relative expression levels of randomly selected 6 DE circRNAs in non-diarrhea and diarrhea lambs by q-PCR and found to be consistent with RNA-seq results. Using Miranda to analyze circRNA-miRNA-mRNA interactions, we found a certain target relationship between 6 circRNAs, 5 miRNAs and 8 mRNAs. q-PCR was used to verify the relative expression of mRNA in lambs without diarrhea and diarrhea, and the results were found to be consistent with RNA-seq results.【Conclusion】 This study explored the expression profile of circRNA in the spleen of lambs without diarrhea and diarrhea, and further understood its regulation in the process of disease resistance in sheep. The discovery of circRNA,which was differentially expressed in the spleen of lambs without diarrhea and diarrhea, could help to find out how the lambs resist the mechanism of diarrhea, and to provide a scientific basis for improving the lamb's ability to resist diarrhea.
引文
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[12]徐兴文.羊大肠杆菌病防治.中国畜禽种业,2017,4:129-130.XU X W.Prevention and treatment of Escherichia Coli in sheep.China Animal and Poultry Industry,2017,4:129-130.(in Chinese)
[13]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.Herbivore,2017,6:76.(in Chinese)
[14]LEWIS S J,HEATON K W.Stool form as a useful guide to intestinal transit time.Scand J Gastroenterol.1997,32:920-924.
[15]P,G,P.P,and R.N,circBase:a database for circular RNAs.Rna-a Publication of the Rna Society,2014.20(11):1666-1670.
[16]GAO Y,WANG J,ZHAO F,CIRI:an efficient and unbiased algorithm for de novo circular RNA identification.Genome Biology,2015.16:4.
[17]ANDERS S.HUBER W..Differential expression of RNA-Seq data at the gene level-the DESeq package.European Molecular Biology Laboratory,2013.
[18]ROBINSON,M D,MCCARTHY D J,SMYTH G K.edgeR:a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics,2010,26(1):139-140.
[19]KANEHISA M,ARAKI M,GOTO S,HATTORI M,HIRAKAWA M,ITOH M,KATAYAMA T,KAWASHIMA S,OKUDA S,TUKIMATSU T,YAMANISHI Y.KEGG for linking genomes to life and the environment.Nucleic Acids Research,2008,36:480-484.
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[21]ENRIGH A,JOHN B,GAUL U,TUSCHL T,SANDER C,MARKS S.MicroRNA targets in Drosophila.Genome Biology,2004,5(1):R1.
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