大白猪仔猪感染流行性腹泻病毒后空肠转录组学变化研究

Study on the Changes in Jejunum Transcriptomics of Yorkshire Piglets Infected with Porcine Epidemic Diarrhea Virus

【目的】猪流行性腹泻(porcine epidemic diarrhea,PED)的广泛流行给猪场造成了严重损失。现有疫苗对其变异毒株的防控作用非常有限,从宿主角度筛选的有效免疫相关分子标记仍然缺乏。本研究旨在鉴定感染和未感染猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)的大白猪仔猪空肠组织差异表达基因(differentially expressed genes,DEGs),为PEDV致病机制研究奠定基础。【方法】以8头未吃初乳的0日龄大白猪为试验动物,使用人工乳饲喂至3日龄,随机挑选4头进行攻毒(感染组),其余4头作为对照组。收集感染后死亡及对照组仔猪的空肠组织进行转录组测序。通过差异表达分析筛选与病毒复制及机体免疫应答相关的基因,对DEGs进行GO功能和KEGG通路注释,并随机挑选5个DEGs进行实时荧光定量PCR验证。【结果】通过对测序数据质控、比对等分析共鉴定到16256个蛋白编码基因(protein coding genes,PCGs),通过差异分析鉴定到1328个DEGs,其中629个表达上调,699个表达下调。GO功能和KEGG通路富集分析显示,上调的DEGs显著参与了106个的GO条目和36条KEGG通路,主要与有机体系统和环境信号过程有关;下调的DEGs显著参与了54个GO条目和23条KEGG通路,主要与代谢反应有关。通过对CCND 2、CREBBP、EGFR、MCL 1、PIK 3 CB进行实时荧光定量PCR检测发现,其表达趋势与转录组测序结果一致。【结论】本研究鉴定到1328个DEGs,其中629个表达上调,699个表达下调,上调基因主要参与环境信息处理、有机体系统通路,下调基因主要参与代谢通路,为探究PEDV致病的分子机制提供了理论依据。

【Objective】The widespread prevalence of porcine epidemic diarrhea(PED)has caused serious losses for pig farms.Existing vaccines provided very limited protection against mutated strains and the effective immune-related molecular markers screened from a host perspective were lacking.This study was aimed to identify differentially expressed genes(DEGs)in jejunum of Yorkshire piglets infected with Porcine epidemic diarrhea virus(PEDV),which would lay the foundation for research on the pathogenic mechanism of PEDV.【Method】Eight 0-day-old Yorkshire piglets without colostrum were selected and rasied by milk powder until 3 days of age,four of them were attacked by PEDV as the infected group,and the other four piglets belong to control group.The jejunum were collected from the eight piglets died after infection and alive in control group for transcriptome sequencing.The genes associated with viral replication and organismal immune response were identified by differential analysis,and the GO and KEGG functional annotation were carried out for DEGs.In addition,five DEGs was randomly selected to perform Real-time quantitative PCR validation.【Result】In total,16256 protein coding genes(PCGs)were identified by quality control and comparative analysis of sequencing data,and 1328 DEGs were detected through the differential analysis,of which 629 were up-regulated and 699 were down-regulated.GO function and KEGG pathway enrichment analysis revealed that up-regulated DEGs were significantly involved in 106 GO terms and 36 KEGG pathways,that were mainly associated with organismal systems and environmental signalling processes.The down-regulated DEGs were significantly involved in 54 GO terms and 23 KEGG pathways,that were mainly associated with metabolic responses.Real-time quantitative PCR results showed that the expression trends of CCND 2,CREBBP,EGFR,MCL 1 and PIK 3 CB genes were consistent with the transcriptome sequencing results.【Conclusion】In this study,1328 DEGs were identified,of which 629 were up-regulated and 699 were down-regulated.The up-regulated DEGs genes were mainly involved in environmental information processing and organismal system pathways,while the down-regulated genes were mainly involved in metabolic pathways,providing a theoretical basis for investigating the molecular mechanism of PEDV pathogenesis.