基于RNA-Seq筛选ALV-J感染汶上芦花鸡肝差异表达致病相关基因

Application of RNA-Seq Technology for Screening the Differentially Expressed Pathogenic Genes in Wenshang Barred Chicken Liver Infected by Subgroup J Avian Leukosis Virus

为挖掘感染ALV-J汶上芦花鸡的肝差异表达致病相关基因。本研究以汶上芦花鸡为试验素材,分别在42和300日龄进行ALV血液病毒分离检测筛选ALV阴性和阳性个体,对ALV阳性个体有病理变化的肝组织和阴性个体肝组织进行PCR检测,分别选取3只ALV阳性(G1组)和阴性(G2组)个体的肝组织,并对G1组PCR产物测序、聚类分析确定ALV亚型,利用RNA-Seq测序技术筛选差异表达基因,并进行功能分析,利用荧光定量qRT-PCR对部分差异表达基因进行验证。结果表明,G1组样品经PCR检测、PCR扩增产物测序和聚类分析确定ALV为J亚型,转录组分析发现,共有42个差异基因在GO和KEGG中富集,其中,上调基因18个,下调基因24个。随机选取的5个差异表达基因qRT-PCR验证结果与RNA-Seq测序结果相一致。GO分析显示,差异表达基因主要涉及细胞过程、代谢过程、对刺激的反应、免疫系统等;KEGG分析显示,差异表达基因主要富集在细胞过程、信号传导、疾病、新陈代谢等信号通路。本研究通过转录组分析发现了影响ALV-J致病性的多个基因和关键信号通路,为深入了解ALV-J致病的分子机制提供了思路。

The aim of this study was to screen the differentially expressed pathogenic genes in liver of Wenshang Barred chicken infected by subgroup J avian leukosis virus(ALV-J).ALV uninfected and infected Wenshang Barred chickens were pre-screened by virus isolation from blood at 42 and 300 days of age.The liver tissue samples from both ALV infected chickens which had the pathological changes and ALV uninfected chickens were identified accurately by PCR detection,then the liver tissues from 3 ALV-positive(G1 group)individuals and 3 ALV-negative(G2 group)individuals were sampled for further study.PCR ampilification products of 3 ALV-positive individuals were confirmed as subgroup J by sequencing and cluster analysis.The liver tissue samples from both 3 ALV-J positive individuals and 3 ALV-negative individuals were selected for screening the differentially expressed genes by RNA-Seq technology,then functional analysis was conducted.The partial differentially expressed genes were verified by qRT-PCR.The results showed that 42 differentially expressed genes were enriched in GO and KEGG databases,which included 18 up-regulated genes and 24 down-regulated genes.Moreover,expressions of 5 differentially expressed genes randomly selected were verified by qRT-PCR,which confirmed that mRNA expression levels were consistent with RNA-Seq sequencing results.GO analysis results indicated that the differentially expressed genes were mainly involved in cellular processes,metabolic processes,response to stimulus,immune system,and so on.KEGG analysis results showed that the differentially expressed genes were mainly enriched in cellular processes,signal transduction,disease,metabolism pathways.This study found many genes and critical signaling pathways which affected the pathogenicity of ALV-J by transcriptome analysis and would provide new ideas for understanding the pathogenic mechanism of ALV-J at molecular level.