ZBED6基因敲除猪肾脏转录组分析  

Transcriptome Analysis of Porcine Kidney with ZBED6 Gene Deletion

作　　者：田文杰 王丹丹 王圣楠 马月辉[2] 蒋琳[2] 宋子仪 TIAN Wenjie;WANG Dandan;WANG Shengnan;MA Yuehui;JIANG Lin;SONG Ziyi(College of Animal Science and Technology,Guangxi University,Nanning 530000,China;Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,China)

机构地区：[1]广西大学动物科学技术学院,南宁530000 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《中国畜牧兽医》2022年第5期1662-1670,共9页China Animal Husbandry & Veterinary Medicine

基　　金：广西自然科学基金面上项目(2020GXNSFAA297043);国家重点研发计划项目(2020YFA0509503)。

摘　　要：【目的】探究锌指BED结构域结合蛋白6(zinc finger BED domain-containing protein 6,ZBED6)基因敲除对猪肾脏组织基因转录表达的影响,并解析ZBED6基因调控猪肾脏代谢的靶基因及其通路。【方法】对8月龄ZBED6基因敲除巴马香猪(ZBED6 KO)和同月龄野生型巴马香猪(WT)的肾脏组织(n=3)进行总RNA提取,利用实时荧光定量PCR检测胰岛素样生长因子2(insulin-like growth factor 2,IGF2)和差异基因的表达量;以Illumina Hiseq高通量测序技术对ZBED6 KO和WT猪肾脏组织mRNA进行转录组测序(RNA-Seq),用猪Sus scrofa 11.1作为参考基因组序列,通过生物信息学软件TopHat、Cufflink、Cuffmerge和Cuffdiff对测序数据进行分析,筛选ZBED6 KO和WT猪肾脏组织中的差异表达基因,对差异表达基因进行层次聚类和KEGG通路富集分析,并通过实时荧光定量PCR验证RNA-Seq结果中差异表达基因的可靠性。【结果】实时荧光定量PCR结果显示,ZBED6 KO猪肾脏IGF2基因mRNA表达量显著高于WT猪(P<0.05)。测序共获得78 G数据量,每个样本比对率在87.3%以上,测序质量良好;ZBED6 KO和WT猪肾脏组织之间共检测到25213个基因,以调整后P<0.05和log2|FoldChange|>2为筛选标准,得到299个差异表达基因,其中上调的基因103个,下调的基因199个。热图和主成分分析(PCA)结果显示,ZBED6 KO和WT组猪肾脏基因组内表达模式相似,组间表达模式不同;KEGG通路富集分析显示,差异基因主要参与视黄醇及疾病代谢相关通路。ZBED6基因敲除后,其中有9个差异表达基因(CYP2C42、AOX1、ENSSSCG00000036274、RDH16、CYP2A19、ENSSSCG00000022724、CYP26B1、CYP1A1、RDH5)被富集到视黄醇代谢通路中,可能参与调控猪肾脏代谢平衡。实时荧光定量PCR检测发现,7个差异表达基因(CYP2C42、AOX1、RDH16、CYP2A19、CYP26B1、CYP1A1、RDH5)的表达模式与RNA-Seq分析结果一致,证实RNA-Seq结果的可靠性。【结论】本试验利用RNA-Seq技术分析了ZBED6基因敲除�【Objective】This study was aimed to investigate the effect of zinc finger BED domain-containing protein 6(ZBED6)gene knockout on gene transcription and expression in porcine kidney,and analyze the target genes and pathways of ZBED6 gene regulating porcine kidney metabolism.【Method】Kidney tissues were collected from 8-month-old ZBED6 gene knockout Bama Xiang pigs(ZBED6 KO)and age-matched wildtype Bama Xiang pigs(WT)(n=3).The total RNA was extracted and Real-time quantitative PCR was used to analyze the expression of insulin-like growth factor 2(IGF2)and other differentially expressed genes.Illumina Hiseq high-throughput sequencing technology was performed to evaluate the mRNA of kidney in ZBED6 Ko and WT pigs.Sus scrofa 11.1 was used as the reference genome sequence,the differentially expressed genes were analyzed and filtered by the softwares TopHat,Cufflink,Cuffmerge and Cuffdiff.Hierarchical clustering analysis and KEGG pathway enrichment of the differentially expressed genes were also performed.The reliability of differentially expressed genes in RNA-Seq results was verified by Real-time quantitative PCR.【Result】Real-time quantitative PCR results showed that the expression of IGF2 gene mRNA in kidney of ZBED6 KO pigs was significantly higher than that of WT pigs(P<0.05).The sequencing results showed that 78 G data was obtained,and at least 87.3%of the reads were mapped to porcine genome,suggesting the quality of sequencing was high.RNA-Seq analysis showed that a total of 25213 genes were detected in kidney of WT and ZBED6 KO pigs.Under the criteria of adjust-P<0.05 and log2|FoldChange|>2,299 differentially expressed genes were obtained,including 103 up-regulated genes and 199 down-regulated genes.Heatmap and PCA results showed that the gene expression pattern was similar within groups,and was different between groups.KEGG pathway enrichment analysis displayed that the differentially expressed genes were mainly involved in retinol and other metabolic pathways,especially,9 differentially expressed gen

关 键 词：猪 ZBED6基因 肾脏 代谢 RNA-SEQ 差异表达基因 

分 类 号：Q78[生物学—分子生物学]