ZBED6基因对巴马香猪脾发育的作用机制分析

Mechanism of ZBED6 Gene on Spleen Development of Bama Xiang Pig

ZBED6基因作为一个转录因子,能够与IGF2结合进而调节肌肉的生长发育。但其在脾生长发育中的作用尚不清楚,本研究利用RNA-seq测序技术比较ZBED6基因敲除巴马香猪(ZBED6-KO)和同日龄野生型巴马香猪(WT)的脾组织转录组,探究ZBED6基因对巴马香猪脾组织的发育影响。利用t检验对ZBED6-KO猪和WT猪脾组织大小的表型差异及ZBED6直接调控的靶基因IGF2的表达量进行显著性分析。提取ZBED6-KO猪和WT猪脾组织的总RNA,在Illumina Hiseq 2500平台进行RNA-seq分析。以猪Sus scrofa11.1为参考基因组,用转录组分析的标准流程筛选ZBED6-KO猪和WT猪脾组织中的差异表达基因。用DAVID在线网站对差异表达基因进行GO和KEGG富集分析。然后,随机选取7个差异表达的基因,利用实时荧光定量PCR技术验证测序结果的准确性与可靠性。结果显示,与WT猪相比,ZBED6-KO猪脾的重量和IGF2基因表达量均显著增加(P<0.05),表明ZBED6基因的敲除对巴马香猪脾组织的生长发育有一定的促进作用。测序结果显示,各样本至少获得4G的数据量,每个样本的Clean Ratio及Q30比率均在90%以上,83.94%以上的reads可比对在猪的参考基因组上,表明测序数据质量良好,真实可靠;对测序数据进行转录组分析,共筛选到161个差异表达基因,其中上调基因90个,下调基因71个;差异表达基因的层次聚类分析显示,ZBED6-KO猪的3个个体(spleen1、spleen3、spleen6)的表达模式相似,WT的3个个体(spleen2、spleen4、spleen5)的表达模式相似,进一步证明测序数据的准确可靠;GO和KEGG富集分析中,富集到10条显著的GO条目以及5条显著的信号通路,2条与肌肉发育相关的通路;实时荧光定量PCR试验随机检测7个差异表达基因的表达模式与RNA-seq分析结果相一致,证实了测序数据的可靠性。以巴马香猪为模型,利用RNA-seq技术研究ZBED6基因的敲除对中国地方猪脾发育的作用影响,为挖掘ZBED6基因的更多功能提供了基础。

As a transcription factor,ZBED6 gene can bind to IGF2 to regulate muscle growth and development.But its role in the growth and development of the spleen is unclear.RNA-seq sequencing technology was used to compare the spleen tissue transcription of ZBED6 gene knockout Bama Xiang pigs(ZBED6-KO)and wild-type Bama Xiang pigs(WT)at the same age,so as to explore the effects of ZBED6 gene on spleen tissue development of Bama Xiang pigs ZBED6 knockout.The t-test was used to analyze the phenotypic differences of spleen tissue,and the gene expression of IGF2 that a target gene directly regulated by ZBED6 transcription factors between ZBED6-KO pigs and WT pigs.The total RNA from the spleen tissues of ZBED6 knockout group and wild-type group of Bama Xiang pigs was extracted,and RNA-seq analysis was performed on the Illumina Hiseq 2500 platform.Using Sus scrofa11.1 as reference genome,the differentially expressed genes in spleen tissues of ZBED6-KO and WT Bama Xiang pigs were screened by the standard process of transcriptome analysis.Using DAVID online website to analyze GO and KEGG enrichment analysis of differentially expressed genes.Seven differentially expressed genes were randomly selected to verify the accuracy and reliability of the sequencing results by the RT-qPCR technique.Compared with WT Bama Xiang pigs,the spleen weight and IGF2 gene expression of ZBED6-KO pigs were significantly increased(P<0.05),indicating that ZBED6 gene knockout could promote the growth and development of spleen tissue of Bama Xiang pigs.The sequencing results showed that at least 4G of data was obtained from each sample,and the ratio of Clean Ratio to Q30 of each sample was more than 90%,and more than 83.94%of the reads could be mapped with the pig reference genome,which indicating that the sequencing data were true and reliable.A total of 161 differentially expressed genes were screened by transcriptome analysis,including 90 up-regulated genes and 71 down-regulated genes.Hierarchical cluster analysis of differentially expressed genes showed that the expression patterns of 3 individuals(spleen1,spleen3,spleen6)of ZBED6-KO pigs were similar,and the expression patterns of 3 individuals(spleen2,spleen4,spleen5)of WT were similar,which further proved that the sequencing data were accurate.In GO and KEGG enrichment analysis,10 significant GO terms and 5 significant pathways were enriched,in which 2 pathways were related to muscle development.The expression patterns of 7 differentially expressed genes were randomly detected by real-time fluorescent quantative PCR experiments consistent with the results of RNA-seq analysis,which confirmed the reliability of sequencing data.Using the Bama Xiang pig as a model,the RNA-seq technology was used to study the effect of ZBED6 gene knockout on the spleen development of domestic pigs,which provided a basis for mining more functions of the ZBED6 gene.