广西麻鸡卵巢组织中差异lncRNA筛选及ceRNA网络构建

Screening of Differential lncRNAs and Construction of ceRNA Network in Ovary Tissue of Guangxi Ma Chicken

【目的】筛选广西麻鸡卵巢中差异表达的长链非编码RNA(long non-coding RNA,lncRNA)、信使RNA(messenger RNA,mRNA)以及微RNA(microRNAs,miRNA),并进一步筛选出与鸡产蛋性状相关的lncRNA,为后期研究lncRNA对鸡产蛋性状的调控提供理论支持。【方法】根据广西麻鸡从开产到41周龄的产蛋情况筛选出高产和低产的广西麻鸡各3只,并以其卵巢组织为材料,进行转录组测序,使用edgeR和DEseq2软件从测序得到的数据中筛选出差异表达的lncRNA、mRNA以及miRNA,利用miRanda软件和lncRNA的顺式作用分别预测miRNA和lncRNA的靶基因,对差异lncRNA的靶基因进行GO功能和KEGG通路富集分析后,利用Cytoscape(v 3.8.2)软件构建竞争性内源RNA(ceRNA)网络图。利用实时荧光定量PCR验证转录组测序结果。【结果】本研究共获得广西麻鸡卵巢组织中438个差异表达的lncRNAs、18个差异表达的miRNAs以及301个差异表达的mRNAs。对上述差异表达的lncRNA的靶基因进行GO功能和KEGG通路富集分析后发现,其显著富集于激素分泌调节、中胚层形成、中胚层发育以及中胚层形态发生等与产蛋有关的生物过程,以及MARK、甘露糖O-聚糖的生物合成、卵母细胞减数分裂、孕激素介导的卵母细胞成熟以及促性腺激素释放激素等信号通路。通过GO功能和KEGG通路富集分析获得6个与产蛋性状相关的lncRNAs,分别是TCONS_00091814、TCONS_00051866、XR_006939520.1、XR_005839807.1、TCONS_00088588、TCONS_00037324。根据测序结果成功构建出包含9个miRNAs、60个lncRNAs和3个mRNAs的ceRNA网络图,且网络中所有的基因均在低、高产组中差异表达。所选差异表达基因实时荧光定量PCR结果与转录组测序的基因表达水平一致,说明转录组测序结果可靠。【结论】通过RNA测序技术从广西麻鸡卵巢中筛选到了一系列差异表达的lncRNA,并筛选出了6个可能与产蛋性状相关联的lncRNAs,为进一步提高鸡的产蛋性能提供了理论支持。

【Objective】This study was aimed to screen differentially expressed long non-coding RNA(lncRNA),messenger RNA(mRNA)and microRNA(miRNA)of ovary in Guangxi Ma chicken,and further select lncRNA related to egg production traits,providing theoretical support for the study of lncRNA regulation of egg production traits in chickens.【Method】Based on the egg production of Guangxi Ma chickens from start of production to 41 weeks of age,three high-yielding and three low yielding Guangxi Ma chickens were selected,and their ovarian tissues were used as materials.RNA sequencing technology was used to sequence them.Differentially expressed lncRNA,mRNA,and miRNA were screened from the sequencing data using edgER and DESeq2 software.the target genes of miRNA and lncRNA were predicted by miRanda software and cis-acting of lncRNA,respectively.GO function and KEGG pathway enrichment analysis were performed on the target genes of differentially expressed lncRNA,and a ceRNA network diagram was constructed using Cytoscape(v 3.8.2)software The transcriptome sequencing results were verified by Real-time quantitative PCR.【Result】RNA sequencing technology was used to obtain 438 differentially expressed lncRNAs,18 differentially expressed miRNAs,and 301 differentially expressed mRNAs in the ovaries of Guangxi Ma chicken.GO function and KEGG pathway enrichment analysis of the target genes of the differentially expressed lncRNAs showed significant enrichment in bioprocesses related to hormone secretion regulation,mesoderm formation,mesoderm development,and mesoderm morphogenesis related to egg production,as well as signaling pathways such as MARK,biosynthesis of glycosaminoglycans,oocyte meiosis,progesterone-mediated oocyte maturation,and gonadotropin-releasing hormone.Through GO function and KEGG pathway enrichment analysis,six lncRNAs related to egg production traits were obtained,including TCONS_00091814,TCONS_00051866,XR_006939520.1,XR_005839807.1,TCONS_00088588 and TCONS_00037324.A ceRNA network map containing 9 miRNAs with 60 lncRNAs and 3 mRNAs was successfully constructed based on the sequencing results,and all the genes in the network were differentially expressed in the low and high yield groups.Real-time quantitative PCR results of the selected differentially expressed genes were consistent with the gene expression levels from transcriptome sequencing,indicating that the transcriptome sequencing results were reliable.【Conclusion】A series of differentially expressed lncRNAs were screened from ovary in Guangxi Ma chicken using RNA sequencing technology,and six lncRNAs possibly associated with egg production traits were identified,providing theoretical support for further enhancing egg production performance in chickens.