鸽F3卵泡转录组构建及卵泡闭锁相关基因分析

Transcriptome Construction of F3 Follicle and Analysis of Genes Related to Follicle Atresia in Pigeons

【目的】通过对鸽F3卵泡颗粒细胞进行高通量测序筛选出与卵泡闭锁相关的关键基因。【方法】选择产蛋间隔第5(LI5)和7(LI7)天的白羽王鸽各3只,构建F3卵泡颗粒细胞转录文库,利用转录组测序筛选差异表达基因(differentially expressed genes,DEGs),并对DEGs进行功能注释,利用实时荧光定量PCR对随机选择的5个DEGs进行表达量验证。【结果】LI5和LI7期鸽F3卵泡颗粒细胞的6个cDNA文库中获得41.96~43.62 Mb clean reads,Q30>92%,比对率为82.21%~85.94%。筛选出LI5和LI7期F3卵泡颗粒细胞DEGs共6587个,其中2318个基因上调,4269个基因下调。GO功能富集分析显示,DEGs主要富集在细胞过程、代谢过程、连接和催化活性功能等条目。KEGG通路富集分析显示,DEGs主要显著富集到聚焦黏附、内质网加工蛋白、卵巢类固醇合成和类固醇激素生物合成等信号通路。结合蛋白-蛋白互作网络和关键通路的DGEs发现,StAR、HSD 3 B_(1)、MARCH 6、BCL 2、BOK、CDCA 7等基因在鸽卵泡闭锁中发挥了重要作用。实时荧光定量PCR结果显示,5个基因在LI5和LI7期鸽F3卵泡颗粒细胞中表达变化趋势与转录组测序结果一致。【结论】试验获得的6个差异表达基因(StAR、HSD3B1、MARCH6、BCL2、BOK、CDCA7)在鸽卵泡闭锁中发挥重要作用,为进一步了解鸽卵泡闭锁的分子机制提供依据,并为抑制鸽卵泡闭锁、提高鸽产蛋量提供理论基础。

【Objective】This study was aimed to screen key genes involved in follicular atresia by high-throughput sequencing of granulosa cell of F3 follicle in pigeons.【Method】Three White King pigeons at laiying interval 5(LI5)and 7(LI7)days were selected to construct the transcriptome library of granulosa cell of F3 follicle,respectively,and RNA-Seq was used to screen and annotate the differentially expressed genes(DEGs),the expression of 5 randomly selected DEGs were verified by Real-time quantitative PCR.【Result】The clean reads of 41.96-43.62 Mb were obtained from 6 cDNA libraries of granulosa cells of F3 follicle in pigeons at LI5 and LI7 stages,the value of Q30 was greater than 92%,and the comparison rate was ranged from 82.21%to 85.94%.A total of 6587 DEGs were obtained from granulosa cell of F3 follicle in pigeons at LI5 and LI7 stages,of which 2318 DEGs were up-regulated and 4269 were down-regulated.GO function enrichment analysis results of DEGs was performed,mainly in important biological pathways such as cell process,metabolic process,binding and catalytic activity.KEGG pathway enrichment analysis results showed that DEGs were mainly involved in focal adhesion,protein processing in endoplasmic reticulum,ovarian steroidogenesis and steroid hormone biosynthesis.Combining protein-protein interaction analysis and key DEGs in critical pathways,it found that StAR,HSD 3 B_(1),MARCH 6,BCL 2,BOK and CDCA 7 genes played an important role in follicular atresia in pigeons.Real-time quantitative PCR results showed that the expression trends of 5 genes in granulosa cells of F3 follicle in pigeons at LI5 and LI7 stages were consistent with the results of RNA-Seq.【Conclusion】Six key genes(StAR,HSD3B1,MARCH6,BCL2,BOK and CDCA7)were obtained,which played an important role in follicular atresia in pigeons.The results provided a basis for further understanding of the molecular mechanism of follicular atresia and a theoretical basis for inhibiting follicular atresia and improving egg production in pigeons.