猪卵泡液外泌体处理卵巢颗粒细胞的SNP/Indel筛选分析

SNP/Indel Screening Analysis of Porcine Ovarian Granulosa CellsTreated with Follicular Fluid Exosomes

旨在分析卵泡液外泌体对卵巢颗粒细胞基因的影响,了解卵泡液外泌体在卵泡发育过程中的调控机理,为母猪繁殖研究提供理论依据。本研究选取6月龄、健康状态良好且体重相近的二元母猪为材料,屠宰后收集卵巢150枚,利用梯度离心法获取卵泡液外泌体与猪卵巢颗粒细胞(porcine ovarian granulosa cells,POGCs),并在体外将卵泡液外泌体与猪卵巢颗粒细胞共培养。通过RNA-seq技术对猪卵巢颗粒细胞(granulosa cell samples,GC,n=3)和与外泌体共培养的猪卵巢颗粒细胞(granulosa-exosome co-culture samples,GCE,n=3)进行测序。结果显示,在GC和GCE组中平均每个样品获得5.54×10^(7)条clean reads,Q20和Q30质量得分均在92%以上。在GC和GCE组的6个样品中共获得1310979个SNPs突变和104498个InDel突变,其中纯合型SNP/Indel突变数量为170426个,杂合型SNP/Indel突变数量为1245052个,突变杂合子数目明显高于纯合子且SNP的发生率较高。另外,在突变类型中,转换类型共计973003个,颠换类型339974个,转换的类型显著高于颠换类型。经基因注释,突变主要发生在基因3′UTR、5′UTR、内含子区域,其次为外显子和基因间隔区。另外,通过与差异基因对比后,够筛选出1583个候选基因,经GO和KEGG功能富集发现,候选基因主要与细胞周期以及细胞增殖/凋亡过程相关。此外,共发现14个与细胞周期、增殖/凋亡通路相关的关键候选基因,其中11个基因存在互作关系。本研究获得的这些SNP/Indel信息可为后续研究外泌体在母猪生殖上的调控奠定科学基础。

The aim of this study was to analyze the effect of follicular fluid exosomes on the genes of ovarian granulosa cells,to understand the regulation mechanism of follicular fluid exosomes during follicular development,and to provide a theoretical basis for the study of sow reproduction.In this study,6-month-old binary sows with good health status and similar body weight were selected as experimental materials,and 150 ovaries were collected after slaughter,and follicular fluid exosomes and porcine ovarian granulosa cells(POGCs)were obtained from follicular fluid exosomes using gradient centrifugation,and co-cultured with follicular fluid exosomes and porcine ovarian granulosa cells was co-cultured in vitro.Porcine ovarian granulosa cells(granulosa cell samples,GC,n=3)and porcine ovarian granulosa cells co-cultured with exosomes(granulosa-exosome co-culture samples,GCE,n=3)were sequenced by RNA-seq.The results showed that an average of 5.54×10^(7) clean reads were obtained per sample in the GC and GCE groups,and the Q20 and Q30 quality scores were greater than 92%.A total of 1310979 SNP mutations and 104498 InDel mutations were obtained in 6 samples in the GC and GCE groups,among which the number of pure homozygous SNP/Indel mutations was 170426 and the number of heterozygous SNP/Indel mutations was 1245052,and the number of mutant heterozygotes was significantly higher than the number of pure heterozygotes homozygotes and with a high incidence of SNPs.In addition,among the mutation types,there were 973003 transformations and 339974 subversions transversion,and the number of transformations was significantly higher than the number of subversions transversion.After gene annotation,the mutations mainly occurred in the 3′UTR,5′UTR,and intron regions of the genes,followed by exons and spacer intergenic regions.In addition,after comparison with the differential genes,enough the 1583 and candidate genes were screened,and it was found by GO and KEGG functional enrichment that the candidate genes were mainly related to cell cycle and cell proliferation/apoptosis processes.In addition,a total of 14 key candidate genes related to cell cycle and proliferation/apoptosis pathways were identified,of which 11 genes had reciprocal interaction relationships.These SNP/Indel information obtained in this study can provide a scientific basis for subsequent studies on the regulation of exosomes on sow reproduction.