梅山猪胚胎附植期EphB2的组织表达及RNA-seq分析

猪产仔数是一个重要的经济和繁殖性状, 而胚胎附植是影响猪产仔数的重要因素。为了研究促红细胞生成素产生肝细胞受体B2 (EphB2)对猪胚胎附植过程中子宫内膜迁移和粘附活动的影响, 文章以太湖流域梅山猪为研究对象, 利用实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹(Western blot)方法, 检测EphB2基因在梅山猪胚胎附植前、中、后期子宫内膜附植点/非附植点和卵巢组织的mRNA和蛋白表达谱, 并用转录组测序(RNA-seq)方法分析了不同附植时期子宫内膜附植点和卵巢组织的差异表达基因。qRT-PCR和Western blot结果表明, EphB2在胚胎附植前、中、后期子宫内膜附植点和非附植点的mRNA和蛋白均呈现先升高后降低的表达趋势, 且附植中期的表达量显著高于前期和后期(P<0.01);EphB2在附植前、中、后期卵巢中的mRNA和蛋白表达趋势则相反, 为先降低后升高, 且不同时期间表达差异显著(P<0.05)。RNA-seq结果表明, EphB2在子宫内膜附植点的mRNA表达, 附植中期极显著高于附植前期(P<0.01);EphB2在卵巢的mRNA表达, 附植中期显著高于附植后期(P<0.05)。综上所述, EphB2很可能在猪胚胎附植过程中发挥着重要的调控作用, 为潜在的猪产仔数性状候选基因。

Tissue expression of EphB2 and RNA-seq analysis during embryo im-plantation in Meishan pigs

Embryo implantation is a key step affecting swine litter size, which is an important economic and reproduction trait in pigs. In order to investigate the effect of erythropoietin-producing hepatocellular receptor B2 (EphB2) on endometrium migration and attachment during swine embryo implantation, the mRNA and protein expression levels of EphB2 in endometrium implantation sites, endometrium non-implantation sites and ovary were detected in Meishan sows during pre-implantation, mid-implantation and post-implantation period using real-time quantitative PCR and Western blot. Differential expression genes were also analyzed in endometrium implantation sites and ovary during different implantation periods by RNA sequencing (RNA-seq) technology. The qRT-PCR and Western blot results showed that EphB2 mRNA and protein expression curve was the same in endomtrium implantation sites and endometrium non-implantation sites during pre-implantation, mid-implantation and post-implantation period, with a first increase followed by a decrease, and its expression level during mid-implantation was significantly higher than pre-implantation and post-implantation (P<0.01). In contrast, EphB2 mRNA and protein expression curve in ovary during pre-implantation, mid-implantation and post-implantation period showed a first decrease followed by an increase, and the expression levels were significantly different among different implantation periods (P<0.05). RNA-seq results indicated that EphB2 mRNA expression during mid-implantation was higher than that of pre-implantation extremely significantly in endometrium implantation sites (P<0.01), and was significantly higher than that of post-implantation in ovary (P<0.05). By and large, EphB2 might play an important role in swine embryo implantation, and it’s a potential candidate gene for litter size in pigs.