KDM2B基因克隆及其在牦牛组织和卵母细胞减数分裂过程中的表达研究

摘要

This research was conducted to clone the Lysine-specific histone demethylase 2B (KDM2B) gene in yak,identify its expression pattern in various tissues and meiosis process of oocytes,respectively,which might provide reliable basis for studying the mechanism of KDM2B in oocyte meiosis of yak.The samples of yak heart,liver,spleen,lung,kidney,brain,small intestine,stomach,muscle,ovary,testicle and uterus were collected after slaughtering.Total RNAs in different samples were extracted.Ovaries from healthy yaks of 3 to 5 years old were selected for collecting the cumulus-oocyte complex (COCs),and then oocytes and cumulus cells were collected by hyaluronidase.The coding sequence of KDM2B gene was cloned by RT-PCR.The mRNA expression of KDM2B in different tissues was determined by quantitative real-time PCR (qRT-PCR).The COCs in 3 stages of GV,MⅠ and MⅡ were cultured in vitro,and the expression of KDM2B in the process of oocyte meiosis was detected by qRT-PCR.The results showed that the CDS region of KDM2B was 3 930 bp,encoding 1 309 amino acids.Compared with the existing predicted yak sequence,it belonged to long-form transcript and had high homology with cattle,sheep and goat,but had lower homology with zabra,fish and chicken.KDM2B gene of yak was widely expressed in various tissues,and its expression was relatively higher in spleen,uterus,testicle and ovary.The expression level of KDM2B in MⅠ stage was significantly higher than that in GV and MⅡ stage in oocytes (P＜0.01).In cumulus granulosa cells,the expression level of KDM2B was significantly increased by the process of oocyte meiosis (P＜0.01).This research provided basic data for further research of oocyte meiosis mechanism and improving breeding efficiency in yak.%本研究旨在克隆牦牛组蛋白去甲基化酶2B(Lysine-specific histone demethylase 2B,KDM2B)基因,检测其在牦牛不同组织及其在卵母细胞减数分裂过程中的表达水平,从而为研究KDM2B基因在牦牛减数分裂中的作用机制提供试验依据.牦牛屠宰后,采集心、肝、脾、肺、肾、脑、小肠、胃、肌肉、卵巢、睾丸和子宫组织样品,分别提取各个样本的总RNA.另选择3～5周岁的健康牦牛,采集卵巢后,抽取卵丘-卵母细胞复合体(Cumulus-oocyte complex,COCs),透明质酸酶作用后,获得卵母细胞和颗粒细胞,利用RT-PCR扩增得到KDM2B基因CDS区序列,实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)检测KDM2B基因在不同组织中的表达水平.体外培养获得GV、MⅠ、MⅡ 3个时期COCs,用qRT-PCR法检测KDM2B基因在卵母细胞减数分裂过程中的表达规律.结果表明,KDM2B基因CDS区长为3 930 bp,编码1 309个氨基酸,与牦牛已有预测序列相比,属于长型转录本.与牛、绵羊、山羊的同源性较高,与斑马鱼和鸡的同源性较低.KDM2B基因在牦牛的各个组织中均有表达,其中脾、子宫、睾丸和卵巢的表达量较高.在MⅠ期卵母细胞中,KDM2B基因的表达水平显著高于GV期和MⅡ期(P＜0.01).在卵丘颗粒细胞中,KDM2B基因的表达水平随减数分裂的进行而显著增加(P＜0.01).本研究为进一步研究牦牛卵母细胞减数分裂机制及改善牦牛繁殖效率提供基础资料.