牦牛KAT8基因在组织及卵泡发育过程中的表达

Expression profiles of KAT8 gene in different tissues and ovarian follicle development of yak

探究组蛋白乙酰化酶8(KAT8)在牦牛卵泡发育及卵母细胞成熟过程中的表达规律,为深入探讨KAT8在雌性牦牛生殖过程中的作用机制提供参考。采集3~5周岁成年未妊娠母牦牛卵巢,分离培养出生发泡时期(GV)、第1次减数分裂中期(MⅠ)和第2次分裂中期(MⅡ)的卵母细胞,分别提取上述3个时期卵母细胞总RNA,反转录获得cDNA。以卵巢cDNA为模板,采用分段扩增方法,利用RT-PCR技术克隆牦牛KAT8基因编码区(CDS)序列,运用相关生物信息学分析软件预测KAT8编码蛋白的结构功能。利用实时荧光定量PCR(RT-qPCR)技术检测KAT8 mRNA在牦牛卵巢、肾脏、胃、小肠、肌肉、心脏、子宫、脾脏、肺脏和肝脏组织及GV、MⅠ、MⅡ期卵母细胞中的相对表达量,采用免疫组织化学技术分析KAT8在牦牛卵泡发育不同时期的表达定位。结果显示,克隆出牦牛1938 bp KAT8基因,其中CDS区全长为1377 bp,共编码458个氨基酸。物种进化树结果表明,牦牛与黄牛、野牛、绵羊及山羊的核苷酸序列同源性较高,分别为99.64%,99.27%,98.33%,98.04%。KAT8在牦牛各组织中广谱表达,肌肉、心脏和卵巢中的表达量相对较高,极显著高于其他7个组织(P<0.01),其中肌肉和心脏中的相对表达量显著高于卵巢组织(P<0.05)。免疫组织化学检测发现,KAT8在各发育阶段的牦牛卵泡中均有表达,主要定位于卵母细胞及颗粒细胞,卵泡膜细胞中表达相对较少。在卵母细胞减数分裂过程中,KAT8表达水平呈下降趋势,GV期卵母细胞KAT8 mRNA表达量相对较高,但与MⅠ和MⅡ期均无显著性差异(P>0.05)。KAT8基因在遗传进化过程中高度保守,并在牦牛卵泡发育及卵母细胞减数分裂进程中呈现动态表达模式,提示KAT8参与卵泡发育及卵母细胞减数分裂成熟过程。

To investigate the expression profiles of histone acetyltransferase 8(KAT8)during the maturation of ovarian follicle,the ovaries of 3-5-year-old non-pregnant female yaks were collected,and the germinal vesicle of oocytes(GV),meiosis metaphase I(MⅠ)oocytes and meiosis metaphase II(MⅡ)oocytes were isolated and cultured.Total RNA of oocytes was extracted and cDNA was obtained by RT-PCR.The coding region(CDS)of KAT8 gene in yak was amplified by RT-PCR using oocyte cDNA as template.The structure and function of KAT8 protein were predicted by bioinformatics analysis software.The gene expression profiles of yak in ovary,kidney,stomach,small intestine,muscle,heart,uterus,spleen,lung,liver,GV,MⅠoocytes and MⅡoocytes were obtained by qRT-PCR.The expression and location of KAT8during the follicles maturation were analyzed by immunohistochemistry.The result showed that the KAT8gene of the yak was 1938bp,the CDS region was 1377bp in length and encoded a total of 458amino acids.The phylogenetic tree results showed that the gene sequence of yak KAT8had high similarity with cattle,bison,sheep and goat(99.64%,99.27%,98.33%and 98.04%,respectively).KAT8was broadly expressed in various tissues of yak,and the expression levels in muscle,heart and ovary were significantly higher than those in other tissues(P<0.01).Furthermore,its relative expression levels in muscle and heart were significantly higher than those in ovary(P<0.05).Immunohistochemical detection showed that KAT8was expressed in yak follicles,and was mainly expressed in oocytes and granulosa cells.During the process of oocytes meiotic maturation,the relative expression of KAT8in GV was the highest,however,there were no significant differences between MⅠoocytes and MⅡoocytes(P>0.05).In conclusion,the above results indicate that KAT8is highly conserved in the process of genetic evolution and shows a dynamic expression pattern during the maturation of follicles and oocyte meiosis process of yak,suggesting that KAT8 is involved in the follicle maturation and process of oocyte meiosis.