牛体外受精胚胎早期发育差异表达基因的筛选及定量检测  

Screening and Quantitative Analysis of Differential Expression Genes in In vitro Fertilized Bovine Pre-implantation Embryos

作　　者：刘冬[1] 沙日娜[1] 温建勋[1] 王瑞[1] 仓明[1] 刘东军[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学及生物技术教育部重点实验室,呼和浩特010021

出　　处：《农业生物技术学报》2012年第1期54-61,共8页Journal of Agricultural Biotechnology

基　　金：国家基础科学人才培养基金(No.J0730648);国家自然科学基金(No.30760168);内蒙古自然科学基金重大项目(No.200711020401)

摘　　要：卵母细胞成熟和母源型-合子型过渡过程中,存在大量基因的表达差异,使得应用mRNA差异显示(DDRT-PCR)筛选对胚胎发育起关键作用的基因成为可能。为了解牛卵母细胞成熟过程及体外受精胚胎不同发育阶段相关基因mRNA表达规律,本研究以牛生发泡(GV)期卵母细胞、体外受精获得的8细胞期胚胎和囊胚期胚胎为检测对象,利用mRNA差异显示技术筛选这三个时期mRNA表达差异基因,并用实时定量PCR分析差异基因在这三个时期以及体外成熟卵母细胞中的mRNA丰度。结果成功筛选得到5个差异片段,经测序和GenBank数据库比对分析,检索到与这5个片段有高同源性的已知基因,分别为无机焦磷酸1基因(PPA1)、ErbB2互作蛋白基因(ERBB2IP)、350kD中心体相互作用蛋白基因(CEP350)、核糖体蛋白L27a基因(RPL27A)和Ⅱ类主要组织相容复合物下调因子基因(IK),实时定量PCR检测和SPSS统计分析结果表明,PPA1、ERBB2IP和CEP350mRNA表达量随胚胎发育进程呈不同程度的降低趋势,RPL27AmRNA表达量在囊胚期由降低转为升高,IK的转录物含量在囊胚期前呈波动变化,到囊胚期显著降低。本研究结果为进一步了解卵母细胞成熟对后续胚胎发育的影响以及合子基因组激活机制提供了实验依据。The large capacity of differential expression genes during oocyte maturation and the transition from maternal to zigotic control makes it possible to screen genes playing crucial roles in embryonic development by mRNA differential display reverse transcription PCR（DDRT-PCR）.In order to explore the gene expression mechanism of in vitro maturation（IVM） of oocytes and in vitro development of pre-implantation embryos derived from in vitro fertilization（IVF） in different developmental stages in cattle,mRNA DDRT-PCR was employed to screen differential expression genes in germinal vesicle（GV） stage oocytes,8-cell stage embryos and blastocysts.Real-time quantitative PCR（Q-PCR） was used to analyze the expression abundance of the differential expression genes in the three stages and in vitro matured MⅡ oocytes.Five differential displayed straps were discovered by DDRT-PCR.The results of sequencing and database analysis showed that the 5 fragments were corresponding to pyrophosphatase（inorganic） 1（PPA1）,erbb2 interacting protein（ERBB2IP）,restin-like（CEP350）,ribosomal protein L27a（RPL27A） and IK cytokine,down-regulator of HLAⅡ（IK）,respectively.Q-PCR and SPSS statistical analysis indicated that the mRNA level of PPA1,ERBB2IP and CEP350 decreased along with the procedure of embryonic development,whereas that of RPL27A turned to increase till blastocyst stage.The mRNA level of IK fluctuated before blastocyst stage and then decreased.Overall,the results of this research provide more information on effects of oocyte maturation on the following embryonic development and the mechanism of zygotic gene activation.

关 键 词：卵母细胞成熟 胚胎发育 差异基因 MRNA差异显示 实时定量PCR 

分 类 号：S823[农业科学—畜牧学]