牛早期胚胎发育中AID基因的表达及其调节区DNA甲基化的动态变化

doi:10.13560/j.cnki.biotech.bull.1985.2016.07.034

摘要/Abstract

摘要： 以具有DNA主动去甲基化作用的活化诱导胞苷脱氨酶（Activation-induced cytidine deaminase,AID,亦称为AICDA）基因为研究对象,检测其在牛卵母细胞及体外受精胚胎发育不同阶段的表达变化及其调节方式,揭示细胞重编程分子机制。应用Real-time PCR、BSP（Bisulfite Sequencing PCR）和免疫荧光化学等方法分析DNA甲基化对牛早期胚胎发育中AID基因表达的影响。结果显示,AID基因在牛早期胚胎发育中受DNA甲基化的调控,AID基因的T-DMR（tissue-dependent and differentially methylated region）位于其转录起始位点-88 bp – -431 bp。在牛卵母细胞成熟过程中,T-DMR第2和第3号CpG位点的DNA甲基化明显去除,而其他位点都未发生变化。卵母细胞在成熟过程中AID基因的积累与DNA甲基化状态变化相关。在牛体外受精胚发育早期的各阶段,尽管AID基因的表达不同,但AID基因T-DMR除第2和第3号CpG位点一直都维持去甲基化状态外,其他位点始终维持甲基化状态。推测其表达的变化可能是胚胎基因组激活有关。以上研究表明,AID基因T-DMR的低甲基化与其表达存在一定的相关性。通过免疫荧光检测发现,从卵母细胞成熟期到桑椹胚期,AID蛋白都是均匀分布于细胞核和细胞质中。而囊胚时期大量AID蛋白集中于内细胞团,这可能对于内细胞团多能性的维持起重要作用。综上所述,牛卵母细胞成熟中积累的AID作用于受精过程,其启动子区的DNA去甲基化与AID基因的表达有关,胚胎基因组激活后AID基因的表达可能与胚胎发育有关。

关键词: AID基因, 牛体外受精胚胎, DNA甲基化

Abstract: DNA methylation plays an important role in mammalian fertilization. The chromosome has undergone complex reconstruction and ultimately obtained totipotent during fertilization. Using activation-induced cytidine deaminase protein（AID,also known as AICDA with DNA active demethylation function,as research target,the expression changes of AID and its regulation mechanisms at different development stages of oocyte and in-vitro fertilization（IVF）embryo were detected for exploring the cell reprogramming mechanism. Real-time PCR,BSP（Bisulfite Sequencing PCR）,and immunofluorescence chemistry were used to analyze the effects of DNA methylation on the AID expression during bovine early embryonic development. The results showed that AID gene was regulated by DNA methylation in the bovine early embryonic development. Combining the expression of AID gene in the different tissues and DNA methylation status of its promoter region,the tissue-dependent and differentially methylated region（T-DMR）of AID gene was located at the transcriptional start site -88 bp--431 bp. During the maturing process of bovine oocyte,No. 2 and 3 CpG sites in the T-DMR region were obviously demethylated,whereas the other sites were not changed,and AID gene was correlated with the DNA methylation. At the different stages of early IVF embryo,though the expression of AID gene varied,only No. 2 and 3 CpG sites in the T-DMR region of AID were in the status of demethylation,while other sites were in methylation continuously,the changes of the expression probably was related to the embryonic genome activation. The above results revealed that low methylation of T-DMR in AID gene was correlated with its expression. Immunofluorescence detection discovered that AID proteins were uniformly distributed in the nucleus and cytoplasm from the oocyte maturation stage to morula stage. And a large number of AID protein concentrated in the inner cell mass at the blastocyst stage,which played the important role in the maintenance of pluripotency of the inner cell mass. In summary,accumulated AID during oocyte maturation presents an effect on fertilization,the DNA methylation in the promoter region is closely related to its expression,and the expression of AID gene,after activation of the embryonic genome,may be associated with embryo development.

Key words: AID gene, bovine in vitro fertilization, DNA methylation

奥旭东,萨如拉,王杰,王会敏,于海泉. 牛早期胚胎发育中AID基因的表达及其调节区DNA甲基化的动态变化[J]. 生物技术通报, 2016, 32(7): 242-249.

AO Xu-dong SA Ru-la WANG Jie WANG Hui-min YU Hai-quan. Expression of AID and Dynamic Changes of Its DNA Methylation in Regulation Region During Bovine Early Embryonic Development[J]. Biotechnology Bulletin, 2016, 32(7): 242-249.

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