Cloning and testicular dmr methylation analysis of b-boule gene 5′-flanking regulation regions | b-Boule基因5′调控序列的克隆与睾丸组织DMR甲基化分析

Chinese. 摘　　要：目的研究b-Boule基因5′调控序列的序列特征,以及牦牛、黄牛与犏牛睾丸组织b-Boule基因DMR甲基化状态的差异,为揭示b-Boule基因的表达调控和犏牛雄性不育的表观遗传机制提供依据。方法采用PCR扩增和克隆测序技术获得牦牛b-Boule基因5′调控序列,利用生物信息学方法分析b-Boule基因5′调控序列的序列特征,采用亚硫酸氢钠测序法检测牦牛、黄牛与犏牛睾丸组织中b-Boule基因DMR的甲基化状态。结果b-Boule基因5′调控序列长度为1 352 bp,核心启动子区含有SP1等甲基化敏感位点,5′端存在一个CpG岛。犏牛b-Boule基因DMR的甲基化水平（17.78%）高于牦牛（7.50%）和黄牛（6.94%）（P〈0.01）,特别是CpG位点33―35的甲基化水平差异更明显。结论犏牛b-Boule基因DMR的甲基化水平高于牦牛和黄牛,结合前期mRNA表达水平和组织学观察结果,认为DMR甲基化在b-Boule基因的表达调控中发挥关键作用,犏牛b-Boule基因可能是通过DMR区的高甲基化抑制其mRNA表达来阻滞精子发生减数分裂过程。

English. Objective In order to reveal b-Boule gene expression and regulation,and to supply evidence for apparent genetic mechanism of cattle-yak male sterile,the 5′-flanking regulatory sequence of b-Boule,and the difference of b-Boule gene DMR methylation status in testes among cattle,yaks and cattle-yaks were studied.Method The 5′-flanking regulatory sequence of b-Boule in yak was cloned and sequenced by the PCR amplification,the 5′-flanking regulatory sequence of b-Boule was analyzed using bioinformatics,and the DMR methylation status of b-Boule gene in testes among cattle,yaks and cattle-yaks were detected using sodium bisulfite sequencing.Result The 5′-flanking regulatory sequence length of b-Boule is 1 352 bp,and there is a CpG island.The core promoter region contains SP1 methylation-sensitive site.The methylation level of b-Boule gene DMR in cattle-yaks（17.78%） was significantly greater than that in cattle（6.94%） and yaks（7.50%）（P0.01）.The difference of the methylation level is obviously,especially the CpG site＇s 33-35.Conclusion The methylation level of yak b-Boule gene DMR is significantly greater than that of cattle-yak.Combined with the previous study results about mRNA expression and histological observation,it was speculated that the DMR methylation played a key role in the expression and regulation of b-Boule,and it might arrest meiotic process of spermatogenesis by its mRNA expression inhibition through the high methylation level of b-Boule gene DMR in cattle-yak.