【摘要】 【目的】研究犏牛与黄牛、牦牛睾丸组织SNRPN基因DMR甲基化状态、mRNA表达水平的差异,为揭示犏牛雄性不育的表观遗传机制提供依据。【方法】根据黄牛SNRPN基因序列设计引物,通过克隆测序获得牦牛SNRPN基因5’端序列,采用亚硫酸氢钠测序法检测犏牛及其亲本睾丸组织中SNRPN基因5’端DMR的甲基化状态,并采用Real-time PCR检测犏牛及其亲本睾丸组织中SNRPN基因的表达水平。【结果】牦牛SNRPN基因5’端序列长为1137bp,与黄牛的同源性达98.2%;生物信息学分析发现含有YY1和SP1等甲基化敏感位点。犏牛SNRPN基因DMR的甲基化水平(42.22%)极显著高于黄牛(21.08%)和牦牛(20.81%)(P<0.01)。黄牛和牦牛睾丸组织中SNRPN基因mRNA表达水平高于犏牛,但未达到显著水平(P>0.05)。【结论】犏牛睾丸组织SNRPN基因DMR的甲基化水平极显著高于黄牛和牦牛,且mRNA表达水平低于黄牛和牦牛,说明犏牛SNRPN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。更多还原

【Abstract】 【Objective】 In order to reveal the apparent genetic mechanism of cattle-yaks about male sterile, DMR of SNRPN methylation status and its mRNA expression in testes between cattle-yaks and their parents were studied. 【Method】 The 5′-flanking sequence of SNRPN in yak was cloned and sequenced by the primers which designed on the basis of sequence of cattle SNRPN DNA. In cattle-yaks and their parents’ testes, the methylation status of SNRPN 5′-flanking was detected using sodium bisulfite sequencing and the SNRPN mRNA expression levels were also detected by Real-time PCR. 【Result】 The 5′-flanking sequence of SNRPN (1 137 bp) in yak was obtained, which the homology between yaks and cattle was 98.2%, and bioinformatics analysis suggested that the region contains the putative methylation-sensitive binding sites YY1 and SP1. The results showed that the methylation level of SNRPN gene in cattle-yaks (42.22%) was very significantly greater than that in cattle (21.08%) and yaks (20.81%) (P<0.01). The SNRPN expression level of cattle-yaks was lower than that of their parents in testes, but there was no significant difference (P>0.05). 【Conclusion】 The testicular methylation level of SNRPN gene in cattle-yaks was very significantly greater than that in cattle and yaks, the testicular SNRPN expression level of cattle-yaks was lower than that of their parents, indicating that the SNRPN gene maybe arrest meiotic process of spermatogenesis by its mRNA expression inhibition through the high methylation level of SNRPN gene DMR in cattle-yaks.更多还原