AANAT基因在滩羊不同繁殖时期卵巢中的转录差异及DNA甲基化程度分析

试验旨在检测5-羟色胺-N-乙酰基转移酶(AANAT)基因在绵羊休情季节和繁殖季节(卵泡期和黄体期)卵巢组织中的转录差异,并分析转录差异是否由DNA甲基化修饰程度改变所导致。试验采用自然环境条件和饲养管理一致,且体重差异在0.5 kg范围内的空怀母滩羊作为试验动物,采集其休情期、卵泡期和黄体期(每个时期3只)的卵巢组织,采用SYBR染料法进行实时荧光定量PCR检测AANAT基因在滩羊不同繁殖时期卵巢组织中的转录水平。随后针对转录水平有差异的两个时期(休情期和卵泡期)的样本,利用MethPrimer 2.0在线软件预测AANAT基因启动子区和第一外显子区的CpG岛;用重亚硫酸盐测序法(BSP法)检测AANAT基因启动子区及第一外显子区的甲基化程度。试验结果显示,滩羊休情期卵巢组织中AANAT基因转录水平显著低于卵泡期的AANAT基因转录水平(P<0.05),休情期与黄体期滩羊卵巢组织中AANAT基因的转录水平差异不显著(P>0.05)。滩羊卵巢组织中AANAT基因启动子区上存在着一个长度为173 bp的CpG岛,第一外显子区存在着一个长度为118 bp的CG岛。然而,两个甲基化岛区内的单个CpG位点甲基化程度在滩羊休情期和卵泡期之间均不存在显著差异,暗示AANAT基因的表达受甲基化修饰外的因素调控。本研究结果可为进一步探讨AANAT基因在季节性发情和卵泡成熟中的功能提供参考资料。

Analysis on Difference of Transcription and DNA Methylation Level of AANAT Gene in Ovary of Tan Sheep at Different Reproductive Stages

The aim of this study was to detect the transcriptional differences of 5-hydroxytryptamine N-acetyltransferase(AANAT)gene in ovine ovarian tissue during the anestrus and breeding season(follicular stage and luteal stage)and to analyze whether the transcriptional differences were caused by changes in the degree of DNA methylation modification.In the study,non-pregnant Tan sheep whose natural environmental conditions and feeding management were consistent and the weight difference was within 0.5 kg were selected.Their ovarian tissues during the anestrus,follicular and luteal stages(3 ewes in each stage)were collected.SYBR dye method was used to carry out Quantitative real-time PCR to detect the transcription level of AANAT gene in the ovarian tissue of tan sheep at different reproductive stages.Subsequently,MethPrimer 2.0 online software was used to predict the CpG island in promoter region and the first exon region of AANAT gene,and the bisulfite sequencing(BSP)method was used to detect the methylation degree of the promoter region and the first exon region of the AANAT gene for the anestrus and follicle stage samples with transcriptional differences.The results showed that the transcriptional level of AANAT gene in the ovarian tissue of Tan sheep during anestrus was significantly lower than that in the follicular period in the breeding season(P<0.05),and there was no significant difference of transcriptional level between anestrus and luteal period(P>0.05).There was a CpG island with a length of 173 bp in the AANAT gene promoter region and a CG island with a length of 118 bp in the first exon region in the Tan sheep ovarian tissue.However there was no significant difference on the methylation degree of the single CpG site in promoter region and the first exon of the AANAT gene in Tan sheep ovary tissue,suggesting that the expression difference of the AANAT gene was regulated by other factors other than methylation modification.The results of this study might provide references for further study on the function of AANAT gene in seasonal estrus and follicular maturation.