miR-30a通过表观调控Per2基因影响苏尼特羊季节性发情性状的分子机制

Seasonal estrus is a main bottleneck restricting the efficient production in sheep industry. The molecular mechanism and major genes for ovine seasonal estrus are still unclear. It is confirmed that alternations between a breeding season and a period of anestrus depend upon the ability of day length (photoperiod) to synchronize an endogenous timing mechanism which called the circadian clock. The circadian clock in mammals depends on interacting transcriptional/translational feedback loops. Clock gene Per2 is an important regulator of feedback loops which has been confirmed that the protein expression changes according to the change of rhythm cycle. It is highly likely that microRNA regulation plays critical roles in the mammalian clock to its target genes. Researches on mice had shown that miR-30a can regulate the expression of clock genes, thus is there a targeting regulation between miR-30a and Per2 gene affecting the estrus of sheep? In order to verify this hypothesis, the expression of miR-30a and Per2 will be detected under different lighting conditions in hypothalamus of Sunite sheep (seasonal breeder). Luciferase-reported system will be applied to test if mRNA of Per2 gene is direct target of miR-30a. Furthermore, whether the exogenous inhibition of miR-30a expression can lead to an altered circadian rhythm will also be checked. We will try to elucidate the mechanism of how Per2 is epigenetically regulated by miR-30a to mediate seasonal estrus and provide idea for genetic alteration of the seasonal estrus in sheep.

季节性发情是绵羊生产效率低下的主要因素之一，但目前绵羊季节性发情的分子机理和主效基因都不明晰。已有研究表明动物通过生物钟协同外界光周期的变化调节发情期和乏情期的转换。哺乳动物的生物钟是由基因反馈环在转录和翻译水平的相互作用调控的。钟基因Per2是基因反馈环的重要调节因子，其蛋白表达量会随节律周期的改变而变化。miRNA对其靶基因的表观调节在哺乳动物生物钟调控中起关键作用；研究发现小鼠miR-30a能调控钟基因的表达，那绵羊miR-30a能否靶向调控Per2而影响其发情活动？为验证这一假设，本项目拟检测miR-30a和Per2在不同光照条件下苏尼特羊下丘脑中的表达差异；双荧光报告素酶系统确定Per2和miR-30a的靶标关系；结合miR-30a对生物节律的影响，初步阐明miR-30a通过表观调节Per2的表达影响绵羊季节性发情的分子机制，为通过分子遗传方法改变绵羊季节性发情性状提供理论基础。

我国大部分地方绵羊都属季节发情品种，绵羊生产效率受限。本研究以常年发情小尾寒羊和季节性发情苏尼特羊为实验对象，研究钟基因在不同情期的表达特征及其和miRNA的靶标关系。主要结果如下：.1.Per基因（Per1、Per2、Per3）在2个不同品种绵羊的松果体、下丘脑、垂体、卵巢、输卵管以及子宫中均表达。Per1表达量在苏尼特羊中均高于小尾寒羊，在松果体、卵巢和输卵管达到显著差异（P<0.05）。由分型结果，Per1基因的2个SNPs g.27342699G>A和g.27346502A>G均存在3种基因型，g.27342699G>A基因型频率和等位基因频率在季节性发情和常年发情的绵羊品种中达到显著差异水平（P≤0.05）。.2.Per2基因在苏尼特羊卵巢和子宫中的表达均显著高于小尾寒羊，在输卵管中相反（P<0.05）；由分型结果，Per2基因的g.2852655T>C位点存在3种基因型，该位点基因型频率和等位基因频率在两种绵羊品种之间有显著差异（P<0.05）。.3.在苏尼特羊中，除输卵管外，Per3基因的表达量在其它组织中均高于小尾寒羊，且松果体和子宫达显著水平（P<0.01）；由分型结果，g.43657503A>G、g.43670012T>C、g.43670807A>G、g.43677259T>C和g.43707227G>A 5个SNPs在2个绵羊品种间基因型频率、等位基因频率均达到显著差异（P<0.05）。.4.3’RACE法扩增Per2的3’UTR和CDS区并测序，预测绵羊miR-30a-5p与Per2基因的CDS区存在靶结合位点，3’UTR结合位点已发生突变。.5.将合成的Per2基因CDS区和3’UTR野生型和突变型的序列构建到双荧光载体中，转染细胞。与对照组相比，miR-30a和Per2-CDS组荧光素酶活性显著降低（P＜0.01），miR-30a和PER2-3’UTR无明显变化。miR-30a-5p与Per2基因的CDS区具靶标关系。.钟基因Per在绵羊多组织表达，较高水平表达的Per基因可能与季节性发情调控有关；Per1基因1个SNP位点、Per2基因1个SNP位点以及Per3基因5个SNP位点可能是季节性发情调控的关键位点。绵羊miR-30a-5p与Per2基因CDS区存在一个靶结合位点。.本项目结果为常年发情绵羊新品种选育提供了分子基础和候选基因。