启动子区-700T/G突变调控NR5A2基因转录和湖羊高繁殖力的分子机制

Hu sheep is a famous high fecundity breeds in China, but till now major genes of prolificacy in sheep have not been identified. NR5A2 gene is the key factors regulating the follicular development, ovulation and fertility in mammals, but no report about the relationship between NR5A 2 and prolificacy in Hu sheep has been found yet. In this project, we are intended to choose NR5A2 as candidate genes, a -700T/G mutation in promoter region of NR5A2 as object which significantly associating with litter size of Hu sheep and affecting its transcription and promoter activity of NR5A2. We will analyze the effects of MTF-1 on NR5A2 transcription and cells apoptosis of different genotypes ovarian granulose cells through vitro and vivo using RNAi, overexpression, analysis of luciferase activity, TUNEL and FACS technology, detect the binding activity of MTF-1 with different genotype promoter region using EMSA and ChIP technology in order to clarify the molecular mechanism of -700T/G mutation in promoter region regulating the high prolificacy traits of Hu sheep by transcription factor MTF-1 influencing NR5A2 transcription and further regulating granulose cell apoptosis of ovarian and follicular atresia. Our research will contribute the important theory significance to the formation of molecular mechanism analysis in Hu sheep prolificacy.

湖羊是我国著名的高繁殖力绵羊品种，目前尚未鉴定出影响其高繁殖力性状的主效基因。NR5A2是调控哺乳动物卵泡发育、排卵和繁殖力的关键基因，关于其与羊高繁殖力关系的研究未见报道。项目拟以NR5A2为候选基因，以前期筛选的与湖羊产羔数显著关联、影响其转录和启动子区活性的SNP位点-700T/G为研究对象，采用RNAi、过表达及荧光素酶活性分析、TUNEL和FACS技术，通过体内、外试验分析转录因子MTF-1对不同基因型卵巢颗粒细胞中NR5A2基因的转录及颗粒细胞凋亡的影响；采用EMSA和ChIP技术检测转录因子MTF-1与不同等位型启动子区结合活性的差异；以阐明启动子区-700T/G突变是通过转录因子MTF-1影响NR5A2基因的转录来调控卵巢颗粒细胞凋亡和卵泡闭锁，进而调控湖羊高繁殖力性状形成的分子机制。本项目对初步解析湖羊高繁殖力性状形成的分子机制具有重要的理论意义。

NR5A2,也叫LRH-1和FTF, 是孤儿核受体家族成员之一，在胚胎发育、卵巢颗粒细胞增殖、性别分化和类固醇合成中都发挥着重要的作用。但是在绵羊中NR5A2基因与繁殖性状关系的研究并不清楚。本研究扩增获得NR5A2基因启动子序列，通过构建缺失荧光素酶载体转染293T细胞，荧光素酶活性实验显示其核心启动子区位于-721~-281 nt。DNA池方法在核心启动子-700处筛选到T>G突变，基因分型和关联分析发现此位点在湖羊群体中有三种基因型，且GG型母羊的二胎产羔数（2.20±0.20; 1.97±0.06）和平均产羔数显著高于TG型（1.68±0.10; 1.74±0.05）和TT型（1.67±0.10; 1.62±0.06）母羊 (P<0.05)。GG型母羊的三胎产羔数（2.10±0.10）显著高于TT型（1.56±0.12）（P<0.05）。构建TT和GG纯合型启动子荧光素酶报告载体转染293T细胞后发现GG型启动子活性显著高于TT型，进一步的预测发现T>G突变增加了MTF-1转录因子结合位点，且竞争性EMSA实验证实MTF-1特异性与GG型NR5A2基因启动子区结合。过表达MTF-1增加NR5A2基因转录活性和NR5A2基因表达水平。干扰和过表达MTF-1后转染绵羊卵巢颗粒细胞后发现MTF-1调控绵羊卵巢颗粒细胞中caspase-3活性，说明其参与调控颗粒细胞的凋亡。结果表明NR5A2基因核心启动子区-700T>G 突变因为增加的MTF-1转录因子结合位点增加了NR5A2基因转录活性和表达水平进而提高湖羊的产羔数。