湖羊和巴什拜羊BMP15基因c.-1760C>A变异与启动子区活性的关系

[目的] 为了解湖羊和巴什拜羊骨形态发生蛋白15(BMP15)基因编码区和5’调控区(5’UTR)序列特征与差异,检测其SNPs(single nucleotide polymorphisms)并探索其功能。[方法] 利用克隆测序技术获得湖羊和巴什拜羊BMP15基因编码区和5’UTR序列,运用生物信息学方法分析其序列特征;采用DNA池测序法筛选湖羊和巴什拜羊BMP15基因SNPs,直接测序法检测SNPs位点多态性;采用荧光素酶报告基因系统检测启动子区活性。[结果] 湖羊和巴什拜羊BMP15基因编码区序列长度均为1182 bp,编码393个氨基酸,编码蛋白含有典型的TGF-β结构域;湖羊和巴什拜羊BMP15基因编码区序列的同源性为99.92%,发现1个SNP位点。获得1806 bp湖羊和巴什拜羊BMP15基因5’调控区序列,发现5个SNPs位点。BMP15基因c.-1760C>A位点多态性分析发现湖羊群体中均为CC基因型,巴什拜羊群体中有CC、CA和AA 3种基因型,等位基因C和A频率分别为0.7981和0.2019。启动子区活性检测显示CC型启动子区活性明显高于AA型。[结论] 湖羊和巴什拜羊BMP15基因编码区序列高度保守,c.-1760C>A变异可能影响BMP15基因启动子区活性。
[Objectives] To identify the characterization of 5’-untranslated region (5’-UTR) and coding region (CDS) sequence of bone morphogenetic protein15 (BMP15) gene between Hu sheep and Bashibai sheep,we analyzed the single nucleotide polymorphism (SNP) in BMP15 gene and explored its function.[Methods] The coding and 5’-untranslated sequence of BMP15 gene were obtained using cloning and sequencing technology and we used the bioinformatics methods to analyze its sequence characterization in the Hu sheep and Bashibai sheep. The SNPs of BMP15 gene were screened using DNA pool technology and were detected using sequencing method. The activity of BMP15 promoter was investigated using dul-luciferase reporter assay system.[Results] The full length coding region of BMP15 were both 1 182 bp in Hu sheep and Bashibai sheep,which encoded 393 amino acids and contained TGF-β domain. The coding regions of BMP15 in Hu sheep shared similarities of 99.92% to Bashibai sheep,in which we detected one SNP in Hu sheep and Baishibai sheep. A total of 1 806 bp sequence of 5’-UTR was obtained,where we identified five SNPs in Hu sheep and Baishibai sheep. There was one genotype (CC) and three genotypes (CC,CA,AA)(c.-1760C>A) in the 5’-UTR of BMP15 gene in Hu sheep and Bashibai sheep,respectively. Further analysis showed that the frequency of C allele and A allele were 0.798 1 and 0.201 9,respectively. The luciferase reporter assay indicated that the promoter activity of genotype CC was obviously high than genotype AA.[Conclusions] The coding region sequences of BMP15 gene are highly conservative between Hu sheep and Bashibai sheep. The SNP (c.-1760C>A) may be involved in the effect on the promoter activity of BMP15 gene