绵羊Myostatin基因5′调控区的生物信息学分析及孕激素对调控区活性的影响

摘要

[目的]克隆并分析绵羊Myostatin基因5′调控区,并在细胞水平研究孕激素对该调控区活性的影响.[方法]利用PCR方法扩增绵羊Myostatin基因5′调控区,采用NatInspector等软件分析并比较其和牛、猪调控区的调控元件和转录因子.以EGFP为报告基因,构建表达载体,转染C2C12细胞,同时添加不同剂量的孕激素(0、10、100和1000nmol·L-1),然后通过RT-PCR方法检测报告基因EGFP和内源性Myostatin的mRNA水平.[结果]获得绵羊约3.2kb Myostatin基因5′调控区(MSTN5′regu),预测了两个对双肌性状可能存在贡献的位点.在绵羊、牛和猪的相应调控区发现了许多可能对该基因转录调控起重要作用的调控元件和相应转录因子,包括E-box、MEF2、MTATA、MTBF、HOMF、PRE、ARE和GRE等.各种元件在不同动物间基本都有,但具体的序列、位置以及数量有所差异.成功构建了绵羊pMSTN5'regu-EGFP表达载体,且转染后100nmol·L1孕激素明显抑制了内源性Myostatin mRNA和报告基因EGFP mRNA的表达.[结论]绵羊Myostatin基因的转录受多种转录因子和相应调控元件的调控,适量孕激素可通过下调Myostatin基因5′调控区的活性而抑制该基因的转录.%[Objective] The aim of this study is to clone and analyze the 5' regulatory region of myostatin gene in sheep and to investigate the effect of progesterone on the activity of the 5' regulatory region. [Method] The 5' regulatory region of myostatin gene in sheep was cloned by PCR, and the regulatory motifs and transcriptional factors, along the region or the similar regions of bovine and porcine, were analyzed and compared by some softwares such as Matlnspector. The EGFP was selected as reporter gene and the expression vectors were constructed. The vector was transfected into C2C12 cells and the different doses of progesterone (0 nmol-L"1, 10 nmoi-L"1, 100 nmol-L'' and 1 000 nmol'L'1) were supplemented, then the mRNA of reporter gene EGFP or endogenous myostatin gene was detected by RT-PCR. [Result] The 5' regulatory region of myostatin gene in sheep was obtained, and two sites that is possibly contributive to double-muscular character were predicted. Many putative regulatory motifs and transcriptional factors, which may play important roles in the transcriptional regulation of myostatin gene, were found along the regulatory region of sheep, bovine and porcine, such as E-box, MEF2, MTATA, MTBF, HOMF, PRE, ARE and GRE. Most of motifs were found in three animals, but the bases, positions and numbers of the motifs were different among the three animals. The expression vectorsrnpMSTN5'Regu-EGFP of sheep were successfully constructed and transfected. 100 nM progesterone obviously inhibited the mRNA of reporter gene EGFP and endogenous myostatin gene. [Conclusion] The transcription of myostatin gene in sheep was regulated by various transcriptional factors and corresponding regulatory motifs, and the appropriate dose of progesterone inhibited the transcription of myostatin gene by downregulating the activity of the myostatin gene 5' regulatory region.