【作者】 邓捷；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2012， 硕士

【摘要】 肌肉生长抑制素（Myostatin）基因是生长转化因子β（transforming growth factor-beta，TGF-β）超家族中的一员，是动物肌肉生长发育过程中一个重要的负调控主效基因。Myostatin基因的编码区在不同物种间相对保守，其编码的蛋白前体由三部分组成：N末端信号肽、N端前导肽和C末端成熟肽。但由于其C末端氨基酸序列与TGF-β家族其他成员的同源性较低（最高45%），因而又可归为新的GDF-8（Growth-differentiationfactor8）家族成员之一。1.猪Myostatin基因启动子上游调控区潜在结合位点的研究在本研究中，首先，利用PCR的方法扩增得到猪Myostatin基因上游最长为1969bp的启动子片段。将不同长度的启动子片段连接绿色荧光报告基因载体后，转染C2C12成肌细胞和NIH3T3成纤维细胞，结果仅在成肌细胞中出现绿色荧光现象，说明Myostatin基因启动子为组织特异性启动子。其次，对该启动子区域采用逐步删除的方法获得5个新的长度不等的启动子片段，利用荧光素酶报告基因实验检测其在C2C12成肌细胞中的活性。实验结果显示，在Myostatin基因启动子转录起始位点上游1bp至-137bp，-137bp至-466bp内存在有两个转录正调控区域，而在-852bp至-1969bp内存在有一个转录负调控区域。再次，对于转录活性上调效果明显的-137bp至-466bp区域，我们进一步细分后最终确定转录正调控作用的顺式作用元件位点位于-137bp至-218bp区域内。最后，根据已有文献报道及序列特征，设计并合成4条生物素标记的双链DNA探针，通过电泳凝胶迁移实验（Electrophoretic Mobility ShiftAssay，EMSA）验证，-206bp至-189bp区域能够与转录因子蛋白结合，并通过特异位点冷探针竞争实验、-206bp至-189bp区域碱基突变实验、-206bp至-189bp区域特异缺失载体的荧光素酶活性检测实验，最终确定该区域为转录因子蛋白的特异结合位点。2.转录因子结合蛋白的分析鉴定首先，根据生物软件预测和文献报道的转录因子结合位点分析，推测该区域为转录因子蛋白MEF2家族成员结合位点。其次，构建真核表达质粒载体pC-MEF2A、pC-MEF2C，以C2C12成肌细胞和诱导的肌管细胞为研究对象，转入含有MEF2结合位点的启动子片段和上述真核表达载体，通过荧光素酶活性检测实验和浓度梯度实验，发现高表达转录因子MEF2C后能够显著增强Myostatin基因启动子活性2~6倍，而高表达另一亚型MEF2A，启动子活性无明显改变，初步证明结合的转录因子蛋白为MEF2C。再次，利用EMSASupershift实验，确定与Myostatin基因启动子上游结合的正调控转录因子蛋白为MEF2C。最后，设计并合成內源MEF2C干扰片段，以C2C12成肌细胞和诱导的肌管细胞为研究对象，分别转入MEF2C真核表达载体及其干扰片段，用实时定量PCR和Western blotting方法检测Myostatin基因转录及蛋白水平的变化，结果发现MEF2C过表达时，Myostatin基因的mRNA转录水平升高了2~4倍，蛋白表达水平也明显升高；当內源MEF2C被干扰沉默后，Myostatin基因的mRNA转录水平和蛋白表达水平明显降低。这些结果显示，MEF2C可以通过激活Myostatin基因，从而在猪肌肉生长发育过程发挥关键性的调节作用。本研究为Myostatin基因的转录调控提供了确切有效的顺式元件作用位点和与之结合的转录因子蛋白，为更加深入的探讨Myostatin的功能和调控网络提供了一种新的研究方向。更多还原

【Abstract】 Myostatin gene is a member of transforming growth factor β (transforming growthfactor-beta, TGF-β) superfamily, an important negative regulator of the animal muscle growthand development process. Myostatin gene coding region conserved among different species,and its encoded protein precursor consists of three components: N terminal signal peptide,N-terminal propeptide and biologically active carboxyl terminus. Because of its low aminoacid homology of C-terminus to the other TGF-β family members (up to45%), it is alsoclassified as a member of new class of GDF-8(Growth-differentiation factor8) family.1. The research of porcine Myostatin promoter upstream regulatory region of potentialbinding sitesIn this study, first we cloned and amplified up to1969kb upstream of the porcineMyostatin gene promoter region. The different length fragments of promoter were connectedto the report vectors, then transfected into C2C12myoblasts and NIH3T3fibroblasts, whichrevealed that the promoter activity was specific found in myoblasts, indicating that theMyostatin gene promoter is tissue-specific promoter. Then, using a serial deletion strategy, wegot five new length fragments of the promoter, according to the luciferase assay, the resultsshowed that the upstream of the transcription start site there exist two positive transcriptionalregulatory regions among1bp to-137bp,-137bp to-466bp, and exist a negativetranscriptional regulatory region within the-852bp to-1969bp. Then, for obvious effect ofincreased transcriptional activity of-137bp to-466bp, we conducted more report vectors andfinally determined the possible positive regulation region of cis-acting elements located siteswithin the-137bp to-218bp. Finally, according to the existing research papers and sequencecharacteristics, we designed and synthesised four biotin-labeled double-stranded DNA probesto do the Electrophoretic Mobility Shift Assay (EMSA), and verified-206bp to-189bp regioncould be the specific sites of transcription factor protein binding to. Then by using themethods of site-specific cold probe competition,-206bp to-189bp region mutation,-206bpto-189bp region-specific deletion vectors of luciferase assay in the detection experiments,and ultimately we determined the region for the transcription factor protein the specificbinding site was in-206bp to-189bp. 2. Identification of transcription factor-binding proteinsFirst, according to biological software and reported research papers, we predicted thetranscription factor binding sites, suggesting that trans-acting factor might be the member ofMEF2protein family. Then, Eukaryotic expression vector pC-MEF2A, pC-MEF2C wereconstructed, by luciferase assay and the concentration gradient experiments, overexpressionMEF2C, but not MEF2A increased Myostatin promoter activity with MEF2motifs by two tosix folds, in both myoblasts and myotubes. The initially evidence proved that the MEF2Ctranscription factor protein should be the one combined to the binding sites. Then, by usingEMSA supershift methods, the identification was proved that MEF2C was the specifictranscription factor binding to the upstream regulatory region of Myostatin promoter.Ultimately, designed and synthesised the fragments of endogenous MEF2C interference, wecarried out real-time quantitative PCR reaction and Western blotting to show thatoverexpression of MEF2C, the mRNA levels of Myostatin gene were significantly increasedtwo to four folds and protein expression levels were meanwhile increased; while silence ofendogenous MEF2C, the mRNA levels and protein expression levels of the Myostatin genewere significantly decreased. These results proved that the transcription factor MEF2C couldmodulate and restrain myogenesis by Myostatin activation and Myostatin-dependent genedevelopment processing in porcine. Our research could provide a potential target site and aneffective transcription factor to regulate Myostatin expression and gave a new researchdirection to explore the functional performance of Myostatin.更多还原