猪骨骼肌发育相关新转录因子MASTR的靶基因及其调控通路研究

It is important to understanding of molecular regulation mechanism of skeletal muscle and accelerated growth and development of the pig quality, cultivation of high-yield varieties (lines) by identified and studied its regulation mechanism and function of the new gene in skeletal muscle development. The project intends to catch the target sequence of transcription factor MASTR using the ChIP-seq technical basis on the previous study results in skeletal muscle of porcine. And then verified of the select terget gene using Luciferase reporter system and EMSA, and also detected the expression levels of target gene by over-expression and inhibition of expression MASTR in cell level, finally excavate the important gene and its signaling pathway in the development of skeletal muscle. And then speculated the role in skeletal muscle growth and development process of MASTR by combined the SNPs scan and trait association analysis results of target gene. The results establish the foundation of reveal the function and regulation mechanism, and further complete the regulation network of skeletal muscle and the more in-depth study function of target gene. At the same time, provid the new genetic material to the molecular breeding of pigs. Therefore, the project have important academic value and application prospects.

寻找鉴定与猪骨骼肌的生长发育相关的新基因，并对其调控机制和功能进行研究，对了解猪骨骼肌生长发育的分子调控机制，加速猪优质、高产新品种（系）的培育具有重要意义。本项目拟以前期对骨骼肌发育中新转录因子MASTR的研究为基础，利用染色质免疫共沉淀-测序（ChIP-seq）技术钓取猪转录因子MASTR的靶序列，通过荧光素酶报告系统和EMSA等方法对钓取的靶基因进行生物学验证，并在细胞水平上对猪MASTR基因进行超表达、抑制表达进而检测靶基因的表达量的变化，挖掘影响猪骨骼肌生长发育靶基因及其重要信号通路，同时结合靶基因SNPs扫描和性状关联分析结果，综合推测MASTR基因在骨骼肌生长发育过程中的作用。研究结果为揭示猪MASTR基因的功能及其调控机制、进一步完善猪骨骼肌生长发育的调控网络、靶基因更深入的功能研究奠定基础，同时为猪分子育种提供新的遗传素材，有重要的学术价值与应用前景。

本项目通过建立猪骨骼肌细胞培养系统，构建重组腺病毒载体系统和ChIP-seq技术，对MASTR转录因子的调控机制进行探索。结果显示：共定位到90456个峰(P＜10－5 )，GO和KEGG分析发现其参与包括线粒体基质，MHC蛋白复合物，丙酮酸代谢过程和蛋白激酶调节活性等过程；对靶基因结合位点进行分析，结果显示13个基因的结合位点位于启动子区域。针对13个目标靶基因，通过Q-PCR、双荧光素酶报告系统和EMSA试验进行验证，结果显示PRKAG1，IL18R1和SLC28基因为MASTR转录因子的潜在下游靶基因；同时构建大白猪肉质性状群体，对候选靶基因进行群体遗传学水平检测，共获得8个编码区SNP位点，结果未发现与肉质性状呈显著关联性，后续将进一步进行验证和分析。本研究通过CHIP-seq技术筛选了猪MASTR转录因子的靶基因，初步揭示了该转录因子的调控机制，为进一步完善猪骨骼肌生长发育分子调控机制奠定了一定基础，同时为猪分子育种提供有用素材。