Effects Of Smad3 On The Transcriptional Regulatory Activity Of Myomaker Gene 5' Regulatory Region In Sheep

Myomaker is a muscle-specific transmembrane protein that controls myoblast fusion during muscle development.At present,the study of sheep myomaker gene is rarely reported.In the previous study,we amplified the 5' regulatory region of sheep myomaker gene and analyzed it by bioinformatics.Smad response elements,SMAD3 and GC_SBE,were found in this region.The purpose of this study is to further investigate the effects of Smad3 protein and SMAD motifs on the transcriptional regulation activity of myomaker gene 5' regulatory region in sheep,which can lay a foundation for elucidating the transcriptional regulation mechanism of myomaker.In this experiment,based on the cloned 5' regulation region of sheep myomaker gene,the SMAD-mutant MyomakerProSMADM-EGFP vectors were constructed by mutating SMAD motifs into a specific restriction site and using EGFP as a reporter gene,and transfected into C2C12 myoblasts respectively.The effects of SMAD motifs mutation on the transcriptional regulation activity of the sheep myomaker gene 5' regulatory region were investigated and the effects of Smad3 on the transcriptional regulation of myomaker gene in sheep were further studied.The quantitative detection by qRT-PCR and EMSA were performed.The results showed that(1)SMAD3 and GC SBE motifs influenced the expression of EGFP mRNA,and the effects of SMAD motifs on the transcriptional regulation activity of sheep myomaker gene 5' regulatory region were related to the status of cells.The tissue-level EMSA results showed that there was a specific transcription factor----Smad3 protein,which bound to SMAD3 motifs,in the nuclear protein of fetal sheep muscle tissue(2)The effects of overexpression or silencing of the Smad3 gene on EGFP mRNA expression were related to the period of myoblast growth and differentiation.Cell-level EMSA results showed that the effect of overexpressing Smad3 on the transcriptional regulation of sheep myomaker gene 5' regulatory region was mediated by the SMAD3 motif.In conclusion,the effect of Smad3 on the transcriptional regulation activity of sheep myomaker 5 regulatory region was related to the status of cells,and was mediated at least partially by the combination of Smad3 and SMAD3 motif.