Study On Interaction Mechanism Of MEF2 Family Transcription Factors And MSTN Promoter In Cattle

Muscle tissue is the most important component of livestock and poultry.Byexploring the regulation mechanism of muscle growth and development,which is great significance for improving the muscle production and the growth traitsof livestock and poultry.Myostatin(MSTN)was regarded as GDF8(Growth differentiation factor 8,GDF8)and it was a negatively regulator of skeletal muscle growth and differentiation.The results of muscle atrophy by over expression of the MSTN in animal,but it could be lead to muscle hypertrophy and appear "double muscle" phenomenon by inhibition or knockout of the MSTN in animal.Myocyte enhancer factor 2(MEF2)belongs to the MADS supergene family and it is mainly composed of four members(MEF2A,MEF2 B,MEF2C,MEF2D).The MEF2 family of transcription factors could specifically recognize and bind to the promoter which it involved in muscle regulation,thereby regulated the growth and development of animal muscle.This paper attempts to elucidate the regulatory mechanism of MSTN gene expression by studying the interaction between MSTN promoter and transcription factor.In this study,the local yellow cattles were used as the research object,and the expression of MSTN in seven tissues(heart,liver,spleen,lung,kidney,adipose and longissimus dorsi)were detected by qRT-PCR of four local cattles in Guizhou.The successfully constructed pGL3-Basic-MSTN and pcDNA3.1(+)-MEF2A(MEF2B,MEF2 C,MEF2D)eukaryotic expression vectors were co-transfected into mouse C2C12 cells.The dual luciferase activity were measured after 24 hours.By rebuilding the recombinant expression vector pEGFP-N3-MSTN-P1-MEF2 C which replaced(CMV)area with MSTN-P1 core fragment,and transiently transfected into mouse C2C12 cells and rat mandibular gland epithelial cells,the expression of green fluorescent of cells was observed after 12~24 hours,and it was preliminarily verified whether the MSTN-P1 promoter core fragment could initiate the expression ofMEF2 C in cells.The expression of MEF2 C in mouse C2C12 cells and rat mandibular gland epithelial cells were detected by qRT-PCR after 48 hours,and to further verify the regulation between MSTN promoter and MEF2 C at transcriptional level.The research results were as follows:(1)The MSTN gene was expressed in 7 tissues of 4 local yellow cattle in guizhou,with the highest expression in the longissimus dorsi.The expression of MSTN gene in some tissues was different due to different breeds.(2)The MSTN promoter has priming activity.The MEF2 C could significantly enhance MSTN promoter activity,while MEF2 A,MEF2B and MEF2 D had little effect on MSTN promoter activity.(3)The MSTN-P1 promoter fragment had positively regulated the mRNA expression of MEF2 C in mouse C2C12 cells and rat mandibular gland epithelial cells,and it was indicated that the MSTN promoter could initiate the expression of MEF2 C gene in cells.(4)The MEF2 C had positively regulated the transcriptional activity of the MSTN promoter.