The Research Of Akirin1 In Bovine Myoblasts Differentiation

The myofiber of vertebrate was formed in the embryonic period and the number of myofiber was determined in this phase.The growth of adult muscle was depended on the proliferation of muscle satellite cells (hypertrophy) but not the increasing number of myoblasts (hyperplasy).It is necessary to elucidate the mechanism of myogenesis for its importance on embryo development, cell proliferation and differentiation.The myogenesis of vertebrate was controlled by classical transfactors such as MRFs,MEF2s,MSTN and other genes.In this article we investigate the effects of akirin1 in the differentiation of bovine myoblasts in order to provide theoretical basis for the enhancement of meat quality and productivity .This research could also benefit for the clinical treatment of muscular dystrophy and muscle regeneration and provide a theoretical foundation for the transgenic cattle.In this study,we cloned the CDS region of bovine akirin1 and constructed akirin1 eukaryotic expression vector which was named pIRES2-EGFP-akirin1 and transfected it into bovine myoblasts for the transient expression of Akirin1.Then detected the relative expression of akirin1 and the labeling genes of muscle differentiation through the real-time PCR and western blot after transfection. Amino acid of akirin1 was analyzed with bio-informatics methods so that the conserved structure and function domain was predicted, so was the transfactor binding site of the promoter of myogenin. Finally we monitored the activity variance of myogenin gene promoter after co-transfected with pIRES2-EGFP-akirin1 through the Dual-luciferase reporter gene detection system.In this article, the akirin1 gene of bovine was exactly cloned and the eukaryotic expression vectors of akirin1 pIRES2-EGFP-akirin1 constructed.The green fluorescence observed in myoblasts which transfected with the plasmid proved that akirin1 can highly express in bovine myoblasts .The mRNA levels of akirin1 and muscle differentiation related genes in the myoblasts after transfected with the plasmid was detected by real-time PCR. The results showed that these genes respectively increased in all detected timepoints. Western blot results showed that, comparing with the control groups, the protein expression levels of myogenin were all increased at the observing timepoints during differentiation. Dual-luciferase assays results indicated the significant increase in myogenin promoter activity when the promoter was co-transtected with pIRES2-EGFP-akirin1,which demonstrate that the potential mechanism of the promotion of myogenic activity by akirin1 is enhance the promote activity of myogenin promoter.Conclusion: Over-expression of akirin1 was able to improve the expression of myoblasts differentiation related genes and the possible mechanism of this enhancement to myogenic differentiation was akirin1 could promote the activity of myogenin promoter.