Studies On Flounder Muscle Regulatory Factors: Myogenin And MRF4

The present study focused on the cloning and expression of myogenin and MRF4 and the analysis of their promoter and function during flounder embryogenesis. The whole genomic sequence of flounder myogenin and MRF4 both contain 3 exons and 2 introns, and the identity of their deduced amino acid sequences were higher with those of several fish species.Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites when 5-6 somites were formed, and expanded later to the lateral region of the somite and expressed in the whole somite. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region, but remained strong within the tail region.The result of RT-PCR demonstrated that Myf-5 and MyoD was expressed earlier than myogenin, while the expression of MRF4 was later than that of myogenin. And myogenin and MRF4 were detected only in skeletal muscle by RT-PCR, which further indicated that they play roles in regulating muscle development.Transient expression analysis showed that the promoters of flounder myogenin and MRF4 were sufficient to direct muscle-specific GFP expression in zebrafish embryos, which indicated that there were conserved regulatory sites in the promoter regions and these elements could act across species. Further study on the promoter MRF4 showed that there must be necessary sites for regulating the expression of MRF4 in the promoter between -181 and -506.Flounder MRF4 recombinant protein was expressed and purified, its polyclonal antibodies was prepared and analyzed. The endogenous MRF4 was detected by the polyclonal antibody. Thus, further MRF4 function studies on protein level can be performed.