Cloning Of Musclin Gene5’ Regulatory Region In Sheep And Transcription Regulation Of Musclin Gene

Musclin is a bioactive factor, which was identified by signal sequence trap of mouse skeletal muscle cDNA by Nishisawa et al. in2004. It plays an important role in nutrition metabolism and development of muscle. The transcription regulatory mechanism of musclin gene has not yet been elucidated till now. Therefore, it has important theoretical and practical significance to clone the5’ regulatory region of musclin gene and then identify some important regulatory elements along the region.The PCR primers were designed according to the sequence from sheep genome (AC1V010109253) published in NCBI and the1.4kb5’regulatory region of musclin gene was amplified for the first time and cloned to T-vector. This cloning vector was named pT-MUSCLINpro (1.4kb). Many putative regulatory elements were first analyzed through bioinformatics, such as TATA box, CAAT box, E-box, MEF2, FOXO, GAGA-box, HMTB and SMAD. Some elements are conservative and some are variable among sheep, bovine, porcine, human and mouse. To better study the mechanism of transcription and expression regulation of musclin, the musclin gene5’ regulatory regions of different length (1.4kb,1.2kb,1.0kb,0.8kb,0.4kb and0.3kb) were amplified from the plasmid pT-MUSCLINpro(1.4kb) and then were successfully subcloned into pEGFP-N1(4.1kb) vector. They were named pMUSCLINpro-EGFP. The pMUSCLINpro-EGFP vectors were transfected into mice C2C12cells. Fluorescence quantitative PCR detection results of EGFP showed that the transcriptional regulation activity of musclin promoters with different lengths were different(1.4kb>0.8kb>1.2kb>1.0kb>0.4kb>0.3kb). The results suggested the importance of some regulatory elements in the regulatory region. In order to identify further the elements, a GA-box and three MEF2-boxes were selected to test by EMS A. EMSA results showed that the four elements play important roles in the function of sheep musclin promoter. The study laid an important foundation for better investigating the transcriptional and expressional regulatory mechanisms of musclin gene in sheep.