Cloning And Function Analysis Of Porcine Skeletal Muscle-Specific Gene Myoz1/tnnc2Promoter

Promoter is a region which can initiate transcription of downstream gene. Right promoter can promote target gene to express in selected organism, for example, phytase gene expression in enteron and fat-related gene expression in muscle. PPARy gene which is mainly related to intramuscular fat deposition can regulate the fat differentiation and deposition. For that, in order to improve the intramuscular fat content, some muscle-related promoters can increase PPARy expression in muscle. In this study, the core functional regions which can be used to regulate swine PPARy gene expression in muscle had been obtain from the skeletal muscle-specific promoters of myozl gene and tnnc2gene, and the study provide some theoretical basis for the construction of intramuscular fat transgenic mouse model. The results as follows:(1) On the basis of phylogenetic footprinting,the result of MEME analysis showed that myozl promoter functional region is close to5’end of exon1, whereas tnnc2promoter functional region is far from5’end of exon1.(2) According to the result of MEME, myozl promoter and tnnc2promoter were regarded from-1885to+170, and-2131to+182respectively. Online software TFSEARCH analysis showed that myozl promoter has TATA-box element typically, whereas tnnc2promoter has not. In addition, multiple binding sites of transcription factors related to muscle function had been found, such as MyoD and MEF-2. Several promoter fragments had been designed though the analysis of TFSEARCH.(3) Transient transfection of deletion fragments reconstruction showed that myozl muscle-specific core promoter which located between-478to+170had3-fold activity compared with negative control, and tnnc2muscle-specific core promoter which located between-2131and-1083had an8-fold activity. Further more, the activity of two core promoters had been identified by observing green fluorescence.(4) PPARy gene expression in C2C12had examed by Q-PCR, their expression level had been increased by2-and3-fold compared with negative control, and PPARy protein expression had been detected by western blot. The reconstructions of swine PPARy had been linearized. The linearized fragments which prepared for microinjection of transgene mice contained core promoters, the PPARy CDS regions with6xhis tag, and two poly (A) elements from pGL3-basic vector.