Functional Analysis Of Porcine Skeletal α-actin And ME1Gene Promoters

Pigs are closely related to people’s daily lives. The consumption of pork is also very highin the world. The improvement in its production and quality is closely associated with humanliving.Using skeletal muscle-specific promoter to allow exogenous genes expressed in skeletalmuscle by transgenic technology is appropriate for improving pork quality traits. A-actin isthe main ingredient of skeletal myofibrillar in pigs and the promoter of α-actin gene is shownto drive transgenes to be expressed uniquely in skeletal muscle. In this study, PCR, restrictionenzyme digestion and site directed mutagenesis approaches were used to construct differentexpression vectors. The promoter activity was compared by means of cell culture, transienttransfection and activity assay using dual luciferase reporter system. The results arefollowing:1. Two segments of2.0kb-2.3kb and0.06kb-0.37kb were critical in regulating theexpression level of porcine α-actin gene.2. A CArG box in the0.06kb-0.37kb segment affects the transcriptional activity ofporcine α-actin gene. When the segment is deleted or CArG box is mutated, the promoteractivity in driving transcription was significantly reduced (P<0.01) to20%or43%comparedto the3.04kb segment.The result may provide reference for vector construction with efficient expression andskeletal muscle specificity in pork.Fat deposition is closely linked to pork quality. ME1is the key enzyme responsible for thesynthesis of endogenous fatty acid in organism, which is closely related to the efficiency offatty acid synthesis and the capacity of fat deposition in pigs. In order to analyze theregulation mechanism of porcine ME1gene transcription, methods of PCR and site directedmutagenesis were used to clone the5′regulatory region of porcine ME1gene and analyze therelationship between the ME1polymorphism and reporter gene expression level in3T3-L1cell line. The function of the5′regulation region of porcine ME1gene was also examined byconstructing different deletion vectors and cell transfection. The results suggested that1. There was a SNP caused by G to A transition at-1068site of the5′regulatory regionof porcine ME1gene. At the-1068site, allele G has positive effect on mRNA expression inbackfat tissues. 2. By deletion approach, The fragment of614bp-717bp was identified to containactivating elements and263bp-405bp was identified to contain repressing elements in porcineME1gene5′regulatory region.In conclusion, the transcriptional regulation of porcine α-actin and ME1genes wereinvestigated in this study, some important elements regulating their expression levels wererevealed. Moreover, the influence of G to A transition in the regulatory region of porcine ME1gene on transcription was analyzed.