Sequence And Function Analysis Of The Porcine Cofilin2Gene Promoter

ObjectivesIn order to study the machinery of transcription of CFL2, Confirm thepromoter of CFL2and Learn more about its role in regulating gene expressionafter cloned, sequence analysis and the preliminary research on the regulationand control function. The relationship of CFL2gene and muscle developmentmay deserve further investigations.MethodsWe used the method of genome walking to clone the promoter of CFL2.Confirm the promoter of CFL2after product purification, ligation, transformationand sequence analysis. The sequence was analyzed by bioinformatics softwareto find the core active area and putative binding sites of main transcriptionfactors of porcine CFL2gene promoter.Twelve DNA fragments of the5’ flank region of the porcine CFL2gene werefurther isolated from porcine genomic DNA via PCR and inserted into theluciferase reporter vector to make12CFL2reporter constructs. All reportervectors were transfected into C2C12, NIH3T3, or Hela cells and their relativeluciferase activity measured respectively to confirm the core active area of theporcine CFL2gene promoter.ResultsThe2.5kb sequence of5’-flanking region from the major transcriptionalstart site was cloned by genome walking. Bioinformatics software analysisrevealed that the promoter region of the porcine CFL2gene features the typicalstructure of eukaryotic promoters: two TATA-boxes, two GC-boxes, oneCAAT-box, four transcription initiation sites and one start codon. Theseelements were all located upstream of the start codon within a range of-1.5kb. We then inserted the promoter fragments into luciferase reporter vectorspGL4to generate reporter constructs which were subsequently transfected intoC2C12, NIH3T3, or Hela cells and their relative luciferase activity measured.Our data show that pGL4.10-1554(-1502bp to+51bp) was significantlyhigher than other promoter fragments (P<0.05).In contrast, the relative activityof pGL4.10-1680was significantly decreased, suggesting that the region from-1628bp to-1502bp might contain an inhibitory cis-acting element. Of the threecell lines studied, C2C12cells had the highest level of transcriptional activityand NIH3T3cells had the lowest, suggesting that cells vary in terms of CFL2expression.ConclusionsThe2.5kb sequence of5’-flanking region from the major transcriptionalstart site and the sequence was analyzed by bioinformatics software.We foundthat the porcine CFL2gene5’-flanking region sequence encompasses atypical type II eukaryotic promoter with a TATA-box and four possibletranscription start sites as well as several CAAT-box and GC-box elements thatcontrol the frequency of transcription initiation. The identification of CpG islandimplies that DNA methylation might also modulate CFL2expression. We foundno mutation sites in5’-flanking region of-1580-0in Landrace pig, Meishan pig,Hebao pig and Duroc pigsThe promoter’s active region was mapped to a region from-1502bp to-1317bp.