The Polymorphism Of Porcine ATF4 Gene And Research Of Promoter Region

Fat is not only essential nutrients to human and animals but also an important component of living organisms. Regulation of its metabolism is affected by many factors, including genetic, nutrition, and environmental factors, which form a complex regulatory network in the regulation of fat metabolism. Pigs as an important source of animal protein whose over fat deposition directly impact on the improvement of lean meat, Therefore, research on genes related with fat deposition become hotspot for experts in animal genetics and breeding.Activating transcription factor 4 (ATF4), belongs to the family of basic zipper-containing proteins. Reccently study identifies a possible new function for ATF4 in regulating lipid metabolism and thermogenesis.Given its important role in fat metabolism and the study in porcine reported less, we select ATF4 as a candidate gene for fat deposition study the molecular mechanism of the differences between lean and fat-type pigs in order to provide more information on this gene for molecular marker-assisted selection breeding.Based on these we clone ATF4 and studied its genetic effects.And the main results are as follows:1 Sequence comparison revealed that an mutation occurred A159G substitution at downstream of initiation codon (ATG).2. We carried out PCR-AluI-RFLP analysis followed by association analysis in F2 "Large white×Meishan" resource family. In all the individuals tested, Large White and Landrace pigs possessed AA genotype, while Meishan and Tongcheng pigs possessed GG genotype. Association analysis in F2 resource family showed that this site was highly associated with buttock fat thickness(BFT) (P<0.01) and had significant effect on thorax-waist fat thickness(TFT), average backfat thickness(ABT), loin eye height(LEH) and loin eye area (LEA)(P<0.05).3.RT-PCR was applied to detect the tissue distribution of the porcine ATF4 gene in different tissues (heart, liver, spleen, lung, kidney, stomach and so on). Result show that the ATF4 gene was the highest expressed in muscle.4. Real-time RT-PCR analysis showed that the expression level of ATF4 gene in the skeletal muscle of Large White and Meishan pigs were no significant difference before postnatal 3 days. And at postnatal 60 and 120 days, the expression level was dominantly higher in Meishan pigs than in Large White pigs.5. Cloning 2007bp in ATF4 promoter region by PCR and using the online bioinformatics software, we predict core promoter region and find cis-acting elements and transcription binding sites, we use this predicted result to desgin primer and. construct 6 repoorter gene with different length of promoters.6. The recombinant constructs were transfected into PK-15 and C2C12 cell. The promoter activity was evaluated by dual-luciferase reporter assay. The 7 promoters all were transcriptional activated and pGL3-1280 had the high activity in C2C12 cell. All the result laid the experimental foundation for further study on the transcriptional regulation of ATF4.7. Sequence comparison revealed that there are 11 sites of potential SNP and insertion/ deletion.we find an 20bp insertion/deletion mutation occurred at 1984 bp of the of initiation codon (ATG). We then carried out PCR analysis.In all the individuals tested, Large white pigs pigs possessed the insertion, while Meishan possessed the deletion.8. Sequence comparison revealed that a mutation occurred C/T substitution at 2570bp downstream of initiation codon (ATG). We carried out BstXI-RFLP analysis.In all the individuals tested, all Meishan pigs possessed the TT type, Large white pigs possessed CC type.9. We construct an eukaryotic expression vector pcDNA3.1 (+)-ATF4 by cloning porcine ATF4 gene,which provide us advantage for further study of gene function at the cellular and individual level.