Analysis Of Transcriptional Regulation Of Porcine APOA2and SEPW1Genes

Pig is the main source of animal protein that we get. Pork quality affects the taste directly. With the improvement of living standards, people have become increasingly demands of pork quality. At present, improving meat quality traits of pigs by breeding is a hot research topic. To this end, we studied the genes "APOA2" and "SEPW1" relating to pig fat and muscle respectively at the molecular level, and focused mostly on transcription regulation analysis of gene promoter regions. The main results were as follows:(1) The study on APOA2and SEPW1expression in different tissues of pig. In this study, quantitative real-time PCR was used to analyze the mRNA expression, established tissue expressional profiles. The results revealed that APOA2is expressed predominantly in liver, in smaller amounts in fat tissue, and in negligible amounts in other tissues. Porcine SEPW1was expressed with different degree in various tissues, it has the highest expression level in heart and gastrocnemius, and it showed the lowest level in the spleen.(2) According to the5’flanking sequences of porcine APOA2and SEPW1gene from NCBI, we designed the primers. With genomic DNA of pig as a template, we cloned porcine APOA2gene1919bp5’flanking sequences and SEPW11993bp5’flanking sequences. Then compared the sequences with NCBI gene bank, they are about99percent the consensus.(3) The study on the methylation statuses of porcine SEPW1promoter CpG islands. We predicted no CpG islands of APOA2promorer and three CpG islands of SEPW1promoter. The CpG islands that located in-751～-483of porcine SEPW1promoter was cloned and sequenced, the result showed that the methylation lever was low.(4) The study of promoter activity. We constructed seven different lengths deletion fragments with dual-luciferase reporter gene of porcine APOA2promoter, and six different lengths deletion fragments of SEPW1promoter. Then, these plasmids were transfected into three kinds of cells, and detected the fluorescence activity. We found that-208～-21and-813～-209of APOA2promoter could transcribe independently, and SEPW1core promoter be likely to-443～+27. (5) Analysis and prediction of transcription factor binding sites. Again, we constructed vector of different lengths deletion fragments with dual-luciferase reporter gene in the region where there may be regulatory elements, and combined with bioinformatics method, we found GATA1binding site of APOA2in-601～-565, SP1binding site of SEPW1in-378～-306.(6) Using the site-directed mutagenesis method to detect transcription factor binding sites. Three bases of GATA1binding site and one base of SP1binding site were changed, then the mutation plasmid was transfected into C2C12and PK cells respectively, and the results showed that the fluorescence activity decreased very significantly.(7) The eukaryotic over-expression vector of transcription factor and deletion fragments vector were transfected into PK cells, and found the fluor activity of promoters increased significantly after the over-expression of transcription factors.(8) First, the small RNA interference fragments of porcine SP1were transfected into PK cells, and detected that the expression of SP1were decreased significantly. Then, the interference fragments and deletion fragments vector were transfected into PK cells, and found the fluorescence activity was decreased significantly.