Cloning And Transcriptional Regulation Analysis Of The Promoter Region Of Porcine O3FAR1Gene

Swine is one of the main livestock,and it’s similar to human in genetic, physiological, and biochemical aspects.For these reasons, pigs become one of the most ideal animal model for some diseases, for instance diabetes, obesity, dyslipidemia and so on. In recent years, the physiological function of free fatty acid has attracted wide attention, because it not only can provide energy for the organization but also can be used as a signaling molecule to mediate many cellular processes. Studies have found that, diabetes, obesity, lipid metabolic disorder are in close contact with elevated peripheral levels of free fatty acids.03FAR1is a new discovery of long chain fatty acid receptor, and it is taking part in the ipogenesis, cell proliferation, hormone secretion and metabolism process in vivo.03FAR1is considered to be related to certain diseases, it can be used as a drug target of some diseases, such as obesity, diabetes mellitus. In the mechanism of gene expression regulation, gene transcription is a very important process, it plays a link role in the genetic information transfer. The frequency and transcription initiation site of the transcription is regulated by the key regulatory element that is promoter. Promoters mainly by interacting with transcription factors to regulate the expression of gene. Identification of promoter, plays an important role in the exposition of gene function. In order to understand the function and the mechanism of transcriptional regulation of porcine O3FAR1gene furtherly, we cloned the promoter region of porcine03FAR1gene, and to study its core area, the results are as follows:(1) In order to draw the tissue expression profile of O3FAR1gene, the expression of03FAR1gene in different tissues of pigs were detected by fluorescence quantitative PCR technique. From the results, the expression of porcine O3FAR1gene is widely expressed in different tissues, but not the same in content.03FAR1gene is highly expressed in the small intestine, while the expression level in adipose tissue is significantly higher than in other organizations.(2)The5’ upstream regulatory sequence of O3FAR1gene was obtained from NCBI, used as a template, a2003bp upstream regulatory sequence was cloned. Using online tools such as PromoterScan, Promoter2, TFsearch to analyze and predict the5’upstream regulatory sequence of the gene, has found many transcription factor binding sites, such as Sp1, C/EBP, GATA-1etc.. At the same time, also found two CpG islands. (3) By comparing the O3FAR1gene in different species can be found that the O3FAR1gene is a conserved gene, and it’s high conservation among some animals, for example rat, human, dog, pig. The similarity of pig and rat, human, dog is respectively86.98%,82.23%,89.47%.(4) By using the PGL3-BASIC dual luciferase report gene vector to construct seven deletion fragments, and then are transfected into three cells to detect the difference fluorescence activity. The results showed that fragment PGL3-P7is with higher activity, speculated that the scope of the core promoter region may be located within the-298bp to+78bp.(5) By the analysis of PGL3-P7sequence structure, binding site of transcription factor C/EBP β is found. In the point mutation experiment wild type and mutant type plasmid are constructed,and the fluorescence activity of mutation plasmid is significantly reduced.(6) The overexpression experiment of transcription factor C/EBPβ by RT-PCR is used to detect the mRNA level of03FAR1gene,and the mRNA level increased significantly (P<0.01); using the technology of siRNA to interfere the endogenous C/EBPβ in cells, and the mRNA level of03FAR1gene was found significantly decreased (P＜0.05).(7)Some probes are designed according to the predicted transcription factor binding sequences, and the transcription factor C/EBPβ is bound with O3FAR1gene promoter is confirmed on protein level by using the EMSA technology.(8) The pcDNA3.1-CDS eukaryotic expression vector of porcine O3FAR1gene was constructed by eukaryotic expression vector pcDNA3.1and obtained03FAR1gene CDS region. Overexpression experiment is accomplished after the transient transfection, then RT-PCR analysis showed that O3FAR1gene mRNA levels increased significantly (Pβ0.01).