Analysis Of Pig Gpihbpl Gene Promoter Fuction

LPL plays an important role in metabolism and transport of lipids. LPL can interact with lipoproteins to anchor them to the vessel wall and facilitate lipoprotein particle uptake, and according to hydrolysis of lipoprotein triglyceride rich lipid can also provide important nutrients for organization. Recent studies have identified glycosylphosphatidylinositol anchored high density lipoprotein-binding protein 1 (GPIHBP1) as the important regulation factor of LPL that serves as a binding platform for lipolysis on the vascular lumen and an endothelial cell transporter transporting LPL from the interstitial spaces to the capillary lumen. But GPIHBP1 gene transcriptional regulation mechanism at home and abroad has not been reported so far. Therefore, the promoter function of GPIHBP1 gene has not been well studied. In this study, we firstly anlysed the gene’s promoter, determined it’s core sequence and the major transcription factors of the GPIHBP1 gene promoter region. Furthermore, we found a single nucleotide polymorphism site in promter region has been significantly correlated with backfat thick. Using the experimental methods in bioinformatics, molecular biology and cytology, we have studied the porcine GPIHBP1 gene promoter function. The results as follows:1. We obtained the 2232 bp DNA sequence of the promoter region of the porcine GPIHBP1 gene. The 5’end deletion fragments of Pig GPIHBP1 gene were cloned and recombined into pGL3-basic plasmids. The recombined vectors were pGL3-95, pGL3-288, pGL3-490, pGL3-1000, pGL3-1299 and pGL3-1966.2. According to analysis the analysis of the different promoter fragments activities with dual luciferase report gene system by transfecting recombined vectors into PK15 cells respectively, we found that-490 -+265 range has the strongest activity. It is likely to be the core promoter is located interval. The sequence -95 up to 265 region has the basic promoter function, which may be related to SOX-9 transcription factor binding element. And in the-95-490 and-1229-1996 intervals there is positive regulation, these STER, ADR1 and c-Ets-1 transcription factor binding elements may contribute to this. The last, from-490 to-1227 range exists negative transfer, which may be related to MyoD, HSF and Barbie Box transcription factor binding elements.3. We sequenced the GPIHBP1 gene promoter region for 56 pigs from six breeds (Tibet, Yanan, Jinhua, Liangshan, Landrace and Duroc).11 mutated sites were found, which are located in the upstream of the transcription start site. And the-903 G>C mutation site can be recognized by Sma I restriction enzyme.4. In order to further analysis of whether these polymorphic loci affect the metabolism of lipids, we carried on analysis the correlation between the GPIHBP1 gene promoter polymorphism loci and the back fat thickness. The results show that, the-703 G>A mutation site was significantly correlated with backfat thick (P< 0.05). Genotype distribution of-703 G>A polymorphic loci has a very significant difference in the six groups (P< 0.01).