Swine DECR1and GPR81Genes Research Of Transcriptional Regulation

As one of the important economic traits in the swine breeding work, pork quality has been the key point focused by breeders. It is known that, fatness trait is an essential factor which affects the pork quality. Making a depth study will help us to find a way to improve the pork quality. For this purpose, the mechanism of transcriptional regulation for two genes affecting fat traits is studied in this experiment, to uncover the veil of theory for transcriptional regulation and lay the foundation for further research. This experiment makes a study of the mechanism of transcriptional regulation for DECR1and GPR81genes mainly from the following several aspects:(1) Making use of QT-PCR to conduct quantitative analysis of different tissues expression level for GPR81gene in large white pig. The results show that GPR81gene was high expressed mainly in fat, liver and kidney tissue. thereinto the expression of GPR81gene was highest in adipose tissue, whereas trace or almost no express in other tissues.(2) By means of NCBI and EBI database, we query and get the5’upstream transcription regulatory regions about2000bp of pig DECR1and GPR81genes, and conduct a PCR clone, then we obtain the2099bp and1798bp sequence respectively. Making a predictive parsing for the5’upstream transcription regulatory regions of the two genes using bioinformatics technology, we find that the5’upstream transcription regulatory regions of DECR1gene exists various of common transcription factor binding sites of the AP1, SP1, MZF1and so on. The CpG islands distribution analysis shows that there is no CpG islands. The5’upstream transcription regulatory regions of GPR81gene have some high score of transcription factor binding sites like the GATA-1, GATA-2, GATA-x, SRY, AML-la and so forth. There is no find the CpG islands in the5’upstream transcription regulatory regions of GPR81gene as well.(3) Designing a series of deletion fragments of DECR1and GPR81gene5’ upstream sequences and constructing the dual luciferase gene reporter vector with deletion fragments and conducting dual luciferase activity detection in C2C12,3T3-L1, PK15cells. Furthermore, constructing the vector with deletion fragments for the DP7of DECR1gene and GP7of GPR81gene separately, and detecting the double fluorescence activity.(4) Analysing the result of double fluorescence activity, we process the sequences between two significant difference for the dual luciferase activity of deletion fragments by bioinformatic prediction and find out the transcriptional regulation factors that play an important role in transcriptional activity of promoters, we discover that the transcriptional regulation factors of SP1, MZF1and GATA1, GATA2, GATAx have significant regulating effects for DECR1and GPR81gene promoters separately.(5) In order to identify the transcription factors binding site and transcription factors in5’upstream transcription regulatory regions of DECR1and GPR81genes, we perform the site-directed mutagenesis for each transcriptional factor binding sites of this two genes, and conduct the transcription factor SP1and GATA1of DECR1and GPR81genes with the Over-expression experiment and in addition, the siRNA experiment was performed for the transcription factor SP1. The results display that after mutation the transcriptional activity of DECR1and GPR81genes5’upstream transcription regulatory regions decreased significantly. The over-expression of transcription factor SP1and GATA1of DECR1and GPR81genes, significantly enhanced the transcriptional activity of two genes5’upstream transcription regulatory regions. The result shows that siRNA experiment decreased the transcriptional activity of DECR1gene5’upstream transcription regulatory regions significantly.