Mechanism Research Of Transcription Factor P53 Regulation Pig ATGL Transcription

Pork is the main source of animal protein in the Chinese diet and occupies an important position in the national economy.A long time ago,pork has been the main meat consumed by the Chinese.Adipose tissue is one of the important component of pork.The adipose tissue in pork is divided to two parts.One part is the pig backfat,which is related to the variety,production efficiency,feed reward,etc.The other part is intramuscular fat,which determines the quality of pork.The impact on pork quality is crucial,so studying the regulatory mechanisms of adipose tissue formation is crucial to improving pork quality.Lipolysis in adipose tissue is a key way to affect fat deposition.Lipolysis is regulated by many lipases.The ATGL(fatty acid triglyceride hydrolase)gene is the starting enzyme for lipolysis.It has been reported that knocking out ATGL causes an increase in the intramuscular fat content in skeletal muscle of mice,and does not impaired the mitochondrial function of muscles.Therefore,ATGL gene may be a high-quality gene target for regulation of intramuscular fat deposition in pigs.However,the mechanism of porcine ATGL gene for intramuscular fat deposition and its upstream transcription mechanism has not been thoroughly studied.In this study,the promoter of porcine ATGL(fatty acid triglyceride hydrolase)gene was cloned successfully.Through online software and construction of a series of truncated report vectors,the transcription factor p53 that had an effect on the activity of porcine ATGL promoter was selected.The cellular level verified the effect of p53 on ATGL gene expression.This study obtained the following main results:1.A 3156 bp upstream fragment of the pig ATGL gene was successfully amplified,cloned into p GL3.0-basic vector,and a dual luciferase reporter system was performed to show that the fragment has transcriptional activity.A series of truncated p GL-3.0 promoter vectors were constructed by homologous recombination.Combined with the results of 5'RACE amplification,it was clear that the core promoter was located in the start codon-220 to +40 region of ATGL.The online software JASPAR and Alggen Promo predicted that there was a conserved p53 binding site in this region.After mutating this site,the ATGL promoter transcriptional activity decreased,indicating that p53 might bind the ATGL promoter and promote ATGL transcription.2.Using the p53 agonist,Triptolide to treat adipogenic differentiated pig primary adipocytes for 6 days,the expression of p53 increased,the lipolysis-related marker genes ATGL and HSL significantly increased,and the content of FFA and TG in the culture medium increased significantly Oil Red O staining and Bodipy staining showed that lipid deposition significantly reduced p53 inhibitor co-treatment and could inhibit ATGL transcription.Similar results were obtained with the 3T3-L1 cell line,suggesting that p53 activation promotes lipolysis by promoting ATGL gene transcription.3.Establish obese mouse models fed with high-fat diets.The mice were injected with p53 activator intraperitoneally for 7 weeks and short-term for 24 hours,and saline was used as a control group.The tissue sections of the mice in the long-term treatment group found that the area of adipocytes of subcutaneous fat and gonad fat was significantly reduced.The results of q RT-PCR and WB showed that the expression of lipolytic marker genes ATGL and HSL was significantly increased,indicating that p53 can also be used in living bodies Enhances ATGL transcription at the level.In the short-term treatment group(24h),the expression levels of p53 and ATGL genes in the adipose tissue and liver of the mice increased significantly.4.P53 activator was used to treat primary subcutaneous adipocytes and 3T3-L1 cell lines of in vitro cultured pigs.Chromatin immunoprecipitation was used to verify the binding of p53 to the ATGL promoter.The results of chromatin immunoprecipitation showed that p53 was activated Treatment of the agent significantly increased the ability of p53 to bind to the ATGL promoter.To sum up,this study successfully cloned the promoter of porcine ATGL gene and screened a new conserved ATGL transcriptional activating factor-p53,which enriched the regulation network of lipolysis and provided new information for genetic improvement of pork quality theoretical basis.