Study On The Effect Of PPARα In Promoting A-FABP Expression And Intramuscular Fat Deposition

The content of intramuscular fat(IMF)is closely related to the tenderness,flavor and juiciness of pork quality.In-depth understanding of the mechanism of regulating intramuscular fat deposition is of great significance to solving problems such as the development of the meat product market.Studies have found that adding conjugated linoleic acid(CLA)to the diet can reduce the subcutaneous fat content and increase the intramuscular fat of Landrace and Large White hybrid pigs.The expression of A-FABP and PPARα genes increased significantly,and different animal experiments showed that the expression of A-FABP was significantly correlated with the content of IMF,suggesting that CLA may affect the expression of A-FABP by stimulating the activity of PPARa.And then regulate the deposition of IMF.This study uses molecular biology,cell biology and other methods to verify at the cellular level that CLA has an activating effect on porcine A-FABP and PPARa promoters.Overexpression of PPARα can significantly up-regulate the A-FABP promoter activity.In this paper,mice were fed with CLA in the diet for three weeks to study the effect’of CLA on the body at the animal level,as well as on the intramuscular fat of the quadriceps muscle,A-FABP,PPARα,fat metabolism and key enzyme synthesis And other expression levels.Finally,in order to further verify the activation effect of PPARα on the A-FABP promoter,siRNA interference technology was used to silence the expression of endogenous.PPARα in cells,and whether there were statistical differences in the effect on the activity of A-FABP promoter was analyzed.The main research results and conclusions of this paper are as follows:1.Recombinant plasmid construction.First,design and synthesize oligonucleotide primers based on the NCBI sequence and related literature.Use the cDNA and genome of pig liver tissue as templates to obtain the target fragments by PCR amplification,connect the expression vector by homologous recombination,and select the recombination PCR,restriction enzyme digestion and sequencing showed that the plasmid construction was completed;plasmid pCMV5-myc-PPAR was transfected into 293T cells,and the expression of PPARα protein was detected;pGL3-Basic,pGL3-PPARα,pGL3-afabp was transfected After 48 hours,the cells were tested with dual luciferase reporter gene system and showed that they had promoter activity.2.The activating effect of CLA on PPARa and A-FABP promoter.In order to verify that CLA can promote the expression of PPRAα and A-FABP genes,the plasmids pGL3-PPARa and pGL3-afabp were respectively transfected in the 293T cell line,and different concentrations(10,25,50,100 M)CLA stimulated cells,and it was found that the final concentration of.100μM CLA can significantly enhance the activity of PPARα promoter and A-FABP promoter(P<0.05 or 0.01).3.Overexpression of PPARα enhances the activity of A-FABP promoter.To verify the promoting effect of PPARα on the expression of A-FABP gene,the plasmids Pcmv5-myc-PPARαand pGL3-afabp were co-transfected in 293T,3T3-L1,and PK15 cells,respectively.The control group was pGL3-Basic and pGL3-afabp.After 48 hours,the promoter activity was tested for statistical difference analysis.Western blot was used to detect the expression of PPARa protein.The results showed that overexpression of PPRAα protein can significantly up-regulate the A-FABP promoter activity(P<0.05).4.The effect of CLA on animal IMF content,A-FABP,PPARα and the expression level of key enzymes in fatty acid metabolism and synthesis.Forty 6-8 week old Kunming male mice were randomly divided into 2 groups(n=20).The experimental group was fed 1.5%CLA-added feed,and ordinary diet without CLA was used as the control group.Weighed for 3 days and sacrificed the mice after 15 days of feeding.Frozen sections of liver and quadriceps femoris tissue were taken for oil red O staining.The content of quadriceps femoris IMF was determined by Soxhlet extraction method,and A-FABP,PPAR(α,γ)expression levels,fluorescent quantitative PCR to detect the expression levels of key enzymes in fatty acid metabolism and synthesis.The results showed that after 15 days in the CLA-fed experimental group,the fat content of muscle and liver increased significantly,forming a "fatty liver" phenomenon;the expression levels of A-FABP and PPAR(α,γ)increased significantly(P<0.05),and fatty acid The expression levels of key metabolic enzymes CPT1,ACOX1,LCAD and CD36 mRNA decreased significantly(P<0.05),and the expression levels of key fatty acid synthesis enzyme genes FASN and ACC increased significantly(P<0.05).5.The effect of siRNA silencing PPRAα gene on the activity of A-FABP promoter.First,culture pig kidney passage cells(PK15)to analyze the optimal transfection concentration and time of siRNA by fluorescence quantitative PCR,and then divide the cells into 3 groups:blank control group(no siRNA interference),siRNA negative control group(Non-specific siRNA,siRNA-Con)and siRNA experimental groups(specific siRNA,PPARa siRNA-1、2),each group was transfected with plasmids pGL3-afabp and Renilla,36 hours after each group of cells were transfected with dual fluorescein The enzyme reporter gene detection system analyzes the A-FABP promoter activity of each group:The results showed that the expression of PPRAa gene in silent cells could significantly reduce the activity of A-FABP promoter(P<0.01).Conclusion:In the process of CLA promoting IMF deposition,C LA promotes the expression of PPARa gene by enhancing the activity of PPARα promoter.Overexpression of PPARα can enhance the activity of A-FABP promoter and thus increase the expression level of A-FABP gene.In animal experiments,CLA up-regulates the content of IMF in muscles and has a significant impact on the factors that regulate IMF,which supports the conclusions of cell experiments.This study explored the molecular mechanism of CLA regulating porcine IMF deposition from the perspective of PPARα signaling pathway regulation,and provided new ideas for improving meat quality.