Study On DGAT1 And DGAT2 Genes Expression Using RNA Interference In Porcine Hepatocytes

Acyl-coenzyme A:diacylglycerol acyltransferase(DGAT)enzymes,including DGAT1 and DGAT2(encoding by DGAT1 and DGAT2 genes,respectively),are the enzyme that catalyze the final reaction in the synthesis of triglycerides(TG).In this study,shRNA sequences were designed according to the mRNA sequences of DGAT1 and DGAT2 genes and combined to vectors.The next step was to transfect the recombinant vectors into hepatocytes and intramuscular preadipocytes of Zongdihua pig.Real-time quantitative PCR was used to quantify the relative expression of DGAT1 and DGAT2 mRNA in hepatocytes and intramuscular preadipocytes.Through the study about the effects of DGAT1 and DGAT2 genes on TG was to explore the fat deposition mechanism of Zongdihua pig.The results as follow:1.According to the mRNA sequences of porcine DGAT1 and DGAT2 genes,6specific interference sequences and a control sequence were designed and connected with pGPU6/GFP/Neo vector,named pGPU6/GFP/Neo-DGAT1-1,pGPU6/GFP/Neo-DGAT1-2,pGPU6/GFP/Neo-DGAT1-3,pGPU6/GFP/NeoDGAT2-1,pGPU6/GFP/Neo-DGAT2-2,pGPU6/GFP/Neo-DGAT2-3 and pGPU6/GFP/Neo-NC.2.The hepatocytes and intramuscular preadipocytes which from the liver and longgissimus dorsi issues of three-day old Zongdihua pig were dissociated by type ?and type ? collagenases,respectively,in vitro.To induce intramuscular preadipocytes to differentiate,the Insulin,Dexamethasone and IBMX were applied.The output and growth of hepatocytes were in good condition,but the intramuscular preadipocytes had slow growth.3.After transfection for 48 hours,the expressions of DGAT1 and DGAT2 was quantified by real-time quantitative PCR.The results showed that the shRNArecombined plasmids suppressed the expression of DGAT1 and DGAT2 in target cells,respectively.The highest suppression ratios of DGAT1 and DAGT2 genes in hepatocytes were 78.45% and 87.52%,and in intramuscular preadipocytes were82.24% and 89.82%.4.The contents of triglyceride and total cholesterol in DMEM medium of hepatocytes and intramuscular preadipocytes were measured.The results showed that inhibition of DGAT1 and DAGT2 genes could reduced the triglyceride and total cholesterol synthesis in hepatocytes.