Overexpression Of Mat Domain Of Fatty Acid Synthase (FASN) Gene In Dairy Goat

Acetyl-CoA and malonyl-CoA transacylase(sMAT)which is one domain of FASN is the key enzyme for short-chain fatty acids synthasis in vivo.It contain the ability to terminate the carbon chain extension of fatty acid to release the short-chain fatty acid.The overpression of MAT will be useful for the future study about the synthesis mechanism of the fatty acids .In this study,we cloned MAT sequence of FASN gene by RT-PCR and then analysed the sequence with bioinformatics. I constructed recombinant adenovirus overexpression plasmid pAdEasy-MAT-HIS through homologous recombination between shuttle vector pAdTrack/CMV and backbone vector pAdEasyâ… .I researched the effect of MAT overexpression on other fatty acid metabolism relative genes in goat mammary epithelial cells,it also provided a basis for regulation research of fatty acid metabolism. The results of this research are as follows:(1).The MAT sequence we coloned in XINONG SANNEN GOAT had a length of 981bp, encoding 327 amino acid residues. The physical property analysis indicated that the molecular weights of goat MAT were 36 kDa with the isoelectric point ( pI ) was 5.83. Both of N-and C sides had shown strong hydrophobic. There were 15 phosphorylation sites, including 13 Serine phosphorylation sites,1 Tyrosine phosphorylation site and 1 threonine phosphorylation site.(2). I constructed the shuttle vector pAdTrack/CMV-MAT-HIS and obtained the recombinant adenovirus vector pAdEasy-MAT-HIS through homologous recombination. Then high-titer rcombinant adenovirus were obtained by packaging and propagation in HEK293 cells.The virus titer was 2×109 U/mL(3).Using gradient methord, I observed the fluorescence conditions and cell morphology of goat mammary epithelial cells which were treated with defferent MOI(50,100,150,200 and 250)for 72h. The data showed that the optimal MOI was 200.The result of western blotting indicted that I overexpressed the MAT successfully. The result of RT-qPCR showed that I overexpressed MAT successfully with a gene expression level 2781-fold that of control group.(5). I incubated the the goat mammary epithelial cells with recombinant adenovirus for 72h.The rusults of RT-qPCR shows that mRNA lever of LPL,LXRα,HSL,ATGL,SCD,H-FABP,SREBP1,LEPR,ADRP,TIP47,GPR41 and GPR43 was reduced by 76.5%,72.1%,51.0%,12.3%,11.9%,51.4%,54.0%,35.0%,40.8%,69.1%,70.8%å'Œ69.6% respectively compared with that of the control gorup. The mRNA expression of FASN and ACACA were 10.20-fold and 1.17-fold that of the control group respectively.In conclusion, I cloned MAT sequence from dairy goat mammary tissue, and got a higher similarity compareing with the goat sequence from NCBI. MAT overpression level increased significantly through treating the dairy goat mammary epithelial cells by the recombinant adenovirus.