Abstract: The aim of this study was to observe the role of the Malonyl/Acetyl transferase (MAT) region of goat fatty acid synthase (FASN) in fatty acids metabolism of goat mammary, and to construct the recombinant adenovirus vector with goat MAT for observing gene overexpression in primary goat mammary epithelial cells. The primers were designed according to the MAT sequence in GenBank, and the gene was cloned and sequenced by RTPCR. The goat gene fragments containing both MAT region and HIS sequence were inserted into shuttle vector pAdTrack/CMV to construct recommbinant shuttle vector pAdTrack/CMVMATHIS. After identifying by digestion and sequencing, pAdTrack/CMVMATHIS plasmid was linearized by PmeⅠ, and then it was transformed into E.coli Bj5183 competent cells containing backbone vector pAdEasyⅠto obtain recombinant vector pAdEasyMATHIS by homologous recombination. pAdEasyMATHIS plasmid was linearized by PacⅠ, and transfected into HEK 293 cells for virus packing, amplification and titer testing by LaSRT. The MAT sequence cloned had a GA mutation at 601 bp by comparing with the corresponding sequence in GenBank, which caused an AlaThr mutation at the 201st amino acid residue. The results of PCR detection and Western blot showed that recombinant overexpression vector containing MAT was successfully constructed. The titer of virus obtained was 2×109 PFU·mL-1. The recombinant adenoviral vector pAdEasyMATHIS was constructed successfully in this experiment.