猪ACSM3基因过表达/敲减质粒的构建

脂肪主要由甘油和脂肪酸组成,酰基辅酶A合成酶中链家族成员3（Acyl-CoA medium-chain synthetase 3,ACSM3）是脂肪酸代谢第一步反应中的关键酶。为了深入研究ACSM3基因的生物学功能,采用克隆、测序、双酶切、连接等分子生物学技术,将ACSM3基因的完整编码区连入pcDNA3.1(+)载体内,构建该基因的过表达质粒;通过生物合成技术,构建该基因的敲减质粒。然后利用体外培养的猪前体脂肪细胞,检测ACSM3基因的过表达/敲减质粒的作用效果。结果表明,成功构建了ACSM3基因的过表达质粒,该质粒的过表达效果可一直持续到转染后的第8天。从3条特异性干扰序列中筛选出1条具有显著敲减效果的序列,敲减作用可持续到转染后的第2天。本研究所构建的过表达/敲减质粒,可为后续的ACSM3基因的功能研究提供有力的技术支持。

Construction of pig ACSM3 gene overexpression / knockdown plasmid

Adipose is mainly composed of glycerol and fatty acids. Acyl-CoA medium-chain synthetase 3 is a key enzyme in the first step of fatty acid metabolism. In order to further study the biological function of ACSM3 gene, the complete coding region of ACSM3 gene was connected into pcDNA3.1(+) vector by molecular biological techniques such as cloning, sequencing, double enzyme digestion and ligation. The knockdown plasmid of this gene was constructed by biosynthesis technology. Then, the effect of overexpression / knockdown plasmid of ACSM3 gene was detected in porcine preadipocytes cultured in vitro. The results showed that the overexpression plasmid of ACSM3 gene was successfully constructed, and the overexpression effect of the plasmid could last eight days after transfection. One sequence with significant knockdown effect was selected from three specific interference sequences, and the knockdown effect lasted two days after transfection. The overexpression / knockdown plasmid constructed in this study can provide strong technical support for the further functional research of ACSM3 gene.