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ACSM3基因过表达/敲减质粒的构建
张冬杰,柳樱子,汪亮,杨秀芹,刘娣
0
(黑龙江省农业科学院畜牧研究所,哈尔滨 150086; 农业农村部种养结合重点实验室,哈尔滨 150086;东北农业大学动物科学技术学院,哈尔滨 150006)
摘要:
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。
关键词:    ACSM3基因  脂肪细胞  过表达  敲减
DOI:10.13610/j.cnki.1672-352x.20220907.013
基金项目:国家自然科学基金(32172696), 国家现代农业产业技术体系(CARS-36)和黑龙江省科研业务费(CZKYF2020A004)共同资助。
Construction of pig ACSM3 gene overexpression / knockdown plasmid
ZHANG Dongjie,LIU Yingzi,WANG Liang,YANG Xiuqin,LIU Di
(Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences, Harbin 150086; Key Laboratory of Combining Farming and Animal Husbandry, Ministry of Agriculture and Rural Affairs, Harbin 150086;College of Animal Science and Technology, Northeast Agricultural University, Harbin 150006)
Abstract:
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.
Key words:  porcine  ACSM3 gene  adipocyte  overexpression  knockdown

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