籽粒苋C4关键酶丙酮酸磷酸双激酶基因的原核表达及酶活性测定

摘要

为了进一步探究籽粒苋丙酮酸磷酸双激酶(AhPPDK)蛋白的作用机制,构建了AhPPDK基因的原核表达载体,通过碱裂解提取质粒,经限制性内切酶酶切,采用1.0%琼脂糖凝胶电泳检测酶切产物,选择正确表达的阳性重组子,将测序正确的重组质粒pEASY-E1-AhPPDK转化菌株Transetta(DE3);利用IPTG诱导蛋白表达.SDS-PAGE凝胶电泳分析表明,重组AhPPDK能在大肠杆菌Transset(DE3)中高效表达,表达的重组蛋白质分子量约为108 kDa,与预期分子量相符,且为可溶性蛋白.利用紫外分光光度法测量结果表明,原核表达的AhPPDK具有酶活性,且上清粗酶液活性高于沉淀粗酶液.研究结果可为进一步探明AhPPDK蛋白的作用机制和转基因利用奠定基础.%To further explore the mechanism of action of AhPPDK protein,the prokaryotic expression vector of AhPPDK gene was constructed.The plasmid was extracted by alkaline lysis,digested with restriction endonuclease,detected with 1.0% Agarose gel.The correct recombinant plasmid pEASY-E1-AhPPDK was transformed into E.coli Transset(DE3),and the recombinant protein was induced by IPTG.SDS-PAGE analysis showed that recombinant AhPPDK could be highly expressed in E.coli Transset(DE3).The expressed recombinant protein had a molecular weight of 108 kDa,which was consistent with the expected molecular weight and was soluble protein.Pyruvate formed by pyruvate phosphodiesterase(PPDK)catalyzes the formation of lactate by excess lactate dehydrogenase and oxidizes reduced coenzyme I(NADH).Using ultraviolet spectrophotometry,the amount of NADH could be calculated according to the change of OD value at 340 nm,and then the activity of PPDK could be deduced.Spectrophotometric method showed that the prokaryotic expression of AhPPDK had the activity of enzyme.These results provide a basis for the further study on the mechanism of action of AhPPDK and the use of transgenes.