【摘要】 除草剂草甘膦 （glyphosate ,N_phosphonomethylglycine ）的靶标酶是由aroA基因编码的EPSP （5_烯醇式丙酮酰莽草酸_3磷酸 ,5_enolpyruvylshikimate_3phosphate）合成酶。采用先进的基因优化技术 ,以鼠伤寒沙门氏杆菌和大肠杆菌的EPSP合成酶基因为模板 ,通过随机结合 ,交错延伸的PCR扩增过程 ,克服定点突变的局限性 ,使基因获得多位点突变 ,并使之在大肠杆菌中得到表达。对纯化后的表达产物的酶动力学性质的测定结果表明 ,获得了与作为模板的野生型相比 ,具有高得多的酶活力 （降低的Km （PEP） ）和大大增强的草甘膦抗性 （升高的Ki（glyphosate） ）的突变基因更多还原

【Abstract】 The herbicide glyphosate inhibits EPSP synthase (5-enolpyruvylshikimate-3-phosphate synthase) encoded by the aroA gene, a key enzyme in the aromatic amino acid biosynthetic pathway in crops and weeds. The staggered PCR extension process has been applied with Escherichia coli and Salmonella typhimurium aroA genes as templates to obtain one variant that exhibits significantly enhanced tolerance to glyphosate. Kinetic patterns of the mutant reveal the successful generation of EPSP synthase glyphosate-tolerant variant with high K i(glyphosate) and low K m(PEP).更多还原