Wang He-Yong, Li Yun-Feng, Xie Long-Xu, Xu Peilin
Biotechnology Research Center, The Key Laboratory of Gene Engineering of the Education Ministry, Zhongshan University, 51027, Guangzhou, People's Republic of China.
J Plant Res. 2003 Dec;116(6):455-60. doi: 10.1007/s10265-003-0120-8. Epub 2003 Sep 4.
Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology. Selected mutant enzymes exhibited high K(i)[glyphosate] and low K(m)[PEP] values compared to the parental enzymes from Escherichia coli ( EcaroA) and Salmonella typhimurium ( StaroA). One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco ( Nicotiana tabacum cv. Xanthi). Transgenic plants with increased resistance to glyphosate were generated.
草甘膦是一种非选择性广谱除草剂，它能抑制5-烯醇式丙酮酸莽草酸-3-磷酸合酶（EPSPS），这是微生物和植物芳香族氨基酸生物合成途径中的一种关键酶。我们之前报道了一种基于交错PCR技术构建抗草甘膦I类EPSPS的策略。与来自大肠杆菌（EcaroA）和鼠伤寒沙门氏菌（StaroA）的亲本酶相比，筛选出的突变酶表现出高K(i)[草甘膦]值和低K(m)[磷酸烯醇式丙酮酸]值。其中一个突变体aroA-M1进一步用烟草叶绿体前导序列进行工程改造，然后置于二元载体pCAMBIA1300中，用于农杆菌介导的基因转移到烟草（烟草品种Xanthi）中。获得了对草甘膦抗性增强的转基因植物。
