Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy1[W][OA]
Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy1[W][OA]
复制标题
DOI：
10.1104/pp.105.068577
复制
发表时间：
2005-12
期刊：
影响因子：
7.4
通讯作者：
Min Zhou;Honglin Xu;Xiaoli Wei;Zhiqiang Ye;Liping Wei;W. Gong;Yongqin Wang;Zhen Zhu
中科院分区：
文献类型：
--
作者：
Min Zhou;Honglin Xu;Xiaoli Wei;Zhiqiang Ye;Liping Wei;W. Gong;Yongqin Wang;Zhen Zhu
5-烯醇丙酮莽草酸 3-磷酸合酶 (EPSPS) 是莽草酸途径中的关键酶，是广谱除草剂草甘膦的靶标。在这里，我们描述了使用基于定向进化的选择系统来选择 EPSPS 的草甘膦抗性突变体。使用该系统，将通过易错聚合酶链式反应诱变的水稻（Oryza sativa）EPSPS基因引入EPSPS缺陷型大肠杆菌菌株AB2829中，并通过功能互补在基本培养基上选择转化体。在三个独立的草甘膦选择实验中鉴定出三个具有高草甘膦抗性的突变体。每个突变体在 EPSPS 编码序列中都包含一个 C317→T 转换，导致蛋白质序列中脯氨酸 106 变为亮氨酸 (P106L)。草甘膦抗性测定表明表达 P106L 突变体的大肠杆菌的草甘膦抗性增加了 3 倍。与野生型EPSPS相比，P106L突变体对草甘膦和磷酸烯醇丙酮酸的亲和力分别降低约70倍和4.6倍。基于动力学模型的分析表明，P106L突变体具有高草甘膦抗性，同时在低磷酸烯醇丙酮酸浓度下保持相对较高的催化效率。使用源自 Michaelis-Menten 方程的数学模型来表征表达水平和选择条件对突变体动力学（Ki 和 Km）变化的影响。这一预测表明表达水平是选择系统的一个重要方面。此外，P106L突变体的草甘膦抗性在转基因烟草（Nicotiana tabacum）中得到证实，证明了在转基因作物中使用P106L突变体的潜力。
5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C317→T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.