Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.
Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.
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DOI：
10.1111/pbi.12074
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发表时间：
2013-09
影响因子：
13.8
通讯作者：
Yong-Sheng Tian;Jing Xu;R. Peng;A. Xiong;Hu Xu;Wei Zhao;X. Fu;Hong-juan Han;Q. Yao
中科院分区：
文献类型：
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作者：
Yong-Sheng Tian;Jing Xu;R. Peng;A. Xiong;Hu Xu;Wei Zhao;X. Fu;Hong-juan Han;Q. Yao
从苹果中克隆了一个新的5-烯醇式莽草酸-3-磷酸合酶（EPSPS）基因（MdEPSPS），并通过cDNA末端快速扩增鉴定了该基因的结构，以筛选出一个适合于转基因抗草甘膦植物的EPSPS基因。然而，野生型MdEPSPS由于其对草甘膦的抗性较差，不适合于转基因耐草甘膦植物的开发。因此，我们对MdEPSPS和一个具有8个氨基酸突变（N63 D，N86 S，T101 A，A187 T，D230 G，H317 R，Y399 R和C413 A）的高度草甘膦抗性突变体进行了DNA改组。经过五轮DNA改组和筛选后被确定。在该突变体上的8个氨基酸替换中，只有两个残基的变化（T101 A和A187 T）被确定为改变草甘膦抗性的必要和累加的定点诱变，这是进一步证实了动力学分析。单位点A187 T突变以前也从未被报道为草甘膦抗性的重要残留物。此外，转基因水稻被用来证实MdEPSPS突变体在开发抗草甘膦作物的潜力。
A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.