Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance
文献类型： 外文期刊
作者： Tian, Yong-Sheng 1 ; Xu, Jing 1 ; Peng, Ri-He 1 ; Xiong, Ai-Sheng 1 ; Xu, Hu 1 ; Zhao, Wei 1 ; Fu, Xiao-Yan 1 ; Han, Hong 1 ;
作者机构： 1.Shanghai Acad Agr Sci, Biotechnol Res Inst, Shanghai Key Lab Agr Genet & Breeding, Shanghai, Peoples R China
关键词： 5-enolpyruvylshikimate- 3-phosphate synthase;Malus domestica;DNA shuffling;glyphosate toleranc
期刊名称：PLANT BIOTECHNOLOGY JOURNAL （ 影响因子：9.803； 五年影响因子：9.555 ）
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年卷期：
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收录情况： SCI
摘要： 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.
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