title: Mutation by DNA shuffling of 5-enolpyruvylshikimate-3-phosphate synthase from Malus domestica for improved glyphosate resistance.
abstract: 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.
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China.
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: Tian
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Hu
authors.initials: H
authors.lastname: Xu
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Fu
authors.forename: Hong-Juan
authors.initials: HJ
authors.lastname: Han
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D018744:DNA, Plant / Q000737:chemistry*; D018525:Germination / Q000235:genetics / Q000502:physiology; D005998:Glycine / Q000031:analogs & derivatives* / Q000235:genetics / Q000494:pharmacology; D007700:Kinetics; D027845:Malus / Q000235:genetics*; D016296:Mutagenesis; D012275:Oryza / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000201:enzymology; D017422:Sequence Analysis, DNA; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D018076:DNA, Complementary; D018744:DNA, Plant; D005998:Glycine; D000097797:Glyphosate
keywords: 5-enolpyruvylshikimate-3-phosphate synthase; DNA shuffling; Malus domestica; glyphosate tolerance; mutation; transgenic rice