Identification Of A Gene Encoding EPSPS With Resistance Of Glyphosate And Development Of Transgenic Glyphosate-resistant Rice Plants

One of the widely used EPSPS inhibitors,glyphosate is a broad-spectrum, nonselective,postemergence herbicide with low risk to humans and the environment due to its low toxicity,lack of residual soil activity,and rare occurrence of resistant weeds.However,glyphosate kills all plants,including crops,and much attention has been paid to the search for glyphosate-tolerant enzymes for transgenic crops.In this research,a bacterica strain was isolationed from serious polluted soil by glyphosate,which can tolerant glyphosate up to 200mM.The strain was identified as Pseudomonas putida by phylogenetic analysis of 16S rDNA sequencing and observation of the strain with scanning electron microscopy.The strain is named by Pseudomonas putida G6 temporarily.G6 gene was cloned by amplication of Pseudomonas putida G6 genome.Amino acid sequence of G6 gene shares more high homology with Classâ…¡EPSPS than Classâ… EPSPS.It contains conserved motif,domain and functionally important amion acid residues of EPSPS.Molecular modeling indicated the tertiary structure of the domain of EPSPS analogy with E.coli EPSPS.pET-G6 plasmid was construced by pET-28 and G6.E.coli BL21(DE3)harboring pET-G6 plasmid was grown in M63 minimal medium supplemented with 1mM IPTG, 50 mg/ml Kanamycin and glyphosate at concentrations ranging from 50 to 150 mM.The cells were grown well in 50mM and 100mM glyphosate,but restrained in 1.50 mM glyphosate.E.coli BL21(DE3)harboring pET-G6 plasmid was induced expression by IPTG.The soluble protein in cell-free extracts was separated by SDS-PAGE to determine the level of EPSPS G6 protein in the cells.A major band of protein is 46 kDa.Polyclonal antibody of Rabbit Anti-G6 was prepared.The full length of maize optimized synthetic glyphosate-resistant G6 gene,including its 5' end DNA fragment encoding the chloroplast transit peptide from the acetohydroxyacid synthase of Zea mays and its 3' end terminator fragment from the maize phosphoenolpyruvate carboxylase,was synthesized by Shanghai Sangon Limited,China.pCAMB1300-ZmUbi-G6sy was constructed with synthesized G6 gene and the Z.mays polyubiquitin-1 promoter,ZmUbi-1.T-DNA plasmid transformation vector pCAMB1300-ZmUbi-G6sy was transformed into Agrobacterium tumefaciens(LAB4404)by agrobacterium-mediated.T₀ transgenic rice plants were identified by PCR,Western blot and Southern blot.Herbicide spray tests were indicated T₀ transgenic rice plants could gown well when it was sprayed 10%glyphosate aqueous solution diluted 16.6x.T₁ transgenic rice plants could gown well when it was sprayed 10%glyphosate aqueous solution diluted 25x.In this study,we have discoverd a novel G6 gene which cannot be inhibited by glyphosate.It was transformed into rice and the T₀ and T₁ transgenic rice is high resistance to glyphosate.It implied that EPSPS G6 gene could be applied to cultivate glyphosate-resistant crops.