Cloning,identification,and application of glyphosate resistant EPSPS derived from Halomonas sp.
A highly glyphosate resistant strain of Halomonas sp.was screened from marine bacteria to breed crops with high glyphosate resistance to cope with the evolution of glyphosate weeds.The gene en-coding EPSPS in this strain was identified through sequencing genome and bioinformatics analysis.The f HoEPSPS,mfHoEPSPS(G384A site mutant),and mHoEPSPS(a mutant with N-end PDT deletion of mfHoEPSPS)were recombinantly expressed and purified in E.coli(DE3).A gene pyramiding strategy mediated by the self-cleaving peptide LP4/2A was used to locate the glufosinate-resistant enzyme(Repat)at the N-end of mHoEPSPS.A dual resistance to glyphosate/ammonium phosphatase(RLH)was con-structed.Tobacco transformed with the RLH gene exhibited simultaneous resistance to glyphosate/glufos-inate compound herbicides.The results showed that the EPSPS gene(fHoEPSPS)of this strain encoded an N-end fused with a bifunctional enzyme of prephenate dehydratase(PDT).The results of analyzing glyphosate resistance showed that the resistance of mfHoEPSPS was 19 times higher than that of fHoEP-SPS.Introducing the coding gene of mHoEPSPS into tobacco endowed tobacco with three times the recom-mended dose of glyphosate tolerance.Tobacco plants transformed with the RLH gene had simultaneous tol-erance 3-5 times the recommended dosage of glyphosate/ammonium phosphine compound herbicides.It is indicated that the glyphosate-resistant EPSPS derived from Halomonas sp.is a new type of glyphosate-tol-erant enzyme.The enzyme activity is further improved through the G384A site mutation.The transgenic to-bacco with RLH gene obtained through gene pyramiding strategy mediated by the self-cleaving peptides shows high glyphosate/glufosinate compound resistance.It will provide ideas for dealing with the evolution of glyphosate weeds.
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