Glyphosate Tolerant Mechanism Of Three Lilyturf Species And Exploring Of New Resistant Genes

Glyphosate(N-(phosphonomethyl)glycine),a non-selective and systemic postemergence herbicide,has been recognized as a unique herbicide for weed control because of its intrinsic properties such as broad-spectrum,low toxicity,and lack of soil residual activity(Duke and Powles 2008).It inhibits 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS,EC 2.5.1.19)in the shikimic acid pathway,an enzyme that is essential for the biosynthesis of the aromatic amino acids,tryptophan,phenylalanine and tyrosine in plants.Because of the unique action way and metabolic mechanism,it was considered impossible of glyphosate-resistant weeds in the field.However,with widespread planting of glyphosate-resistant(GR)crops and extensive reliance on glyphosate,resistant weed biotypes have evolved worldwide.What’s more,there are some plants species have been reported with tolerance to glyphosate.This poses a great threat to the safety of the sustainable use of glyphosate.Therefore,detection of glyphosate resistant/tolerant levels of plant and strengthen the research on the action and resistance mechanism will promot the progress of weed science in China and safety in agricultural production.It has important significance for the securation of the herbicide safety and the sustainable development of agriculture to detect glyphosate resistant/tolerant levels of plant and strengthen the research on resistance/tolerance mechanism.The thesis presents the research on the high glyphosate tolerant level and mechanism of three lilyturf species.What’s more,error-prone PCR was conduct on LS-EPSPS for higher glyphosate resistant gene.In addition,EPSPS of glyphosate resistant and tolerant plants was analyzed systematically to discovery more conserved motifs and mutanted sites.The main results of this study are as follows:1.Bioassay of glyphosate tolerant level in three lilyturfsEven though glyphosate has been widely used as a non-selective herbicide,there are some plants species have been reported with tolerance to glyphosate.In this paper,we report that Lilyturfs(Ophiopogon japonicas(OJ),Liriope spicata(LS)and Liriope platyphylla(LP)are naturally resistant to glyphosate.Dose-dependent response of plants to glyphosate revealed that the ED50 values of OJ,LS and LP were 7000,7360 and 8230 g ae/ha,respectively.This result shows that these plants have resistance to glyphosate about 5-8 fold of the recommended field rate.A spectrophotometric assay was evaluated as an inexpensive and rapid procedure to measure shikimate accumulation in the three lilyturfs.They all accumulated little shikimate at 3000 g ae/ha glyphosate does.There are significant differences in stomatal density and morphological structure of leaf epidermis in the three lilyturfs,which may be one of the reasons for high tolerance to glyphosate.2.Molecular mechanism of glyphosate tolerant in three lilyturfsOur previous study showed that three lilyturf species had naturally tolerant to glyphosate and then further experiments were conduct to determine their molecular tolerance mechanisms.These species tolerate glyphosate at about five times the commercially-recommended field dose.They share three unique amino acids in their 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)that affect glyphosate binding.These correspond to Asp71Met,Ala112Ile and Val 201 Met amino acid substitutions compared to other 231 published plant EPSPS.Glyphosate-treated plants accumulated little shikimic acid but had significantly higher levels of EPSPS mRNA.The IC50 of LS-EPSPS was 14.0 μM,much greater than the 5.1μM of Arabidopsis thaliana.The higher Km and Ki values of LsEPSPS kinetics showed that LsEPSPS had lower substrate binding affinity to glyphosate.Overexpression of LsEPSPS in the recombinant E.coli BL21(DE3)strain enhanced its tolerance to glyphosate.The calculated EC50 value of transgenic A.thaliana was 220 g ae ha-1,about 3.6 fold that of the wild-type.Both OJ and LS had two copies of the EPSPS gene while LP had three copies.Therefore,a combination of unique EPSPS structure and increased gene copy number and expression contribute to natural glyphosate-tolerance in the three lilyturf species.3.Directed evolution of LS-EPSPS based on error-prone PCRLiriope spicata(Thunb.)Lour has a unique EPSPS structure contributing to the highest-ever-recognized natural glyphosate tolerance.The transformed EPSPS can enhance resistance of E.coli and A.thaliana to glyphosate.However,the increased glyphosate-tolerant level is not strong enough to be commercially utilized.Thus,we performed error-prone PCR on LS-EPSPS to select increased resistance of mutant EPSPS gene.One highly glyphosate-resistant mutant named LSE-EPSPS with mutations in five amino acids(37Val,67Asn,277Ser,351Gly and 422Gly)was identified after orientedly screened.The gene expression of LSE-EPSPS in the recombinant E.coli BL21(DE3)strains enhanced tolerance to glyphosate in comparison with control.Furthermore,transgenic LSE-EPSPS A.thaliana demonstrated about 5.45 folds glyphosate-resistance compared with that of the wild-type and 2 fold increases of LS-EPSPS-transgenic plants.Those results indicate that mutated-LSE-EPSPS has a potential to be applied to development of glyphosate-resistant crops.4.EPSPS gene structure analysis of various glyphosate resistant and tolerance plantsThe mutations of EPSPS,EC 2.5.1.19(5-enolpyruvylshikimate-3-phosphate synthase),as unique target of glyphosate,impact its compatibility with plyphosate and eventually cause tolerance/resistance to this herbicide.The systematic analysis of amino acid component and gene sequence of plant EPSPSs will have important theoretic significance to elucidate molecular mechanism of glyphosate-tolerance/resistance and explore novel glyphosate-tolerance/resistance gene resources.We cloned 10 species and collected 101 species of plant EPSPSs and their counterpart genes to systematically study the relationship between the information and tolerance/resistance-glyphosate.It was found that the ED50 ranged from 300 to 4000 ae g/ha in glyphosate-resistant weeds while from 787 to 8229 ae g/ha in glyphosate-tolerant weeds.Based on the phylogeny and sequence conservation,the cloned and collected 111 plant EPSPS genes from NCBI were divided into three groups,monocotyledons,dicotyledons and other plants.Furthermore,we searched the previously reported motifs and studied its structural importance on the basis of homology modelling.Fifteen conserved domains were obtained and four of them are patented.Six conserved motifs were first recognized,CD2,CD5,CD9,CD10,CD11 and CD12.Amino acid mutations with high frequence were all in CD1,CD3,CD4 and CD6,Ile29Thr,Phe96Ser,GlylOlAla,Thr102Ala,Pro106Ala/Leu/Ser/Thr,Met128Ile,Gly134Glu,Lys141Gln,Gly144Asp/Asn,Thr183Ala/Ser,Ala188Thr and Asp351Gly.Different mutations at the same position had different effects on EPSPS structure.During the four mutations of EPSPS in Lolium rigidum,Pro106Ala and Pro106Ser had similar H band change,both extended by 0.14 A,compared with S type.Pro106Thr extended by 0.15 A.Pro106Leu may had the biggest H band change between 101Gly and glyphosate in EPSPS,extended by 0.24 A.In addition,LSE-EPSPS and AM-EPSPS all had mutaions both in the conserved and non-conserved domains.LSE-EPSPS had the mutation of Asp351Gly in CD 12 and Glu37Val,Arg422Gly mutations in non-conserved regions.AM-EPSPS had the mutation of Gly175Asp in CD6 and Ala415Ser mutation in non-conserved regions.These two kinds of mutations may cooperate to endow them with glyphosate resistance/tolerance.However,the substitutions in EPSPS of three lilyturfs and Cayratia japonica(CJ-EPSPS)were in non-conserved motif.There were 91Glu deletion,Ser229Ala and Glu390Val substitutions in lilyturf EPSPS while Ser229Ala and Glu390Val substitutions in CJ-EPSPS.They all have effects on EPSPS activity.The H band between 131Arg and glyphosate was lenghthed by 0.1 A.Consequently,besides conserved motifs,mutation that is not located in conserved motifs but at a position distant from the active site can also alter glyphosate resistance.This result may provide a new perspective of the molecular mechanism of glyphosate resistance/tolerance.