| Topramezone([3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(meth ylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone)is a new 4-hydroxyphenylpyruvate dioxygenase(HPPD)inhibitor herbicide that is widely used on corn for control of annual grass weeds and broadleaf weeds.Due to its broad-spectrum weed control capacity,improved activity,low mammalian toxicity and high environmental safety,topramezone is considered as an ideal target herbicide for transgenic engineering of herbicide tolerance.Up to now,HPPDs from a variety of plants,animals and microorganisms have been identified and characterized.However,only a small part of these HPPDs show strong resistance to HPPD inhibitors,and HPPD with topramezone resistance has not been reported.Thus,screening of highly topramezone-resistant isolate and cloning its topramezone-resistant HPPD gene have important application value in topramezone-resistant transgenic engineering.This study screened and identified topramezone-resistant isolate,cloned topramezone-resistant HPPD gene,and improved the topramezone resistance of the gene through pressure acclimation.1.Screening and identification of topramezone-resistant isolate and cloning of BkhppdIn this study,the suspension of sample was serially diluted and spread on MSM agar supplemented with L-tyrosine and topramezone(as selective pressure),and finally we screened a topramezone-resistant strain,Burkholderia sp.BW-1.from the soil samples.Strain BW-1 could tolerate up to 600 ?M topramezone.The HPPD gene was cloned from the genome of strain BW-1 using a pair of degenerate primers.The complete gene sequence is 1098 bp.encoding 365 amino acids.Sequence analysis showed that BkHPPD shared low similarity(only 25-55%)with reported HPPD inhibitor herbicide-resistant HPPDs.The Km,kcat and Kcat/Km values of BkHPPD for HPPA were 143.3 ?M,10.1 s-1 and 70.5 s-1 mM-1,respectiveely,and the optimal temperature and pH value for the activity of BkHPPD were approximately 30? and 7.4.respectively.The activity of BkHPPD was obviously enhanced by Fe2+,Fe3+ and Mg2+ and strongly inhibited by Hg2+,Zn2+,Co2+ and Ni2+.The specific enzyme activity value for BkHPPD was 12.5 U·mg-1.The IC50 value of topramezone against BkHPPD were 572.0.BkHPPD displayed relatively high activity and highly resistant to HPPD inhibitors,thus is a good candidate gene for construction of HPPD inhibitor herbicide-resistant crop.2.Pressure acclimation of the wild-type strain BW-1 and resistance assayIn order to improve the topramezone-resistant of BkHPPD,strain BW-1 was acclimated by continuously increasing the topramezone concentration in the TMSM broth.After several rounds of transfer,two mutants,designated BWt31 and BWt76,were obtained through pressure acclimation.Strain BWt31 could tolerate 1200 ?M topramezone,whereas strain BWt76 could tolerate up to 2000 ?M topramezone.HPPDs of mutant BWt31(designated BkHPPDt31)and mutant BWt76(designated BkHPPDt76)were cloned.Sequence analysis showed that BkHPPDt31 contained three amino acid substitutions(replacement of histidine 65 with aspartic acid,replacement of asparagine 160 with threonine,replacement of asparagine 258 with serine).In BkHPPDt76,an additional mutation causing the conversion of asparagine to threonine at position 343 was also discovered except for the above mutations.The Km,kcat,kcat/Km and specific enzyme activity values for BkHPPDt31 were 165.8 ?M,12.5 s-1,77.2 s-1 mM-1 and 18.1 U·mg-1,respectively,while for BkHPPDt76 were 224.7 ?M,3.8 s-1,16.9 s-1 mM-1 and 6.3 U·mg-1.BkHPPDt31 displayed higher activity and catalytic efficiency than that of wild-type BkHPPD.In contrast,the activity and catalytic efficiency of BkHPPDt76 were significantly lower than that of the wild-type BkHPPD.In addition,the topramezone-resistant of mutants BkHPPDt31 and BkHPPDt76 were 112%and 232%higher than that of the wild type,respectively.3.Site-directed mutagenesisTo clarify the roles of each amino acid mutation identified in the BkHPPDt31 and BkHPPDt76 mutants,four single-site mutants,designated H65D,N160T,N258S and N343T were constructed.The kinetic parameters.HPPD activity and resistance of these mutants were assessed.The Km and kcat values of H65D for HPPA were 113.6 ?M and 10.5 s-1 respectively,in terms of the catalytic efficiency,the of H65D was 92.4 s-1 mM-1.the specific enzyme activity of H65D was 15.9 U·mg-1:The Km,kcat,kcat/Km and specific enzyme activity values for N160T were 156.8 ?M,9.9 s-1,63.3 s-1 mM-1 and 14.4 U·mg-1,respectively,for N258S were 137.5 ?M,11.4 s-1,123.6 s-1 mM-1 and 17.0 U·mg-1,respectively,for N343T were 241.9 ?M,3.4 s-1,20.3 s-1 mM-1 and 4.9 U·mg-1,respectively.The above results indicated that the increased specific enzyme activity of BkHPPDt31 was resulted from the synergistic effects of all three site mutations(H65D,N160T and N258S),while the significantly decreased catalytic efficiency and affinity of BkHPPDt76 were mainly due to the substitution of asparagine at position 343 with threonine.In addition,the IC50 values of topramezone against H65D,N160T,N258S and N343T were 863.0,421.2,500.7 and 1334.0 nM,respectively.Compared with that of wild-type BkHPPD,the topramezone-resistant of H65D was increased,while N160T and N258S were slightly decreased.However,the IC50 values of the three single-site mutants H65D,N160T and N258S were all smaller than that of mutant BkHPPDt31(containing these three amino acid substitutions).Although the affinity and catalytic efficiency of the N343T mutant were dramatically decreased,the topramezone IC50 of N343T was significantly increased from 572.0 nM to 1334.0 nM.The results site-directed mutagenesis indicated that the increased resistance in BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than from a single site mutation,while the increased resistance in BkHPPDt76 resulted from the single site mutation N343T.In addition,the N343T single-site substitution dramatically decreased catalytic efficiency but increased topramezone resistance,indicating that the N343 was a key residue in the enzyme. |
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