Research On Molecular Mechanisms Of Herbicide DTP Resistance Of CjHPPD

4-Hydroxylphenylpyruvate dioxygenase(HPPD)catalyzes the formation of homogentisate(HGA)from 4-hydroxyphenyl pyruvate(HPP).This is the second step in the pathway of tyrosine catabolism.HPPD as a unique target enzyme has been made a great deal of research since last century because of its biochemical pathways.Multiple types of HPPD inhibitor have been developed,which were widely used in treating hereditary tyrosinemia and managing weeds.An cDNA encoding Coptis japonica HPPD was isolated from cultured Coptis japonica cells,it is resistant to HPPD-inhibitor herbicide DTP.At present,the molecular mechanisms of the resistance still need further study.This study aims at exploring the molecular mechanisms of the DTP resistance of CjHPPD.The results will lay the foundation for enriching the diversity of resistance genes,for developing herbicide-resistant genetically modified crops,and for improving the quality of genetically modified crops.In the paper,first of all,the physicochemical properties,signal peptide,transmembrane spaning domain,subcellular localization,hydrophobicity or hydrophilicity,secondary structure,motifs and tertiary structure of Coptis japonica HPPD were analyzed by bioinformatics.And then using molecular docking technique explored the interactional conformation between DTP and C.japonica HPPD.Secondly,sequences of HPPD were alignmented among C.japonica,A.thaliana,Z.mays,D.carota,P.fluorescens,P.aeruginosa and AM-H4(Pseudomonas sp.),and what's more,the resistance targets of C.japonica HPPD amino acid sequence were protocolled and mutated by site-directed mutagenesis.Thirdly,the heterogeneous expression of recombinant Coptis japonica hppd was developed in E.coli BL21(DE3),in addition,defining the OD400 of Coptis japonica HPPD supernatant as one enzyme activity unit measured other mutants' activity.Finally,combining with the results of bioinformatics and molecular docking,the molecular mechanisms of activity and resistance were analysed.The main results are as follows:Structure prediction results show that the gene ORF could encode a 430 amino acid polypeptide.The polypeptide had no signal peptide,no transmembrane region,and which was a hydrophilic soluble protein that located in microbody and cytoplasm.Random coil and alpha helix were main secondary structure components.There is 75.41% identity between Coptis japonica HPPD and A.thaliana HPPD(1sp9)in homology modeling.14 mutants were protocolled,they were H209 G,A210I,L224 F,T244A,I251 M,P263G,M264 L,Y280F,E282 D,H291G,L292 M,L294V,P319 A and E409 Q.The secondary structure and hydrophilic of these mutants were very different from which of CjHPPD.The results of HPPD enzyme activity and resistance analysis showed that the activities of L244 F,P236G and L292 M drop to 53%-56%,the activities of H209 G and H291 G drop to14.00% and 20.35%,respectively.The activities of several other mutants are correspondingly increased 40%-570%.The resistance to DTP of mutants except H209 G and H291 G were measured,the results showed that resistance of A210 I,L224F,L292 M and P319 A drop to70.33%-98.95%,on the contrary,the others' is correspondingly increase to 112%-150%.In conclusion,the resistance to DTP of Coptis japonica HPPD is connected with its amino acid sequence,H209 and H291 are necessary for enzymatic activity.The twelve sites which are A210,L224,L292,L294,T244,I251,Y280,E282,P263,M264,P319 and E409 play important roles in the herbicide DTP resistance of CjHPPD.Changing these sites' residues will affect the active pocket and its adjacent residues' secondary structure,hydrophobicity or steric hindrance.