Insecticides Detoxification Of Carboxylesterase Wh001g And Its Mutant Enzymes From Helicoverpa Armigera

Helicoverpa armigera is an important and polyphagous agricultural pest,and can feed more than 60 crops and nearly 70 species of wild plants,thus causing serious damage with their generations overlap.In the traditional control of H.armigera,there was a large dependence on using organophosphorus and pyrethroid insecticides,and the long-term unreasonable use of such chemical pesticides has led to serious insecticide resistance in H.armigera.Carboxylesterase is an critical class of detoxification enzyme in insects,and is also a class of the key enzymes involved in insecticide resistance in H.armigera and other insect species.In this study,carboxylesterase gene WH001G was cloned from the H.armigera WH strain and analyzed,which has a lenth of 146 aa sequence glycine-rich-repeat region（GRR）,and thus,it was the longest carboxylesterase and the most complex sequence structure.The WH001G was successfully expressed in Eschrichia coli and purified,with optimized four types of prokaryotic expression vector.The metabolic activities of 001G against seven types of insecticides were determined by High Performance Liquid Chromatography（HPLC）.Further more,variant enzymes of WH001G with different lengths of the simple tandem repeats were expressed by using the same expression system,and the fusion proteins with natural enzymatic activity were obtained.To understand the effect of sequence variation on the catalytic activity of carboxylesterase,the metabolic activity of different purified proteins of mutant enzymes against insecticides were compared with the wild type WH001G.The main results of this study are summarized as follows:1.The full lenth of open reading frame of the carboxylesterase WH001G was obtained by PCR amplification and it was registered in the NCBI database（Gen Bank accession number KT345938.1）.It had 2244 nucleotides,encoding 747amino acid,and the protein molecular weight was 78.9 KDa with theoretical isoelectric point of 4.19.Paticularly,the001G amino acid sequence contained 22 simple tandem repeats including 12 copies of an8aa motif"EGDGGDGG"and 10 copies of a 5aa motif"EGDGG",which was a total length of 146aa and located adjacent to the C-terminal.In addition,the genomic DNA sequence of WH001G was also isolated,which had 3130 bp including 3 exons and 2 introns.2.The carboxylesterase WH001G was expressed in E.coli expression system at a low temperature of 18℃with final concentration of 0.2 m M of IPTG.The p ET30a was shown the most suitalbe vector for expression of WH001G by comparision with the enzyme activities of the eapression in p ET28a,p ET30a,p ET32a and p ET42a.The E.coli was induced at a low temperature for 48 h,and then the fusion proteins were purified by Ni-NTA²⁺column from the supernatant of lysis of cell pellets.Most of the target proteins were eluted by elution buffer of imidazole at the concentration between 50 m M and 100 m M.The SDS-PAGE showed that the purified protein bands were 80 KDa and 70 KDa,which were all identical with the results of Western-Blot.In addition,the results of Native-PAGE also showed that both bands had esterase activity.3.The catalytic activity of the purified protein WH001G toward the model substrates1-naphthyl acetate（1-NA）and 2-naphthyl acetate（2-NA）were determined at 450 nm and508 nm using a full-wavelength microplate reader.The results showed that the WH001G has catalytic activity toward both 1-NA and 2-NA,with the Km values of 9.6±1.8μM and 16.2±3.7μM,respectively.However,WH001G showed better affinity and higher enzymatic efficiency toward 1-NA than those of 2-NA.In addition,based on the above method,the triphenyl phosphate（TPP）used as a positive control and 1-NA was used as a substrate,the inhibition of seven different insecticides（includingβ-cypermethrin,fenvalerate,lambda-cyhalothrin,chlorpyrifos,methyl parathion,ethyl paraoxon,and imidacloprid）on the activity of enzyme were determined.The half inhibitory concentrations(IC₅₀)of the three pyrethroid insecticides（β-cypermethrin,fenvalerate,lambda-cyhalothrin）against WH001G were 501.2±22.7μM,612.3±28.4μM and 578.2±17.4μM,respectively,indicating that the pyrethroid insecticides had a relatively weak inhibitory on WH001G compared with the positive control triphenyl phosphate（TPP）with the IC₅₀value of 360.3±15.9μM.However,the IC₅₀of three organophosphorus insecticides（chlorpyrifos,methyl parathion,ethyl paraoxon）aginst WH001G were 80.7±4.5μM,4.7±0.4μM and 0.02±0.00μM,respectively,suggesting that three organophosphorus insecticides had strong inhibition on the activity of 001G.The above results suggested that WH001G was more sensitive to organophosphorus insecticides.4.The metabolism of the seven insecticides by WH001G were performed by HPLC assay,and the amount of substrate loss after incubation at 30°C for 2 h were detected and the activity was expressed as nanomolar substrate loss/min/mg protein.The inactivated protein was used as a blank control.The specific activity of WH001G againstβ-cypermethrin and lambda-cyhalothrin were 1.69±0.05 and 1.39±0.03 n M/min/mg,respectively,which slightly higher than the specific activity of WH001G against fenvalerate（0.45±0.05 n M/min/mg）.The results showed that pyrethroid insecticides could be metabolized by WH001G at a lower rate.In contrast,001G had no significant difference in the metabolism of organophosphate insecticides,imidacloprid and the control.The 3D model showed that the cypermethrin molecule can bind with the active site of WH001G,and the distance between the serine residue（Ser205）of enzyme active center and the ester-based central carbonyl carbon（C）of cypermethrin was 2.6（?）.5.The prokaryotic expression of variant enzymes of WH001G,including G6,Gx-4,Gx-8 and G1,showed that the mutant enzyme had higher catalytic activity on the substrate1-NA,and its Km values were 2.68±0.69μM,4.21±1.22μM,5.78±2.27μM and 2.54±0.70μM,respectively.The rate constants（kcat/Km）were 2.48±0.54,1.11±0.19,0.90±0.31 and 1.31±0.35 s^-1·μM^-1,which were higher than that of 001G.It suggested that variant enzymes had higher substrate affinity and enzymatic efficiency than 001G.Furthermore,the metabolic specific activities of the mutant enzyme G6,Gx-4 and Gx-8 toward the insecticideβ-cypermethrin were 4.42±0.57,3.99±0.51,and 2.29±0.87 n M/min/mg,respectively,which were higher than the specific activity of 001G.However,they were not obvious hydrolysis toward chlorpyrifos.In summary,in this work,the new carboxylesterase allele WH001G was amplified and obtained from H.armigera,and the GRR region was observed in its protein sequence through the bioinformatics analysis,thus,which indicated WH001G had unique structure.The fusion protein were obtained from prokaryotic expression which had high affinity and catalytic metabolic activity on the model substrate 1-NA,and it had the metabolic activity toward the pyrethroid insecticides.Furthermore,the variant enzymes G6,Gx-4 and Gx-8showed higher affinity and catalytic efficiency toward the model substrate 1-NA,and exhibited higher metabolic activity towardβ-cypermethrin insecticide.Totally,the results revealed that the WH001G could be succesfully expressed in E.coli system to yield active fusion protiens,and 001G can metabolize pyrethroid insecticides.The sequence variation in glycine-rich region of the 001G could affect its metabolic activity toward pyrethroid insecticide.This layed a foundation for the comprehensive understanding the function of the individual members of carboxylesterase family of H.armigera in the insecticide metabolism,and also provided a theoretical basis for revealing the roles and mechanism of carboxylesterase in the development of metabolic resistance in H.armigera in the future.