Degradation Effect Analysis Of Four Detoxification Enzymes Of Locusta Migratoria On Pesticides And Its Application In Astragali Radix

Topic Basis : Pesticide residues refer to the general term for pesticides,toxic metabolites,degradants and impurities that remain in organisms,agricultural by-products and the environment after the use of pesticides.The problem of pesticide residues in Chinese herbal medicines has become a major factor restricting the safe use of traditional Chinese medicines and their exports.Among the various methods for removing pesticide residues,the physical and chemical methods are not suitable for the removal of pesticide residues in medicinal materials because of poor removal effects or secondary residues.The bioenzymatic degradation method based on genetic engineering technology is an alternative way which can solve the problem of coexistence of multiple pesticide residues.At the same time,the bio-enzymatic degradation method will become a new and green method for removing pesticides from Chinese herbal medicines due to high degradation efficiency and no damage the shape of Chinese herbal medicines.Insect detoxification enzymes are mainly involved in the metabolism and detoxification of various substances in insects.Previous studies have shown that carboxylesterase and glutathione S-transferase can participate in the detoxification process of various pesticides in insects.Astragalus membranaceus is derived from the dry root of Astragalus membranaceus(Fish.)Bge.var.mongholicus(Bge.)Hsiao or Astragalus membranaceus(Fisch.)Bge.It has the effect of “bu qi” and fixing the surface,diuresis and detumescence,removing toxic sores and repairing damaged skin.As one of the main medicinal materials in Shanxi Province,it has become one of the economic pillars of the Chinese herbal medicine industry in Shanxi.Astragalus is a root medicinal material.Thus,organochlorine pesticides remaining in the soil are easily immersed in the roots and extremely difficult to degrade.The first edition of the 2015 edition of the Chinese Pharmacopoeia specifically regulates the restrictions on pesticides residues in Astragalus.We found that there is a certain pesticide residue problem in Astragalus based on the literature.Therefore,the research on the removal method of root medicinal materials using Astragalus as the carrier has important scientific significance for the safe useage of Astragalus in the future.Based on the background,this project aims to screen insect carboxylesterase and glutathione S-transferase,which can efficiently metabolize pesticides.We investigated the IV application feasibility of bio-enzymes for pesticide residue degradation in medicinal materials by using the Astragalus as a research object.The results can provide new ideas and methods for the removal of pesticide residues from medicinal materials.Objective: We screen detoxification enzymes with high metabolic activity of pesticides in Locusta migratoria by in vitro overexpression,UPLC and RNA interference techniques.We explore the metabolic efficiency of detoxification enzymes on pesticide residues on the surface of medicinal materials by using the Astragalus as a research object and also evaluate of the effect of detoxification enzyme on the content of index components in Astragalus.Methods:(1)The in vitro eukaryotic expression of the Lm Ces A1 was carried out by Bac to Bac insect baculovirus expression system,and its enzymatic properties and optimal reaction conditions were analyzed.Three kinds of Lm GSTs were expressed by Escherichia coli(E.coli)in vitro and the optimal reaction conditions were tested.(2)The metabolic activities in vitro of four Locusta detoxification enzymes on malathion,malathion,p,p'-DDT and deltamethrin were detected by UPLC;(3)The Lm GSTs gene were silenced in the Locusta by RNA interference(RNAi),and the pesticide assays after ds RNA injection were used to detect the sensitivity of the Locusta to pesticides to verify the function of Lm GSTs in vivo;(4)Lm GSTE4 with the most strong activity was selected to detect the degradation effect of malathion on the surface of Astragalus membranaceus,and the content of isoflavone glucoside(Calycosin-7-O-?-D)as the one of the index components of Astragalus membranaceus was determined to studied the effect of Lm GSTE4 on the medicinal components of Astragalus.And evaluate of the effect of Lm GSTE4 on the content of index components in Astragalus.Results:(1)The Lm Ces A1 eukaryotic expression vector was successfully constructed,and the active protein was expressed in Sf9 cells.The Lm Ces A1 was purified to a single target band(60 k Da)by using Nickel column combined with anion exchange column;The enzyme properties of Lm Ces A1 were analyzed by using ?-NA and ?-NA as substrates.The results found that the optimum reaction conditions are p H 8,27 °C.The fusion protein Lm Ces A1 and significantly inhibited by malaoxon in vitro.UPLC results showed that Lm Ces A1 was not involved in the metabolism of malathion,malaoxon and deltamethrin in vitro under the experimental reaction system;(2)Three kinds of Lm GSTs were expressed in vitro by E.coli,and the fusion Lm GSTs were purified in 250 m M imidazole elution buffer by nickel column.Three enzymes had higher activity in the range of 20-30 °C and at p H 6-8 with CDNB as substrate.Insecticide inhibition experiments found that malathion can inhibit the activities of Lm GSTE4 and Lm GSTS2;UPLC results found that Lm GSTE4 and Lm GSTS2 can metabolize malathion simultaneously,and Lm GSTE4 can metabolize p,p'-DDT to p,p'-DDE;(3)RNAi combined with pesticide bioassay experiments showed that the sensitivity of the locust to malathion was significantly increased after Lm GSTe4 and Lm GSTs2 repression;the sensitivity of the locust to p,p'-DDT has no significant change after depressing the Mrna expression of Lm GSTe4;(4)The application of purified Lm GSTE4 to the degradation ability of malathion on the surface of Astragalus was studied.The results showed that the content of malathion was decreased by 28.3% after treatment with Lm GSTE4;the content of isoflavone glucoside has not affected after the application of Lm GSTE4.Conclusion: This study screened the detoxification enzymes Lm GSTE4 and Lm GSTS2 with high efficiently metabolizing malathion by in vitro and in vivo experiments.We explore the metabolic efficiency of detoxification enzymes on pesticide residues on the surface of medicinal materials by using the Astragalus as a research object.