Recombinant Expression And Biochemical Characterization Of The Carboxylesterase NlCarE From The Brown Planthopper,Nilaparvata Lugens(Hemiptera:Delphacidae)

The brown planthopper,Nilaparvata lugens,is one of the main pests of rice in Asia.It often breaks out and causes serious yield and economic losses,seriously threatening the sustainable development of rice production in the world.There are many esterase genes in N.lugens,which are related to insecticide resistance.To explore the esterase genes of N.lugens can provide gene resources for the monitoring and management of insect resistance to insecticides,the development of new and high-efficiency pesticides,and the detection and degradation of pesticide residues in the environment.In this paper,the gene cloning,prokaryotic expression and enzymatic characteristics of the carboxylesterase NlCarE from N.lugens were studied,and the following main results were obtained.The full-length gene of the carboxylesterase NlCarE was cloned from N.lugens,and the recombinant expression vector was constructed to induce expression in E.coli.The molecular weight of the recombinant expression product is about 70 k D,and the esterase activity can be detected in the supernatant.The polyclonal antibody of NlCarE was obtained and used for expression detectionin N.lugens.The expression level of the carboxylesterase NlCarE is high in older nymphs and adults,the abdomen and digestive tract of N.lugens.The recombinant NlCarE protein has a substrate preference for α-NA and β-NA.The optimal temperature for the enzymatic reaction of the recombinant NlCarE protein is around 37°C,and low or high temperature seems to have an adverse effect on the activity.The optimal p H for the enzymatic reaction of the recombinant NlCarE protein is around7.5,and acidic or alkaline conditions may have an adverse effect on the activity.The recombinant NlCarE protein has higher activity on substrates with shorter C-chains,while there is no detected activity towards the substrates with a C-chain length greater than 10 carbon residues.The acivity of the recombinant NlCarE protein is inhibited by organophosphorus,in which the inhibitory ability of paraoxon is significantly higher than that of chlorpyrifos.The metabolic ability of the recombinant NlCarE protein towards different insecticides was detected using HPLC.The recombinant NlCarE protein has a strong metabolic degradation ability for permethrin,which Km is 30.48 mmol/L and Vmax is46.91 mmol/min/ mg.Through molecular docking simulation analysis,it was found that all tested pesticides can bind to the NlCarE recombinant protein,among which permethrin,deltamethrin and fenvalerate have strong binding effects.These results provide the basis to understand the molecular functional mechanism of carboxylesterase for the resistance management of N.lugens,to develop environmentally friendly and efficient new insecticides for the field control of N.lugens,and to develop new enzyme formulations for the degradation of pesticide residue pollution in the environment.