Metabolism Of Haedoxan A By Three P450 Genes Of Aedes Aegypti And Their Catalytic Activity On Model Substrates

Aedes aegypti is an important vector of arbovirus diseases such as yellow fever and Zika,and its prevention and control mainly relies on pesticides.However,due to the long-term and extensive use of chemically synthesized insecticides,Aedes aegypti has developed serious resistance to them,damaging the environment and ecology.With the enhancement of people’s awareness of environmental protection,there is an urgent need for"efficient,environmentally friendly,safe and economical"green pesticides.Due to the strong environmental compatibility,environmental friendliness,and safety for humans and livestock,botanical pesticides have become the main source of alternative chemical synthesis insecticides.Haedoxan A（HA）is a highly insecticidal lignan compound with a bifuran ring skeleton extracted and isolated from the perennial herb Phyryma leptostachya L.Its insecticidal activity is comparable to the current commercial insecticides permethrin and indoxacarb.The research team conducted proteomic analysis of the HA-resistant Aedes aegypti strain（resistance increased by 20times）in the early stage and selected three P450 enzymes CYP9f2,CYP4g15,and CYP6a14 that may be related to HA metabolic resistance based on protein upregulation.However,it is unclear whether these three P450 enzymes can actually metabolize HA.Therefore,this article intends to determine whether Aedes aegypti CYP9f2,CYP4g15and CYP6a14 participate in the detoxification metabolism of HA through RNAi analysis,and find that CYP9f2 and CYP6a14 may have the role of metabolizing HA,while CYP4g15 may not participate in the metabolism of HA.The heterologous expression and in vitro enzyme activity determination of these three genes were further verified by using the insect cell baculovirus expression system to further verify their metabolic activity on the model substrate,and it was found that CYP9f2 enzyme had the highest activity against TMBZ,PNA and EC,followed by CYP6a14 enzyme,while CYP4g15 enzyme was basically inactive against PNA and EC.The main results obtained in this paper are as follows:1.RT-qPCR was used to detect the expression of three P450 genes in Aedes aegypti at different developmental stages.The results showed that the expression level of CYP9f2 gene was significantly different at different developmental stages.The expression level of CYP9f2 gene was pupal stage>3rd instar>adult stage>4th instar>1st instar;The expression level of CYP4g15 gene was 2nd instar>4th instar>adult>1st instar>pupa>3rd instar;The expression level of CYP6a14 gene was 1st instar>2nd instar>4th instar>3rd instar>pupa>adult.Bioinformatics analysis revealed that there are transmembrane domain domains at the N-terminal of CYP9f2、CYP4g15 and CYP6a14 proteins.2.A chitosan/ds RNA nanoparticle RNAi system was constructed,and the effects of CYP9f2、CYP4g15 and CYP6a14 genes of Aedes aegypti on the insecticidal activity of HA were verified using RNAi.The results showed that after feeding the test worms with ds CYP9f2 and ds CYP6a14 alone,and then treating them with HA,the 24-hour mortality rate increased by 31%and 32%compared to the undisturbed control,respectively,indicating that CYP9f2 and CYP6a14 may have the effect of metabolizing HA;However,feeding ds CYP4g15 alone and then treating with HA did not significantly increase the mortality of the test worms,indicating that CYP4g15 may not participate in the metabolism of HA.3.By heterologously expressing the NADPH cytochrome P450 reductase of Aedes aegypti,and using cytochrome C and K₃Fe（CN）₆ as model substrates,in vitro enzymatic measurements were carried out using the NADPH promoter reaction.The results showed that the enzymatic reaction of the NADPH cytochrome P450 reductase of Aedes aegypti against different model substrates conformed to the Michaelis equation,and its catalytic efficiency was 3-10 times higher than that of the control,indicating that its expression was successful and had catalytic activity,It can participate in the redox reaction of cytochrome P450 enzymes and play an electron transfer function.4.To construct an insect baculovirus expression system,Aedes aegypti cytochrome P450 reductase,CYP9f2、CYP4g15 and CYP6a14 were co expressed in sf9 cells.The quality（P420 enzyme ratio）and content of recombinant P450 enzymes in the enzyme solution can be detected by reduced CO differential spectroscopy.The results show that the three P450 recombinant expression proteins have absorption peaks at 450 nm,indicating that all three P450 genes can be expressed in sf9 cells.Subsequently,the metabolic activity of P450 enzyme against the model substrates TMBZ（tetranitrobenzidine）、PNA（p-nitroanisole）and EC（7-ethoxycoumarin）was analyzed.The results showed that CYP9f2 enzyme had the highest activity against TMBZ（peroxidation）、PNA（oxygen demethylation）and EC（oxygen demethylation）,followed by CYP6a14 enzyme,while CYP4g15 enzyme had no activity against PNA and EC.The above research work and results lay the foundation for further elucidating the mechanism of action of P450 enzymes on HA metabolism,and also provide reference for studying the metabolic function and mechanism of P450 enzymes on other exogenous substances such as pesticides in insects.