Studies On The Function And Regulation Of Three Cytochrome P450 Genes Involved In Endogenous Metabolism In Locusta Migratoria

Cytochrome P450 monooxygeneases(CYPs)belong to a widely distributed gene superfamily and play major roles in metabolism of endogenous hormones and xenobiotics.Several conserved CYPs have been shown to be essential in insect growth and development.Sequencing of insect genomes has revealed large numbers of CYP genes in many species.The migratory locust(Locusta migratoria)is an important agricultural pest in the world,and at least 78 cytochrome P450 genes have been identified.However,the current research mainly focuses on the mechanism of exogenous metabolism and detoxification,and there is almost no research on its roles in endogenous metabolism.Therefore,it is very vital to study the physiological functions and molecular regulatory mechanisms of cytochrome P450 genes involved in endogenous metabolism of migratory locusts.In this paper,we deeply explored the physiological functions of the two Lm CYP4 G genes and Lm CYP303A1 genes and the regulation mechanism of mi RNA on Lm CYP303A1.The main contents are as follows: 1.Functional study on Lm CYP4G62 and Lm CYP4G102 in lipid metabolismRT-q PCR and Western blot were used to analyze the expression levels of the two CYP4 G genes of locusts in different developmental days and tissues,and the results manifested that Lm CYP4G62 was mainly expressed in the middle stage,whereas Lm CYP4G102 was expressed in all stages;but both Lm CYP4 Gs were highly expressed in the integument and fat body.Immunohistochemical localization results showed that Lm CYP4G62 and Lm CYP4G102 were both expressed in oenocytes.However,the two enzymes had a different subcellular distribution,with Lm CYP4G62 localized on the plasma membrane and Lm CYP4G102 dispersed throughout the oenocyte cytoplasm;RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents,in all classes for Lm CYP4G102,but mostly shorter chain CHCs for Lm CYP4G62.Silencing of both genes resulted in increased insecticide penetration through the cuticle,and increased locust susceptibility to desiccation and insecticides.Our studies suggest that both Lm CYP4G62 and Lm CYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration.2.Molecular characteristics and functional study on Lm CYP303A1RT-q PCR was used to analyze the expression of Lm CYP303A1 gene at different developmental stages and tissues in L.migratoria,and the results showed that Lm CYP303A1 had a high expression level before ecdysis and was highly expressed in integument,wing pads,foregut and hindgut;silence Suppression of Lm CYP303A1 expression by RNA interference(RNAi)caused a lethal phenotype with molting defect from nymph to nymph and nymphs to adults;In addition,knockdown of Lm CYP303A1 efficiently suppressed the transcript level of key genes responsible for cuticular hydrocarbons(CHCs)synthesis,which led to a decrease in some CHC levels;Furthermore,Lm CYP303A1 RNAi resulted in locusts being more susceptible to desiccation and to insecticide toxicity;Meanwhile,silencing of Lm CYP303A1 inhibited the degradation of old cuticle and synthesis of new cuticle,and affected the expression of epidermal protein and chitinase.3.Study on the biological function of Cyp303a1 in DrosophilaIn order to further study the conservation of CYP303A1 in different insect species,we studied the physiological functions of Cyp303a1 in Drosophila.It was been found that Cyp303a1 was mainly expressed at the second day in the pupal stage and the ring glands in the white prepupa stage;Knockdown of Cyp303a1 transcripts by RNAi using the Gal4/UAS system with a ubiquitous driver(tubulin-Gal4)and epidermal driver(69B-Gal4)in Drosophila resulted in severe defects in eclosion and lethality during and after adult emergence.Wing-specific silencing of Cyp303a1 also resulted in the failure for wing extension;Stage-specific RNAi of Cyp303a1 using Gal80 ts for thermal-dependent-suppression found that the expression of Cyp303a1 at the middle pupal stage was absolutely required.Furthermore,Cyp303a1 mutants exhibited more than 80% lethality at the late embryonic development stages.Embryonic lethality of the Cyp303a1 mutants was fully rescued by the ubiquitous overexpression of Drosophila Cyp303A1,but it was not rescued by the ubiquitous overexpression of Lm CYP303A1.The result indicates that Drosophila Cyp303a1 is indispensable for embryonic development.CYP303A1 has some functional differences during embryonic development in Drosophila and locust.4.Regulation mechanism of mi RNA on Lm CYP303A1 in locustThe bioinformatic prediction showed mi R-184 has a binding site in Lm CYP303A1.The RT-q PCR analyses showed that the negative correlation of the expression of mi R-184 and Lm CYP303A1.Furthermore,the luciferase assay in vitro and RNA immunoprecipitation(RIP)analysis showed that mi R-184 is directly interacted with Lm CYP303A1.The locusts treated with mi R-184 agomir or antagomir resulted in overexpressing or silencing mi R-184 and displayed a distinct molting defect phenotype,compared with the control group.It was consistent with the results of silencing Lm CYP303A1.The results suggested that mi R-184 regulates the expression of Lm CYP303A1,which makes a deformed molting and final death in locust.In summary,we systematically studied the physiological functions of the three cytochrome P450 genes involved in the endogenous metabolism of L.migratoria and the regulatory mechanism of mi RNA on the locust Lm CYP303A1.These research results not only provide new contents for the related research of paurometabola insects,but also help to find new targets for pest control and insecticide resistance.