Studies On Cytochrome P450-CYP4 Family And NADPH-cytochrome P450 Reductase In Nilaparvata Lugens

The brown planthopper,Nilaparvata lugens St?l(Hemiptera: Delphacidae)is the most destructive pest of ricein Asia.The humidity in the living environment of N.lugens varies greatly.The relative humidity of paddy field is very high.During migration,the high-altitude environment is complex and changeable and the humidity changes more dramatically.Therefore,the waterproofing and water-retention functions of the N.lugens integument are crucial for development and migration.The insect cuticle is covered by a thin layer of hydrophobic lipids,which forms a barrier to prevent the movement of water.Cuticle hydrocarbons(CHC)play a major functional role,reducing water permeability of insect cuticle by approximately 92%.The last step of hydrocarbon synthesis is mainly catalyzed by the enzymes of CYP4 G subfamily and a cytochrome P450 reductase(CPR).Previous studies have identified two genes belonging to the CYP4 G subfamily,which wereinvolved in the synthesis of cuticle hydrocarbons of N.lugens.As the CYP4 G subfamily occured gene duplications during evolution,the other P450 enzymes function in hydrocarbon synthesis may present in N.lugens but remain undiscovered.In addition,current studies on insect CPR genes were limited to the resistance mechanisms to pesticides,the function of CPR genes involved in synthesis of cuticle hydrocarbons has not been understood yet.The function of other P450 genes related to the synthesis of the lipid layer on the cuticle of N.lugens,especially the synthesis of hydrocarbons,is still unclear.In this study,we reported the waterproof and water-retention functions of CYP380C10 and CPR genes by searching the genome and transcriptome database of N.lugens,conducting the phylogenetic analysis and spatial-temporal expression pattern analysis,combined with experiments of RNA interference(RNAi),morphological observation,desiccation tolerance,oviposition analysis,scanning electron microscope(SEM)and Gas ChromatographyMass Spectrometry(GC/MS).The mainly results were as follows:1.CYP380C10 gene of brown planthopper CYP4 family is the ancestor of CYP4 G subfamily.In this study,alignment and phylogenetic relationship analysis of the deduced amino acid sequences of CYP380C10 gene and the CYP4 G subfamily genes revealed that CYP380C10 and CYP4 G subfamily protein contained the conserved activity domains.It suggests that CYP380C10 is an intermediate type during the evolution from CYP4 C to CYP4 G,and an ancestral gene of the CYP4 G subfamily.In addition,the spatial-temporal expression pattern analysis determined that CYP380C10 gene was highly expressed in the newly laid eggs,newly hatched nymphs,newly molted nymphs and newly emerged adults,and showed a specifically high expression level in integuments.2.CYP380C10 gene of N.lugens provides important waterproofing and waterretention functions.In this study,based on the transcriptome database of N.lugens,we synthesized the ds RNAs of 18 genes in the CYP4 family of N.lugens,and used RNAi to knockdown the transcription levels of these genes individually.Except previously reported CYP4G76 and Nl CYP4G115 genes that had lethal phenotype after knockdown,the RNAi results showed that the expression silence of CYP380C10 gene also generated lethal phenotypes.The insects were unable to molt,or lost the waterproof function as observed in the curticles retaining by water droplets or honeydew.In addition,the insects lost the water-retention function.Further analysis using SEM and GC/MS showed that the lethal phenotypes of N.lugens were caused by the destruction of the lipid layer on the body surface and the reduction of CHC content.In addition,this study revealed that there are different functions between the CYP380C10 and CYP4 G subfamily genes of N.lugens.Silencing CYP4G76 gene did not affect the waterproof function of N.lugens curticle.This observation indicates that CYP380C10 gene did not miss its functions in CHC biosynthesis during evolution.CYP380C10 plays the more important role than CYP4G76 gene.3.CPR gene of N.lugens is necessary to the biosynthesis of cuticle hydrocarbons and the development of reproductive system.In this study,spatialtemporal developmental-stage expression analysis determined that CPR gene was highly expressed in eggs,the first-instar nymphs and adults.Tissue expression analysis revealed that CPR gene had the the highest expression levels in ovaries and oocytes,indicating that CPR gene is involved in the maturation of oocytes and the development of ovaries,thus plays an important role during the development of oocytes.RNAi combined with morphological observation,desiccation tolerance,oviposition analysis,SEM and GC/MS analysis revealed that the reduction of CPR gene transcription level led to changes of the CHC content of N.lugens.The total CHC contents decreased,and the proportion of each CHC component in the total CHC also were changed,which caused thinning and destruction of the lipid layer in N.lugens curticle.As a result,the CPR gene knockdown led to the waterproofing and water-retetion function loss of N.lugens cuticle,and impaired the ovary development and the maturation of oocytes of N.lugens female adults.In this study,a N.lugens cytochrome P450 gene CYP380C10 was found as the intermediate type during the evolution from CYP4 C to CYP4G;revealed CYP380C10 that and CPR gene participated in CHC biosynthesis and the waterproofing and waterretetion function of N.lugens cuticle;indicated CPR gene is essential in the ovary development and the maturation of oocytes of N.lugens.These results enriched the understanding of insect CHC biosynthesis pathway and the biological function of CPR gene.