Genome-wide Analysis Of Cytochrome P450 And The Functions Of CYP4G Subfamily Genes In Bactrocera Dorsalis(Hendel)

Cytochrome P450(P450),an important class of detoxification enzymes,is involved in the feeding,growth,development,reproduction and defence in insects by metabolizing endogenous and exogenous substances and plays essential roles in adaptation of insects to environmental circumstances and insecticide resistance.CYP4 G,one of the P450 subfamilies originating from land colonization period of insect ancestors,exhibits important physiological function of the biosynthesis of cuticular hydrocarbon(CHC)to maintain water balance and thus is crucial for adaptation to low humidity.Recent reports revealed that function differentiation of CYP4 Gs occurred in some insects,probably associated with insect development.However,the mechanism is still unclear.Bactrocera dorsalis is infamous for the substantial economic losses it causes on a variety of commercial fruits and vegetables worldwide.This pest possesses a strong environmental adaptation capacity and is expanding to high latitude and low humidity areas,suggesting a strong adaptation capacity to dry environment in the species.Therefore,to explore the relationship between CYP4 G and low-humidity adaptability in B.dorsalis is important to decipher the mechanism of rapid expansion of B.dorsalis and to develop efficient control strategy for it.Additionally,CYP4 G genes evolve new function in insect development and some members may be potential control targets for the pest.In the current study,P450 genes were systematically identified at genome-wide levels and their spatiotemporal expression patterns were analyzed;a series of molecular techniques including RNAi,RNA-seq,GC/MS,and digital microscope system are utilized comprehensively to explore the conserved function of CYP4G100 in CHC biosynthesis;to establish the grading standards of intrapupae on the basis of systematical description about intrapuparial developmental morphology;to clarify the molecular mechanism of CYP4G188-mediated molting hormone in the pupal-adult apolysis of B.dorsalis.The main results were as follows: 1 Genome-wide identification and expression-profiling analyses of P450 genes in B.dorsalisBased on the whole genome and transcriptome databases of B.dorsalis,a total of 101 putative P450 genes were identified.All five P450 domains(Helix-C,Helix-I,Helix-K,PERF,and the Hemebinding motif)can be found in these 101 P450 s.Generally,all 101 P450 genes were divided into four major clans,CYP2,CYP3,CYP4,and Mitochondrial(mito).These four clans could be further divided into 25 families and 57 subfamilies with the largest subfamily consisting of 31 genes and smallest one of only 1 gene.Localization analysis of P450 s in genome scaffolds found that 59 genes existed on scaffolds in gene clusters,suggesting gene amplication from duplication occurred during P450 evolution in B.dorsalis.Subsequently,expression patterns of these P450 s in development stages and tissues were conducted by qPCR(real-time quantitative PCR).More than half P450 s were highly expressed in adults;some P450 s were highly expressed in larvae and pupae;only 9 P450 s exhibited high expression levels in eggs.A majority of P450 s were abundant in the metabolic tissues(fatbody,midgut and malphagian tubules),and a few P450 s were ovary-specifically highly expressed.Three CYP4 G members(CYP4G100,CYP4G101 and CYP4G188)were identified from B.dorsalis genome and their cDNA sequences were cloned.They all have the unique insertion sequence of CYP4 G subfamily genes.The subsequent phylogenetic analysis showed that all insect CYP4 Gs can be divided into two clusters.CYP4G100 and CYP4G101 were in the same cluster and they exhibited similar expression patterns.Both were highly expressed before emergence and in adults with extremely low expression levels of CYP4G101 recorded.CYP4G188 was in another cluster and specifically expressed in the mid-term pupae(5-day-old)and it meant CYP4G188 may evolve novel function.2 CYP4G100 participated in the cuticular hydrocarbons biosynthesis of B.dorsalisCYP4G100 was significantly up-regualted upon desiccation treatment(RH<10%).The transcript silence of CYP4G100 by RNAi impaired the biosynthesis process of CHC and almost all CHC contents were significantly reduced.Meanwhile,the substrates of CHC synthesis,glycerides were increased significantly in B.dorsalis.Besides,water loss and mortality of B.dorsalis were increased significantly under desiccation stress condition after CYP4G100 RNAi.All these results indicated that CYP4G100 affected the adaptability of B.dorsalis to desiccation by participating in the biosynthesis of CHCs.3 CYP4G188 involved in the pupal-adult apolysis of B.dorsalisCYP4G188 was specifically highly expressed at the fifth day of intrapuparial stage,suggesting that CYP4G188 was related to the intrapuparial development.After comprehensive observations and descriptions about morphological features of intrapupae at different day ages in B.dorsalis,the intrapuparial stage of B.dorsalis could be divided into six substages: post-feeding larval,larval-pupal apolysis,cryptocephalic pupal,phanerocephalic pupal,pharate adult,and emergence adult stages based on the changes of larval-pupal apolysis,head evagination,pupal-adult apolysis and compound eye color.Using High throughput RNA sequencing,those genes with opposite and similar expression patterns to CYP4G188 were screened.Gene Ontology and KEGG analysis showed that CYP4G188 was probably involved in pupal-adult apolysis.Based on the successful establishment of RNAi technique by injection in intrapupae,knockdown of CYP4G188 in pupae led to the up-regulation of 20-hydroxyecdysone(20E).Moreover,compared to control,the pupal-adult apolysis was finished earlier and the color changes of compound eyes were observed earlier as well.However,RNAi of CYP4G188 exhibited no significant effects on the emergence time and rate of B.dorsalis.Furthermore,20 E injection led to the phenotype same as RNAi treatment.4 Heterogeneous expression and enzymatic activity of CYP4G100 and CYP4G188Prokaryotic(Escherichia coli)and eukaryotic(Bac-to-Bac)expression systems were utilized to study the catalytic activity of recombinant CYP4G100 and CYP4G188 proteins.Soluble proteins of CYP4G100 and CYP4G188 were obtained successfully from host cells Rosetta(DE3)by using the prokaryotic expression vector p-Cold II.However,CO-difference spectrum analysis showed that these recombinant proteins showed no the typical absorption at 450 nm.Besides,no soluble protein was obtained using Bac-to-Bac eukaryotic expression system(pFast-HT A vector).In summary,CYP4G100 exhibited the conserved function of CYP4 G subfamily and it affected the adaptability of B.dorsalis to desiccation condition by regulating the CHC biosynthesis.In contrast,CYP4G188 was involved in the pupal-adult apolysis of B.dorsalis by regulating 20 E titer negatively.The current results provide valuable data for understanding the mechanism how B.dorsalis adapt to desiccation condition and slowdown the expansion of B.dorsalis.CYP4G188 is involved in the metamorphosis of B.dorsalis and therefore it can be considered as a potential target to control this pest.Besides,the classification standard of intrapuparial development of B.dorsalis provides technical index for accurate prediction of adult emergence and control guide.