Cytochrome P450 Enzyme System And Its Functions In Malathion Resistance In The Oriental Fruit Fly,Bactrocera Dorsalis(Hendel)

The oriental fruit fly,Bactrocera dorsalis,is an important economic pest in tropic and subtropic regions,causing a huge loss in fruits and vegetables.The oriental fruit fly has been resistant to common insecticides because of the long-time pressure from using those insecticides.Previous studies showed that cytochrome P450 s played an important role in the resistance to organophosphate insecticides in insects.Cytochrome P450 s in the oriental fruit fly are associated with the resistance to malathion based on the synergistic effects and assays of enzyme activities.In order to understand the metabolic resistance mediated by P450 s in the oriental fruit fly,the current study cloned the c DNAs of P450 from this pest and characterized the sequences of those cloned P450 s.q PCR was used to analyze the expression levels of P450 s and their responses to exogenous compounds in this pest.NAPDH-cytochrome P450 reductase was cloned from the oriental fruit fly.Relative levels of Bd CPR were analysed by q PCR and its functions in malathion resistance were explored by RNAi and eukaryotic expression system.Using RNA-Seq,libraries of malathion resistant and susceptible strains were established and levels of genes were compared between the libraries,identifying a number of genes associated with malathion resistance.Finally,one P450 gene from the libraries was analyzed using RNAi and eukaryotic expression system.These studies will provide a solid foundation for the mechanisms researches on metabolic resistance in the oriental fruit fly.The main results in the thesis are as follow:1 Cloning and sequence analyses of P450 s from B.dorsalisUsing degenerate primers,RNA-Seq data and RACE PCR,38 P450 s containing full-length open reading frames(ORF)were cloned and identified from B.dorsalis.These P450 s come from four different clans,including 14 families and 27 subfamilies.CYP2,CYP3,CYP4 and mitochondrial clans have 2,19,11 and 6 P450 s,respectively.Among the genes,11 P450 s belong to CYP4 family,12 are in CYP6 family,4 are in CYP12 family,and one P450 in each of the remaining 11 families.The lengths of the ORFs are between 1,350 and 1,665 nucleotides,encoding 489-554 amino acids.The molecular weights of those encoded proteins are from 57 to 64 k Da,with theoretical isoelectric points from 5.88 to 9.62.Predictions based on the sequences showed that 32 out of the 38 P450 s are microsome P450 s,and the other 6 are mitochondrial P450 s.38 P450 s of B.dorsalis were named by the Cytochrome P450 Nomenclature Committee,namely CYP4AC4,CYP4AD1,CYP4D46,CYP4D47,CYP4D48,CYP4E9,CYP4G100,CYP4G101,CYP4P5,CYP4P6,CYP4S18,CYP6A41,CYP6A48,CYP6A49,CYP6A50,CYP6A61,CYP6A62,CYP6D9,CYP6D12,CYP6EK1,CYP6G6,CYP6G8,CYP6GX1,CYP9F6,CYP12A18,CYP12B3,CYP12C2,CYP12N1,CYP28F1,CYP302A1,CYP304A1,CYP305A1,CYP309B1,CYP310C1,CYP314A1,CYP317B1,CYP437A3,and CYP3074A1.They were submitted to Gen Bank,with accession numbers as ADC44464,KX708477,ADC44459,ADP22308,ADP22303,ADP22302,KX708478,KX708479,ADC44461,AFH54172,KX708480,ADP22304,KX708481,KX708482,KX708483,KX708484,KX708485,ADO24531,KX708486,ADP22309,KX708487,KX708488,KX708489,KX708490,KX708491,KX708492,AFH54190,AFH54188,AFH54196,AFH54200,KX708493,KX708494,KX708495,AFH54207,AFH54210,KX708496,KX708497 and KX708498,respectively.Multiple sequences alignment showed that the identities of each pair of 11 CYP4 genes are from 25% to 67%,and the identities from 26% to 60% for 12 CYP6 genes.P450 s from the same family share higher identities,but have lower identities when they are from different families.These P450 s have the conserved domains in P450 superfamily,including Helix C,Helix I,Helix K,Meander and heme-binding domain.P450 s of B.dorsalis share high identities,79%-97%,74%-95% and 70%-94% with their homologous genes in B.oleae,B.cucuribitae,and Ceratitis capitata,respectively.They also have high identities,from 35% to 78%,with the homologous P450 s in Drosophila melanogaster.Phylogenetic analyses showed that 38 P450 s of B.dorsalis and 87 P450 s of D.melanogaster clustered into four different branches,representing four different clans.CYP3 and CYP4 clans have more members.Genes from same families and those homologuos P450 s are closer in the phylogenetic tree.2 Expression levels of P450 s in B.dorsalisUsing q PCR,the study analysed the expression levels of P450 s of B.dorsalis among different geographical populations,developmental stages,tissues,females and males,and their midguts.Among Yunnan,Hainan,Dongguan and Guangzhou populations,CYP6P5 has higher levels in Dongguan and Guangzhou populations than in the other two populations.The levels of CYP6EK1 are higher in Guangzhou population than the other three ones.The levels of the other six tested P450 s have no difference among these populations.We collected the data about P450 s from the digital gene expression profiling(DGE)at different developmental stages.Each P450 has 1 to 4 fragments in DGE and those fragments of the same gene own similar expression patterns during development,which are similar with the q PCR data.Eight tested P450 s have higher levels in larvae and adults,followed by pupae and eggs.CYP4D46,CYP4D47 and CYP4D48,from the same subfamily,have different expression levels during development.Tissues like midguts,fat bodies and Malpighian tubules were used to test the different expression levels of P450 s.The levels of CYP4D46,CYP4D47 and CYP4D48 were highest in midguts,CYP4AC4 and CYP4E9 were highest in fat bodies,and CYP4P5 and CYP6EK1 were highest in Malpighian tubules.CYP6A41 had similar expression levels among these three tissues.No difference was detected in the expression levels of fifteen tested P450 s between 3-d-old females and males.However,CYP12N1 had lower levels,as low as 0.28-fold,in the midguts of 3-d-old females than males at the same age.Moreover,the responses of P450 s to exogenous compounds were tested using q PCR.None of fifteen tested P450 s changed their expression levels in the adults treated with LD50 of beta-cypermethrin for 24 hours.Differently,the levels of CYP4AC4,CYP4D46,CYP4P6 and CYP302A1 decreased in the midguts of those treated adults.Mortalities of adults were 2% and 30% when adults were feeded with the food containing 3 g/L and 10 g/L citral,respectively.Interestingly,the levels of CYP4D46 decreased in the adults feeded with 3 g/L citral,and CYP12N1,CYP302A1 and CYP314A1 decreased in the adults feeded with 3 g/L and 10 g/L citral.However,CYP28F1 increased in the adults feeded with 3 g/L citral,and CYP4D48 and CYP6D9 increased after a feed with 10 g/L citral.3 Functional characterization of NADPH-cytochrome P450 reductase in B.dorsalisIn this study,two transcripts Bd CPR-X1 and Bd CPR-X2 of B.dorsalis CPR(Bd CPR)were cloned.The ORFs of Bd CPR-X1 and Bd CPR-X2 have 2,019 and 1,674 bp,encoding 672 and 557 amino acids.The molecular weights of deduced proteins of Bd CPR-X1 and Bd CPR-X2 were 76 k Da and 64 k Da,with theoretical isoelectric points 5.51 and 7.23,respectively.The C terminal regions of two transcripts are the same,but their N terminal regions are different.Bd CPR-X1 contains three conserved functional domains FMN,FAD and NADPH,and N-terminus membrane anchor.Bd CPR-X2 contains FMN(incomplete),FAD and NADPH,but no N-terminus membrane anchor.The identities between Bd CPR and CPRs from other species are very high and Bd CPR shared more than 90% identity with B.cucuribitae CPR(Bc CPR)and C.capitata CPR(Cc CPR).The phylogenetic analysis showed that Bd CPR was close to Bc CPR and Cc CPR.Bd CPR-X1 is the main CPR existed in B.dorsalis.Bd CPR-X1 expressed highly in adults and lowly in pupae.The levels of Bd CPR-X1 were similar between 3-d-old females and males,but were higher in 9-d-old males than females.Bd CPR-X2 expressed similarly between 3-d-old females and males,and 9-d-old females and males.The levels of Bd CPR-X1 were similar among head,thorax and abdomen.Bd CPR-X1 expressed highly in midguts,fat bodies and Malpighian tubules,but lowly in reproductive organs in females and males.Distributions of Bd CPR-X2 were very similar to Bd CPR-X1.The levels of Bd CPR-X1 and Bd CPR-X2 were similar in malathion resistant strain compared to susceptible strain.ds RNAs of Bd CPR were synthesized and injected into 3-d-old adults.The levels of Bd CPR decreased 52% at 72 h post injection,while injection of PBS did not affect Bd CPR expression.The susceptibility to malathion,indicated by mortality,of adults injected with ds Bd CPR was significantly higher than in the control and in adults injected with PBS.Mortality of adults injected with ds Bd CPR reached 89.9%,while it was 45.0% and 50.5% in the control and in adults injected with PBS,respectively.To detect a functional protein encoded by Bd CPR,the complete ORF of Bd CPR-X1 was cloned in the p Fast Bac HT A vector.Western blot analysis of cell extracts with anti-His antibody showed that the cells successfully expressed either e GFP or Bd CPR.Bd CPR had a high level of cytochrome c reduction activity.MTT assay results revealed that the cells expressed with Bd CPR had slightly higher viability against different concentrations of malathion than the e GFP-expressing cells.4 Comparative transcriptome analyses of malathion resistant and susceptible B.dorsalisBased on the transcriptome datasets of B.dorsalis,we generated four DGE libraries for malathion resistant and susceptible adults and their midguts.We also generated two new transcriptome libraries for fat bodies of malathion resistant and susceptible adults.All libraries were in good quality,with more than 93% clean reads in raw reads.Expression levels of genes between resistant and susceptible adults,midguts from two strains,and fat bodies from two strains were compared respectively.Compared to susceptible adults(SWB),141 genes were up-regulated,and 278 genes were down-regulated in resistant adults(RWB).50 up-regulated genes and 988 down-regulated genes were identified in midguts of resistant adults(RMG),compared to midguts of susceptible adults(SMG).Similarly,349 genes were up-regulated and 1,129 genes were down-regulated in fat bodies of resistant adults(RFB)relative to fat bodies of susceptible adults(SFB).GO analyses of DEGs showed that these DEGs are in different biological processes and cellular components,with different molecular functions.These DEGs involved in different pathways,with the most genes in metabolic pathway.Two P450 genes CYP6G6 and CYP6A2-like,and a GST gene were up-regulated in RWB compared to SWB,with levels up to 9.8,2.2 and 2.8-fold,respectively.4 P450 s and 2 Car Es were down-regulated in RWB.In RMG,5 GSTs,2 Car Es and 15 P450 s were down-regulated.Differently,15 P450 s had higher levels in RFB than in SFB and were up-regulated to 2.3-4.3-fold.These genes were CYP6G6(4.3-fold),CYP6A61(4.0-fold),CYP4E1-like(3.8-fold),CYP6G8(3.4-fold),CYP437A3(3.3-fold),CYP4D46(3.0-fold),CYP309B1(3.0-fold),CYP4AC4(2.9-fold),CYP12B3(2.0-fold),CYP6A13-like(2.8-fold),CYP12A4-like(2.7-fold),CYP4S18(2.6-fold),CYP6D9(2.4-fold),CYP12N1(2.4-fold),and CYP12A5-like(2.3-fold).Besides,cytochrome b5 and two GST genes were up-regulated to 2.1-,2.7-,and 2.8-fold,respectively.Another 6 P450 s were down-regulated in RFB.5 Over-expression of CYP6G6 might be associated with malathion resistance in B.dorsalisCYP6G subfamily is closely related with insect resistance.Amino acid sequence of CYP6G6 shared 47%,51% and 48% identities with CYP6G1,CYP6G2 and CYP6G4,respectively.Their substrate recognition sites SRS1-SRS6 had high similarities,with several conserved amino acids.q PCR results showed that the levels of CYP6G6 in malathion resistant strain were 5.2-fold to its levels in susceptible strain,which was similar to the data in DGE and transcriptome.Additionally,CYP6G6 was highly expressed in the heads of adults and fat bodies,without difference between females and males.CYP6G6 had low levels in reproductive organs in both females and males.The levels of CYP6G6 increased gradually from 3-d-old adults.dsCYP6G6 was injected into 3-d-old adults and the levels of CYP6G6 were checked at 24 h post injection.Compared to the reference gene ?-tubulin,the levels of CYP6G6 were 0.63,0.24 and 0.13 in control,adults injected with PBS and adults injected with ds CYP6G6,respectively.Mortality of each group was 28.4%,33.2% and 39.6%,with a slightly increase in the adults injected with ds CYP6G6.Because of the unstability of different biological repeats,there was no significant difference among groups.CYP6G6 was cloned into p Fast Bac HT A vector and expressed in Sf9 cells.Recombinant proteins were isolated and tested with CO difference spectrum assay,with the absorbance peak at 420 nm rather than 450 nm.There was no absorbance peak in the control.Thus,CYP6G6 expressed in the cells,but with no P450 enzyme activity.