Molecular Characterization And Functional Analysis Of Cytochrome P450S In Locusta Migratoria

Cytochrome P450monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. The diverse functions of the P450superfamily primarily were due to the diversity of cytochrome P450genes. In insects, more than1000cytochrome P450genes have been identified and the number is increasing with the sequencing of new insect genomes. Locusta migratoria is a world-wide agriculture pest, now as a model, it has been used for the study of insect physiology, biochemistry, developmental and immue and so on. In this study, we carried out a genome-wide analysis of cytochrome P450genes to identify putative cytochrome P450genes and characterize their expression responses to insecticide exposures, their detoxification roles by using RNA interference (RNAi) to silence each of eight genes followed by insecticide bioassays. Our findings shed new light on functional importance of each of cytochrome P450genes in the detoxification of insecticides.1. Identification of cytochrome P450-like genes from Locust EST databases and characterization of these genesWe carried out a genome-wide analysis to identify putative cytochrome P450genes and characterize their expression responses to insecticide exposures.15cytochrome P450-like genes were identified from locust EST database (LocustDB). Reverse transcription PCR (RT-PCR) analysis showed that most cytochrome P450-like genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgut, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450was differentially affected by three different insecticides. Deltamethrin caused significant inductions in12h at LD30(dose to kill30%of the tested individuals) in the nymphs, whereas malathion and carbaryl did not have significant effect on cytochrome P450enzyme activity. Further RT-PCR analysis showed significant increases of transcriptions of several cytochrome P450genes in deltamethrin treated locusts. Thus, the increased cytochrome P450enzyme activity is likely due to increased transcriptions of multiple cytochrome P450genes in response to deltamethrin exposures. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels.2. Cloning of cytochrome P450genes from L. migratoriaTwo full-length cDNAs putatively encoding cytochrome P450proteins were sequenced by using5' and3' RACE with the primers designed based on the two putative cytochrome P450cDNA fragments from LocustDB. These cytochrome P450genes were named CYP409A1and CYP408B1by the P450Nomenclature Committee. Based on the database of transcriptome of locust, another six complete cDNA sequences were obtained and verified with the coding region of each gene by RT-PCR. They were named CYP6FD1, CYP6FD2, CYP6FE1, CYP6FF1, CYP6FG1and CYP9AQ2, respectively. Comparisons of the deduced amino acid sequences of these eight P450genes with those of cytochrome P450genes from D. melanogaster indicated that these new cytochrome P450genes of L. migratoria fall into the CYP3clan that includes CYP6, CYP9, CYP28, CYP308-310and CYP317families, which was associated with the metabolism of insecticides and other xenobiotics.3. Stage-dependent and tissue-specific expression patterns of cytochrome P450genes from L. migratoriaThe expression patterns of eight cytochrome P450genes at different developmental stages of the locust were examined in whole body by using real-time PCR. Among the eight genes, three genes (CYP409A1, CYP6FG1and CYP9AQ2) showed significantly fewer expressions in eggs than other developmental stages. No expression was detected in eggs of three genes (CYP6FD1, CYP6FD2and CYP6FE1). The expression of CYP6FD1and CYP6FE1was relatively high in the fourth-instar nymphs and CYP6FD2predominantly expressed in adult. CYP408B1and CYP6FF1transcript can be detected and remained to be highly expressed in all developmental stages.The relative expression of eight cytochrome P450genes was determined in different tissues of the fifth-instar nymphs of L. migratoria by using real-time PCR. The relatively high expression of CYP409A1was observed in the Malpighian tubules, followed by the gastric caecum and fatbodies of the nymphs, but its expression was relatively low in the muscle and hemolymph. The expression of CYP408B1was relatively high in the foregut, hindgut, fatbodies, muscle and brain. CYP9AQ2was expressed in all tissues examined except for hemolymph. The expression of five genes of CYP6family was higher in the fatbodies and Malpighian tubules of the nymphs than other tissues.4. Expression response of cytochrome P450genes of L. migratoria to deltamethrin exposuresReal-time quantitative RT-PCR showed that deltamethrin influenced the expression of cytochrome P450genes, ranging from induction to repression. Deltmethrin at the lower dose induced the expression of CYP409A1, CYP6FD2, CYP6FF1and CYP6FG1by1.7-to2.0-fold. Deltamethrin at all tested concentrations increased gene expression of CYP9AQ2by2.26-to2.55-fold (P<0.05). Exposures of deltamethrin at the concentrations of LD10, LD30and LD50for12h did not show significant effect on the expression of the CYP408B1and CYPFE1. However, exposures of deltamethrin at the concentration of different doses for12h suppressed the expressions of CYP6FD1.In contrast, the transcript levels of eight cytochrome P450genes varied among different insecticide exposure times. For example, the six genes were induced at various levels in deltamethrin treated locusts as compared with acetone treated control locusts. Significant increases (1.2-to6.7-fold) were observed when the locusts were treated with deltamethrin at all the exposure times. However, deltamethrin at the concentration of LD10suppressed the expression of the CYP408B1and CYP6FD1in L. migratoria.5. The function analysis of cytochrome P450from L. migratoria by RNAiThe nymph mortalities after deltamethrin treatment increased16.7to27.1%after CYP409A1, CYP408B1, CYP6FF1and CYP9AQ2were silenced. The nymph mortalities after carbaryl treatment increased40.6%and34.7%after CYP6FD2and CYP6FE1were silenced. Neither malathion nor DDT showed significant changes in nymph mortalities after all the seven P450genes were silenced. These results indicated different P450genes were involved in different insecticides detoxification in the locust. As validated by RNAi followed by insecticides bioassay, CYP409A1, CYP408B1, CYP6FF1and CYP9AQ2played a significant role in conferring deltamethrin detoxification, and CYP6FD2and CYP6FE1were involved in carbaryl detoxification.6. Heterologous expression of cytochrome P450genes from L. migratoriaIn the present study, we want to construct the heterologous expression of cytochrome P450genes, which can provide direct evidence to understand the P450genes function. The results showed than we constructed prokaryotic expression vectors, but the recombinant proteins of pET28a-CYP409Al and pET28a-CYP408B1were not successfully expressed in E. coli.In summary, we carried out a genome-wide analysis of cytochrome P450genes of the locust to identify putative cytochrome P450genes and characterize their expression responses to insecticide exposures. We identified15cytochrome P450-like genes from LocustDB. In order to study the molecular characteristics of these genes, we performed different tissue and developmental stage expression analysis by RT-PCR. Based on these studies, the full-length cDNAs were carried out by RACE-PCR and the sequences were performed using bioinformatics methods. For the eight new genes, we investigated heterologous expressions We performed real-time PCR to evaluate the response of the new genes to insecticide exposure. We used RNAi to analyze their detoxification roles and their functional importance of each of the new genes in the detoxification of insecticides. These reseach may provide necessary genetic information to assess potential consequences of exposure in different insecticides and help us better understand the functions of the insect cytochrome P450genes and their interactions with insecticides at molecular levels, which may facilitate the development of new strategies for pest control.