Molecular Characterization And Functional Analysis Of Three Cytochrome P450Genes From Locusta Migratoria

Locusta migratoria is one of major agricultural pests in China, and its plague is a serious threat to agricultural production. At present, the main control method is the use chemical insecticides. The extensive uses of insecticides has led to the development of insecticide resistance in certain locust populations. Cytochrome P450’s belong to a major class of detoxification enzymes in insects, and are often responsible for or contribute to insecticide resistance in insects. Currently, that the known P450genes associated with insecticide resistance are mainly in the gene families of CYP4, CYP6and CYP9. This study focused on molecular characterization and functional analysis by RNAi of three cytochrome P450genes from L. migratoria.1. cDNA cloning and sequence analysis of three cytochrome P450genes from the locustBased on the locust expressed sequence tag (EST) database and transcriptome, we searched putative cytochrome P450genes by bioinformatics approaches and identified three sequences from CYP4, CYP6and CYP9families. Bioinformatics analysis showed that two sequences from CYP6and CYP9families already had their full-length cDNA sequences with conserved regions, whereas the sequence from CYP4family was a partial sequence, which required further sequencing after the5’ end of this gene was amplified by RACE. The full-length cDNA sequences of the three cytochrome P450genes were verified by further PCR amplifications of their full-length cDNA’s followed by DNA sequencing. These three genes were then named as LmCYP4G62, LmCYP6ELl and LmCYP9AQl by International Cytochrome P450Nomenclature Committee.The cluster analysis was performed among the three locust P450genes and other CYP4, CYP6and CYP9genes from Drosophila whose annotation of CYP genes has been completed. Specifically, the LmCYP4G62cDNA contains1992nucleotides, including an open reading frame (ORF) of1662 nucleotides that encode554amino acid residues,66nucleotides at5’-untranslated region (5’-UTR) and264nucleotides at3’-untranslated region (3’-UTR). The LmCYP6ELl cDNA contains2103nucleotides, including an ORF of1530nucleotides that encode510amino acid residues,84nucleotides at5’-UTR and489nucleotides at3’-UTR. The LmCYP9AQl cDNA contains2103nucleotides, including an ORF of1578nucleotides that encode526amino acid residues,57nucleotides at5’-UTR and935nucleotides at3’-UTR. The molecular masses of the three genes were predicted to be63.5,58.0and60.4kDa, respectively. Their predicted isoelectric points were6.80,6.84and7.20, respectively.2. The expression patterns of three cytochrome P450genes in locusts The expression of the three cytochrome P450genes in different tissues in locusts was analysed by real-time quantitative PCR. Our results displayed that LmCYP4G62was mainly expressed in muscle and fatbodies; LmCYP6EL1was mainly expressed in the midgut, gastric caeca and fatbodies, whereas LmCYP9AQl was mainly expressed in the brain and fatbodies. The expression patterns of the three cytochrome P450genes were also analyzed at different development stages of the locust. Our results indicated that expression levels of LmCYP4G62and LmCYP9AQl were higher at nymphal stages than those at egg and adult stages. In general, the expression of these two genes increased gradually from the2nd to5th instar nymphs, but dramatically decreased in adult stage. In contrast, the relative expression of LmCYP6ELl was higher in egg stage than other stages. Futhermore, we investigated possible induction of these genes by different concentrations of two commonly used insecticides in locusts. Malathion can induce the expression of the three cytochrome P450genes in various degrees, whereas carbaryl showed no effect on the expression of the three cytochrome P450genes.3. Functional analysis of three P450genes in locustsTo further explore possible insecticide detoxification roles of the three cytochrome P450genes in locusts, RNA interference (RNAi) was performed followed by insecticide bioassay. Double-stranded RNA (dsRNA) was synthesized in vitro from each of the three genes and injected into the locusts. After the locusts were treated with an insecticide, the change in locust susceptibility to the insecticide was compared between the locusts injected with the cytochrome P450dsRNA and those injected with dsRNA of GFP. Our results showed that the mortalities of the locust nymphs injected with dsCYP6ELl and dsCYP9AQl were29.35and17.35%, respectively, higher than the controls when chlorpyrfos was used in our bioassays. The mortalities of the nymphs injected with dsCYP6ELl and dsCYP9AQ1were19.94and31.04%, respectively, higher than the controls when carbaryl was used in our bioassays. Furthermore, the mortalities of the nymphs injected with dsCYP4G62, dsCYP6ELl and dsCYP9AQl were26.88,33.78and20.53%, respectively, higher than the controls when deltamethrin was used in our bioassays.In summary, we sequenced and characterized the full-length cDNAs of three cytochrome P450genes (CYP4G62, CYP6EL1and CYP9AQ1) from L. migratoria. We determined the expression patterns of the three genes in different tissues and developmental stages. We also evaluated the transcriptional responses of the three genes when the locusts were exposed to commonly used insecticides. We further assessed the detoxification reloes of these genes by RNAi followed by insecticide bioassays. Our studies provide detailed information on the molecular characteristics and insecticide detoxification roles of these genes in the locust. All such information is expected to help with rational selection and application of insecticides for controlling of this agriculturally important insect pest.