Cytochrome P450s From The Locusta Migratoria: Responses To Plant Allelochemicals And Pesticides

cytochrome P450s (or CYP) is an old family with a variety of biological functions, existing in a lot of organisms. P450s are important detoxification enzymes in insects involved in insecticide resistance. In addition, They might be involved in the biosynthesis pathway of endogenous compounds, such as molting hormone (20-hydroxyecdysone) and juvenile hormone (JH), as well as catalysis hydroxylation of fatty acids. NADPH-Cytochrome P450 Reductase (CPR) is an important content of cytochrome P450 Enzyme system, and it transfer electrons from Nicotinamide Adenine Dinucleotide Phosphate (NADPH) to the P450 heme center.in this study, regard Locusta migratoria as objects,we focus on the tissue-specificity and the induction of 11 drugs to P450 genes from Locusta migratoria. In addition, we investigate the molecular characterization, including expression patterns and inductive effect of LmCPR. This research enrich the relationship between the insect resistance and plant secondary metabolites. It also provides theory basis to effective prevention and control of the locusta and agricultural planting structure adjustment.1. Tissue-specificity analysis of P450 genes from Locusta migratoria.All P450 genes we got from Locusta migratoria. and D. melanogaster were involved in the phylogenetic relationship analysis then we selected ten P450 genes dispersed in different sub-famies form Locusta migratoria, including LmCYP6HLl、 LmCYP6FE1、LmCYP6FF1、LmCYP6FG1、LmCYP4C73、LmCYP4C79、 LmCYP4C80、LmCYP4G62、LmCYP4FD1 and LmCYP302A1. The expression of these genes in different tissues (antenna, foregut, midgut, hindgut, gastric cecum, malpighian tubule, fat body, spermary, ovary,muscle, Integument) in fifth-instar nymphy of Locusta migratoria. was analysed by real-time quantitative PCR. Quantitative tissue expression analysis showed that mRNA levels of P450s widely expressed in the different tissues of fifth-instar nymphy.LmCYP6FE1 showed relatively higher expressions in antenna; LmCYP6FEl and LmCYP6FF1 showed relatively higher expressions in malpighian tubule; LmCYP6EL1、LmCYP4C79、LmCYP4C80 and LmCYP4FDl showed relatively higher expressions in fat body; LmCYP4FD1 and LmCYP4C79 showed relatively higher expressions in midgut and gastric cecum; LmCYP302Al showed relatively higher expressions in ovary; LmCYP4G62 showed relatively higher expressions in integument.2.The induction of drugs to P450 genes from Locusta migratoriaThrid- instar nymphy were trated by 11 drugs, and relative expression of the ten P450 genes from Locusta migratoria were tested. Durgs including insecticides (carbaryl, chlorpyrifos, malathion), plant secondary metabolites (indole, indole-3-carbinol, quercetin,2-tridecanone and xanthotoxin), and;others inducers (clofibrate and barbital)LmCYP4G62 is induced 1.8 fold by carbaryl; LmCYP6HL1 is induced 1.5 fold by chlorpyrifos; LmCYP6HL1 and LmCYP4G62 are induced by malathion (1.9 and 1.4) And LmCYP4C73 is suppressed in the trated of chlorpyrifos and malathion.Quercetin didn’t make difference to ten genes. Indole significant induce three P450 genes from Locusta migratoria, such as LmCYP6HL1, LmCYP6FG1 and LmCYP4C79, but suppressed the expression of LmCYP6FFl, LmCYP4G62 and LmCYP302A1.2-tridecanone induce the most P450 genes,the inductive effect of LmCYP6HL1 (2.6 fold)、LmCYP6FF1 (2.0 fold)、LmCYP6FGl (1.6 fold)、 LmCYP4C73 (1.8 fold)、LmCYP4C79 (1.8 fold) are significant. The expression of LmCYP6HL1 is highly induced 4.3 fold by xanthotoxin;and xanthotoxin also induce the expression of LmCYP6FE1. Indole-3-carbinol only induce two genes, including LmCYP6HL1 (1.9 fold)、LmCYP6FE1 (2.0 fold)Methoprene, an analogue of JH, can impact on six P450 genes from Locusta migratoria. It can induce the expression of LmCYP6HL1、LmCYP6FE1、 LmCYP4C73, but it suppress the expression of LmCYP6FF1、LmCYP4C79 and LmCYP302A1.including the 2.7 fold induction of LmCYP6HL1. Precocene I, a kind of inhibitor of JH, can induce only two genes including LmCYP6HL. (2.6 fold) and LmCYP6FE1 (1.7 fold). A Cholesterol-lowering drug called clofibrate and a downer called barbital didn’t effect most of expression of P450 genes from Locusta migratoria. But its impcatioin is interesting that clofibrate induce LmCYP302Al (2.0 fold), but it suppress LmCYP6HL1. On the contrary, barbital induce LmCYP6HL1 (1.6 fold), but it suppress LmCYP302A1.3.The molecular characteristics of LmCPRTo investigate the tissue distribution and developmental variation of LmCPR, the relative levels of the mRNA among various tissues were determined by qPCR. The result indicate that the expression of LmCPR achieve the highest level in the last day of Embryonic period. After the incubation, the expression of LmCPR has a peak in its life.the peak is fifth-instar nymphy. mRNA levels of P450s widely expressed in the different tissues of fifth-instar nymphy. LmCPR showed relatively higher expressions in antenna and ovary, Integument and hindgut followed.Among above drug, chlorpyrifos can induce the expression of LmCPR significantly with 1.5 fold raised. all the plant secondary metabolites induce the expression level. Among them, Inductive effect of Indole,2-tridecanone and xanthotoxin are significant. In addition, precocene I suppress the expression of LmCPR, and barbital induce the expression of LmCPR.