Cloning And Characterization Of 4 Cytochrome P450 Monooxygenases In Leptinotarsa Decemlineata (SAY)

Cytochrome P450 monooxygenases are the most common biocatalysts in nature, they can catalytic thousands of reactions. CYPs affect insect ability to adapt to diverse habitats. On one hand, some CYPs catalyze the oxidation of an extremely diverse range of chemicals such as insecticides, plant allelochemicals and other environmental chemicals. On the other hand, CYPs have very important physiological functions during all life stages of insects. They are involved in the biosynthesis of molting hormone (ecdysteroids) and juvenile hormone. Some insect CYPs are also involved in the degradation of pheromones, which are critical for chemical communication and behavior. Therefore, insect CYPs have important research value and potential economic value.Based on the transcriptomic data of L. decemlineata and sequences in the GenBank,4 full-length cytochrome P450 monooxygenase cDNAs(CYP314A1?CYP412A1?CYP412A2 and CYP413A1) were obtained. By RNAi and eukaryotic expression, their functions were characterized. The main results were listed as follows:1. Cloning of 4 cytochrome P450 monooxygenases in L. decemlineataBased on the transcriptomic data of L. decemlineata and sequences in the GenBank,74 cytochrome P450 monooxygenase genes were identified. These genes were classified into 19 families and 35 subfamilies. By RACE, the full-length cDNAs of 4 CYP genes (CYP314A1, CYP412A1. CYP412A2 and CYP413A1) in I. decemlineata were cloned. The temporal and spatial expression profile revealed that the 4 CYP genes were expressed in all developmental stages and in all tissues. Moreover, each of the 4 CYP genes has its own specific expression profiles. These results indicated that the 4 genes had very important and differential physiological functions, and deserved further study.2. RNA interference-mediated knockdown of the 4 CYP genes in L. decemlineataDietary introduction of bacterially expressed dsRNAs successfully silenced the 4 CYP genes. Knockdown of these genes caused the lethality of the second instars and other abnormal phenotypes, negatively affected pupation rates, larval weights and developmental durations. Moreover, knockdown of the 4 CYP genes caused similar but less serious impacts on the fourth-instar larvae. Furthermore, the molting hormone analog halofenozide could partially or complete rescue the negative impacts caused by CYP314A1 gene silencing.3. Functional verification of CYPs in L. decemlineataTo verify the functions of the 4 L. decemlineata CYPs, these CYP genes were transfected into insect baculovirus expression vector pFastBacl and successfully expressed in Sf9 cells. Western blot showed the cell lysates containing the 4 recombinant proteins showed an obvious band of about 55 kDa, but no band of GFP or empty vector. The enzyme activity test and RP-HPLC analysis revealed that the cell lysate containing CYP314A1 could converte E to its product 20E.