Heterologous Expression Of Cytochrome P450Gene CYP4CE1from Nilaparvata Lugens By Baculovirus Expression System

The brown planthopper (BPH), Nilaparvata lugens, is an important pest of rice. People began to study the usage of chemical pesticides and to develop new resistant rice varieties in order to control this pest, and apply some defensive effect. However, the use of common chemical pesticides can be effective in the short term pest control, but could easily cause environmental pollution and pest resurgence. Confrontational rice will also be tolerated brown planthopper’s food. Therefore, the study of new resistance genes and elucidating the molecular mechanism of interaction between the brown planthopper and rice are good for provide clues for finding effective environmentally friendly methods and the theory of reference to control brown planthopper.P450genes play an important role in the metabolism of secondary metabolites and adaptation of insects to host plant resistance. In this work, the open reading frame (ORF) of cytochrome P450gene CYP4CE1in brown planthopper and NADPH cytochrome P450reductase (NCPR) gene in housefly (Mcusca dometica) were cloned and sequenced. The confirmed DNA fragments were inserted into MH-vector. The recombinant plasmids MH-CYP4CE1and MH-NCPR were transformed into DH10β,as the donor strain. The baculovirus shuttle vector containing the target genes were constructed by a homologous recombination technique (mating-assisted genetically integrated cloning, MAGIC). The recombinants were screened by antibiotics and PCR. Positive recombinant plasmids were transfected into insect Sf9cells and getting P1generation recombinant baculovirus. To achieve a high-titer of the recombinant baculovirus, Sf9cells were repeated infection. The heterologously expressed CYP4CE1and NCPR proteins were detected by western hybridization.In metabolism of substrates, CYP needs the participation of the NADPH cytochrome P450reductase and cytochrome b5. However, the expression level of the NADPH cytochrome P450reductase in insect cells Sf9is relatively low, and no hemin in insect cells Sf9is usable. In this work, recombinant baculovirus of CYP4CE1and NADPH cytochrome reductase together with hemin were transformed into Sf9, which resulted in the catalytic activity of CYP4CE1. After incubation of CYP4CE1with the universal substrate Phenobarbitalum. the enzymatic activity of CYP4CE1was analyzed by High performance liquid charomatography (HPLC). The result revealed that CYP4CE1can metabolize Phenobarbitalum.In this study, CYP4CE1from brown planthopper and NADPH cytochrome P450reductase gene from housefly were coexpressed successfully in Sf9cells by mating-assisted genetic integrated cloning (MAGIC) and Bac-to-Bac baculovirus expression system. The expressed CYP4CE1was cofirmed to metabolize Phenobarbitalum preliminariely. The data laid a theoretical basis for revealing the molecular mechanism of interaction of BPH and rice, and will facilitate in finding more reasonable control strategies.