cDNA cloning and expression of two novel cytochrome P450 genes CYP6CS1 and CYP6CW1 in Nilaparvata lugens

Two novel genes, CYP6CS1 and CYP6CW1, were cloned from the fourth instar nymphs of brown planthopper Nilaparvata lugens Stål (Hemiptera: Delphacidae) reared on its susceptible rice variety Taichung Native 1 (TN1) plants. The deduced proteins are typical microsomal P450s sharing conserved structural and functional domains with other insect CYP6 members. Temporal expression analysis by Northern blot hybridization indicated pre-exposure to N. lugens moderately resistant rice Minghui 63 (MH63) seedlings caused a time course-dependent induction of CYP6CS1 which peaked after 24 h of treatment; in contrast, CYP6CW1 was induced and remained at a constant time course from 0-72 h. CYP6CS1 and CYP6CW1 are dramatically induced in gut tissues and, slightly upregulated in carcass and fat bodies as revealed in spatial gene expression analysis. Whole mount in situ hybridizaion revealed that the two genes are expressed at a basal level in gut tissue and Malpighian tubules in nymphs fed with TN1 rice. After exposure to MH63, the expression of CYP6CW1 was found to be high in the whole gut, including Malpighian tubules. Expression of CYP6CS1 was significantly increased in midgut, and slightly increased in foregut, hindgut and Malpighian tubules. Fresh insect Sf9 cells were infected with the recombinant baculovirus containing the housefly Mcusca dometica NADPH-Cytochrome P450 reductase gene (NCPR), and CYP6CW1 to coexpress the target proteins. The specific protein bands (about 76.4kD and 61.8kD) were detected by SDS-PAGE and confirmed by Western blot analysis separately. The enzyme CYP6CW1 activity was monitored by using phenobarbitalum as a substrate by high performance liquid chromatography (HPLC). The result revealed that CYP6CW1 can metabolize phenobarbitalum. These data suggest potential roles of the two P450 genes in determining patterns of N. lugens-rice relationships through allelochemical detoxification.