NADPH-cytochrome P450 reductase potentially involved in indoxacarb resistance in Spodoptera litura

Abstract

NADPH-cytochrome P450 reductase (CPR) plays a central role in the metabolism of insecticides. Numerous studies have shown that CPR is associated with insecticide resistance in insect. In this study, two transcripts of Spodoptera litura CPR (SlCPR-X1 and SlCPR-X2) were identified and cloned, and the deduced protein of SlCPR-X1 contains all the conserved CPR structural features (N-terminal membrane anchor, FMN, FAD and NADP binding domains, FAD binding motif, and catalytic residues). However, no N-terminal member anchor and a shorter FMN binding region have been identified in the deduced protein of SlCPR-X2. The specific expression patterns showed that SlCPR-X1 and SlCPR-X2 were detected in all tested developmental stages and tissues, but highly expressed in third-, fourth-, and fifth-instar larvae, and in midgut and fat body. In addition, compared with the susceptible strain, SlCPR-X1 and SlCPR-X2 were up-regulated and more inducible when treated with indoxacarb in the indoxacarb-resistant strain. However, the relative expression, up-regulation and induction of SlCPR-X1 were all higher than those of SlCPR-X2 in the indoxacarb-resistant strain. Furthermore, RNA interference and baculovirus expression system combined with MTT cytotoxicity assay demonstrated that only SlCPR-X1 with the N-terminal membrane anchor as the major CPR potentially involved in S. litura indoxacarb resistance. The outcome of this study further expands our understanding of the important role of insect CPR in xenobiotics detoxification and resistance development, and CPR could be a potential target for insecticide resistance management mediated by RNAi or CRISPR/Cas.

Keywords: Heterologous expression; Indoxacarb resistance; NADPH-cytochrome P450 reductase; RNA interference; Spodoptera litura.