Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

Z Hu,H Chen,Z Li,X Feng,Q Lin,F Yin

doi:10.1017/s0007485314000510

Abstract

Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

Highlights

Cytochrome P450 monooxygenases (P450s), is a superfamily of heme-containing proteins, found in almost all living organisms from bacteria to human (Feyereisen, 2006)
It has often been mentioned that insecticide metabolism is enhanced by overexpression of cytochrome P450s and it appears to be a frequent type of resistance mechanism in insects (Liu & Scott, 1998; Li et al, 2006)
The putative amino acid sequence of CYP321E1 has less than 50% similarity to all other amino acid sequences that already existed in GenBank, only shared optimal homology to three cDNA clones: Helicoverpa zea CYP321A1 (48% identity), Spodoptera littoralis CYP321A11 (47% identity) and Spodoptera litura CYP321B1 (44% identity)

Summary

Introduction

Cytochrome P450 monooxygenases (P450s), is a superfamily of heme-containing proteins, found in almost all living organisms from bacteria to human (Feyereisen, 2006). Cytochrome P450s constitute an important metabolic system and this system plays a central role in the oxidative metabolism of both xenobiotic and endogenous compounds (Feyereisen, 1999) In insects, they are best known for their roles in the metabolism of insecticides, which often results in the development of insecticide resistance (Zhou et al, 2010). Many cytochrome P450 genes, especially belonging to the families of CYP4, CYP6, CYP9 and CYP12, are frequently involved in insecticide metabolism and resistance (Feyereisen, 2006; Li et al, 2007). It has often been mentioned that insecticide metabolism is enhanced by overexpression of cytochrome P450s and it appears to be a frequent type of resistance mechanism in insects (Liu & Scott, 1998; Li et al, 2006). It is important to study the expression profiles of cytochrome P450 genes and examine their inducibility by the particular insecticides, the findings may help researchers better understand functions of the genes and their interactions with insecticides at molecular levels and provide useful genetic information to assess potential consequences of insecticide exposures in insects

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