Abstract
Glutathione S-transferases (GSTs) play an essential role in the detoxification of xenobiotic toxins in insects, including insecticides. However, few data are available for the bird cherry-oat aphid, Rhopalosiphum padi (L.). In this study, we cloned and sequenced the full-length cDNA of an omega GST gene (RpGSTO1) from R. padi, which contains 720 bp in length and encodes 239 amino acids. A phylogenetic analysis revealed that RpGSTO1 belongs to the omega class of insect GSTs. RpGSTO1 gene was highly expressed in transformed Escherichia coli and the protein was purified by affinity chromatography. The recombinant RpGSTO1 displayed reduced glutathione (GSH)-dependent conjugating activity toward the substrate 1-chloro-2, 4-dinitrobenzene (CDNB) substrate. The recombinant RpGSTO1 protein exhibited optimal activity at pH 7.0 and 30°C. In addition, a disk diffusion assay showed that E. coli overexpressing RpGSTO1 increased resistance to cumene hydroperoxide-induced oxidative stress. Real-time quantitative PCR analysis showed that the relative expression level of RpGSTO1 was different in response to different insecticides, suggesting that the enzyme could contribute to insecticide metabolism in R. padi. These findings indicate that RpGSTO1 may play a crucial role in counteracting oxidative stress and detoxifying the insecticides. The results of our study contribute to a better understanding the mechanisms of insecticide detoxification and resistance in R. padi.
Highlights
Glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of multifunctional phase II enzymes that play a crucial role in the detoxification of many exogenous and endogenous xenobiotics compounds and have been widely found in almost all living organisms (Booth et al, 1961; Tu and Akgül, 2005; Li et al, 2007)
The full-length complementary DNA (cDNA) sequence of RpGSTO1 gene was obtained from R. padi and deposited in GenBank (Accession Number: MG709032)
A domain analysis revealed that the RpGSTO1 monomer includes 9 α-helics and 4 β-strands
Summary
Glutathione S-transferases (GSTs; EC 2.5.1.18) are a family of multifunctional phase II enzymes that play a crucial role in the detoxification of many exogenous and endogenous xenobiotics compounds and have been widely found in almost all living organisms (prokaryotic and eukaryotic) (Booth et al, 1961; Tu and Akgül, 2005; Li et al, 2007). Cytosolic insect GSTs can be classified into six major classes: delta, epsilon, omega, sigma, theta, and zeta; there are several unclassified genes (Ranson et al, 2001). The omega class of GSTs (GSTO) is one of the largest GST subfamilies, with multiple functions identified in various species. GSTOs have unique structures and play essential physiological roles that differ from other GST classes (Meng et al, 2014). Insect GSTs display different substrate specificities, catalytic activities and have unique N-terminal and C-terminal extensions that are not observed in the other GST classes (Board, 2011). As GSTs can play roles in detoxification of various insecticides, a change in the GST activity is one mechanism of metabolic resistance to insecticides (Ranson and Hemingway, 2005; Li et al, 2007)
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