GSTs al., 2015). Inhibition of the growth

GSTs aremultifunctional enzymes that play a central role in the detoxification of bothendogenous and xenobiotic compounds. The different classes of GST enzymes arefound in a variety of insect species (Boothet al.

, 1961; Tu and Akgül, 2005; Li et al., 2007). The omega class of GSTs (GSTO) isa class of cytosolic GSTs with structure and characteristics that differ fromother GST class (Whitbreadet al., 2005; Burmeister et al.

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, 2008). In this study, a novel GST of theomega class (RpGSTO1) was identified from the bird cherry-oat aphid R.padi, a serious winter wheat pest in China (Wanget al., 2006). A phylogenetic analysis comparingRpGSTO1 to GSTs from different classes and insects revealed that belongsinto the omega class. RpGSTO1 has high identity with the GSTO1 from peaaphid A.

pisum. The deduced protein sequence of RpGSTO1 includesconserved functional domains, including the G-site and H-site, which werehighly conserved and located at the C-terminal region and N-terminal region. Themost important function of GSTs is to catalyze the conjugation of GSH tovarious endogenous and exogenous compounds (Hayeset al., 2005). The synthetic substrate CDNB iscommonly used in GST activity assays (Kettermanet al., 2011). We observed the ability ofrecombinant RpGSTO1 to catalyze CDNB substrate in the presence ofreduced GSH. GSTs from different insects showed high activity at differenttemperatures and pH values.

We determined that the recombinant RpGSTO1enzyme had optimal activity at a pH of 7.0 and a temperature of 30°C, which isconsistent with previous reports. In previous studies, the enzyme activity wasstable, and high enzyme activity was observed at pH 5.0 to 8.0 from differentinsect GSTs (Samraet al., 2012; Yamamoto et al., 2013; Wan et al.

, 2016). GSTs from insects had an optimalactivity at a temperature range between 25°C to 40°C (Samraet al., 2012; Zhang et al.

, 2013; Tan et al., 2014; Wan et al., 2016; Liu etal., 2017).

Weinvestigated the involvement of RpGSTO1 in the oxidative stressresponse. To perform disc diffusion assay, we cultured E. coli with recombinant RpGSTO1 and the vector for acontrol to achieve the same cell density.

Cumene hydroperoxide is a knownoxidative stress inducer (Burmeisteret al., 2008; Yan et al., 2013; Meng et al.

, 2014; Chen et al., 2015). Inhibition of the growth of thebacteria was observed following overnight exposure to cumene hydroperoxide.GSTs have a key functional role in the detoxification process involved inintracellular transport, synthesis of bio-hormones, and protection againstoxidative stress of both endogenous and xenobiotic compounds (Armstrong,1997; Enayati et al., 2005). Previousstudies indicated that GSTO1 was involved in antioxidant defense (Burmeisteret al., 2008; Wan et al.

, 2009; Yamamoto et al., 2011; Zhang et al., 2016). In this study, cumenehydroperoxide induced oxidative stress in cells expressing recombinant RpGSTO1but showed the zone was decreased compared to cells expressing the vector. Ourresults provide evidence that RpGSTO1 is an antioxidant enzyme thatprotects cells from oxidative stress. InsectGST can detoxify many synthetic insecticides and plant allelochemicals (Liet al., 2007). Synthetic insecticides can causephysiological changes in insects.

Currently, R. padi has developedresistance against various insecticides (Zuoet al., 2016). To explore and characterize theputative roles that RpGSTO1 mightplay, we analyzed the expression patterns of the gene under different insecticidetreatments. We treated insects with the pyrethroids ?-cypermethrin, carbamateisoprocarb, organophosphorus malathion and neonicotinoids sulfofoxaflor andthen measured the mRNA expression level of RpGSTO1.The relative expression of RpGSTO1was affected by these insecticides, and the pattern varied among the differentinsecticide treatments. An omega class GST in B. mori has been reportedto be induced by treatment with various environmental stresses, such asdiazinon, permethrin, imidacloprid, ultra violet-B (UV-B) and bacteria (Yamamotoet al.

, 2011). In previous reports,up-regulation of GST genes following exposure to pyrethroid, organophosphate,carbamate and neonicotinoid were found in insecticide-resistant strains (Hemingwayet al., 1991; Yang et al., 2013; Wei et al., 2015).

Down-regulation of GSTOs werereported in Cnaphalocrocis medinalis exposed to chlorpyrifos (Liuet al., 2015). GSTO gene expression was inducedby different stress conditions, such as different temperature, UV, H2O2,cyhalothrin, phoxim, pyridaben and paraquat in Apis cerana (Zhanget al., 2013). In this study, the mRNA level of RpGSTO1 responded to differentinsecticide challenges, and the responses maybe associated with the oxidativestress caused by insecticide treatment, which were positively correlated withthe previous studies, including that omega GSTs can be induced by insecticidesand could play a part in detoxification of insecticides in R. padi. Inconclusion, our study demonstrated the unique functional characterization,expression pattern and physiological roles of a novel GSTO gene from R.

padi.To our knowledge, this is first time that an omega class GST has been clonedand characterized from the bird cherry-oat aphid. This study also revealed thatrecombinant RpGSTO1 possesses antioxidant activity in response tooxidative stress. The expression level of R. padi RpGSTO1 can be induced under the stresses caused by differentinsecticides.

Our findings provide valuable insight into the functions of theGSTO in this serious pest. 


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