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GSTs al., 2015). Inhibition of the growth

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GSTs are
multifunctional enzymes that play a central role in the detoxification of both
endogenous and xenobiotic compounds. The different classes of GST enzymes are
found in a variety of insect species (Booth
et al., 1961; Tu and Akgül, 2005; Li et al., 2007). The omega class of GSTs (GSTO) is
a class of cytosolic GSTs with structure and characteristics that differ from
other GST class (Whitbread
et al., 2005; Burmeister et al., 2008). In this study, a novel GST of the
omega class (RpGSTO1) was identified from the bird cherry-oat aphid R.
padi, a serious winter wheat pest in China (Wang
et al., 2006). A phylogenetic analysis comparing
RpGSTO1 to GSTs from different classes and insects revealed that belongs
into the omega class. RpGSTO1 has high identity with the GSTO1 from pea
aphid A. pisum. The deduced protein sequence of RpGSTO1 includes
conserved functional domains, including the G-site and H-site, which were
highly conserved and located at the C-terminal region and N-terminal region.

The
most important function of GSTs is to catalyze the conjugation of GSH to
various endogenous and exogenous compounds (Hayes
et al., 2005). The synthetic substrate CDNB is
commonly used in GST activity assays (Ketterman
et al., 2011). We observed the ability of
recombinant RpGSTO1 to catalyze CDNB substrate in the presence of
reduced GSH. GSTs from different insects showed high activity at different
temperatures and pH values. We determined that the recombinant RpGSTO1
enzyme had optimal activity at a pH of 7.0 and a temperature of 30°C, which is
consistent with previous reports. In previous studies, the enzyme activity was
stable, and high enzyme activity was observed at pH 5.0 to 8.0 from different
insect GSTs (Samra
et al., 2012; Yamamoto et al., 2013; Wan et al., 2016). GSTs from insects had an optimal
activity at a temperature range between 25°C to 40°C (Samra
et al., 2012; Zhang et al., 2013; Tan et al., 2014; Wan et al., 2016; Liu et
al., 2017).

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We
investigated the involvement of RpGSTO1 in the oxidative stress
response. To perform disc diffusion assay, we cultured E. coli with recombinant RpGSTO1 and the vector for a
control to achieve the same cell density. Cumene hydroperoxide is a known
oxidative stress inducer (Burmeister
et al., 2008; Yan et al., 2013; Meng et al., 2014; Chen et al., 2015). Inhibition of the growth of the
bacteria was observed following overnight exposure to cumene hydroperoxide.
GSTs have a key functional role in the detoxification process involved in
intracellular transport, synthesis of bio-hormones, and protection against
oxidative stress of both endogenous and xenobiotic compounds (Armstrong,
1997; Enayati et al., 2005). Previous
studies indicated that GSTO1 was involved in antioxidant defense (Burmeister
et al., 2008; Wan et al., 2009; Yamamoto et al., 2011; Zhang et al., 2016). In this study, cumene
hydroperoxide induced oxidative stress in cells expressing recombinant RpGSTO1
but showed the zone was decreased compared to cells expressing the vector. Our
results provide evidence that RpGSTO1 is an antioxidant enzyme that
protects cells from oxidative stress.

Insect
GST can detoxify many synthetic insecticides and plant allelochemicals (Li
et al., 2007). Synthetic insecticides can cause
physiological changes in insects. Currently, R. padi has developed
resistance against various insecticides (Zuo
et al., 2016). To explore and characterize the
putative roles that RpGSTO1 might
play, we analyzed the expression patterns of the gene under different insecticide
treatments. We treated insects with the pyrethroids ?-cypermethrin, carbamate
isoprocarb, organophosphorus malathion and neonicotinoids sulfofoxaflor and
then measured the mRNA expression level of RpGSTO1.
The relative expression of RpGSTO1
was affected by these insecticides, and the pattern varied among the different
insecticide treatments. An omega class GST in B. mori has been reported
to be induced by treatment with various environmental stresses, such as
diazinon, permethrin, imidacloprid, ultra violet-B (UV-B) and bacteria (Yamamoto
et al., 2011). In previous reports,
up-regulation of GST genes following exposure to pyrethroid, organophosphate,
carbamate and neonicotinoid were found in insecticide-resistant strains (Hemingway
et al., 1991; Yang et al., 2013; Wei et al., 2015). Down-regulation of GSTOs were
reported in Cnaphalocrocis medinalis exposed to chlorpyrifos (Liu
et al., 2015). GSTO gene expression was induced
by different stress conditions, such as different temperature, UV, H2O2,
cyhalothrin, phoxim, pyridaben and paraquat in Apis cerana (Zhang
et al., 2013). In this study, the mRNA level of RpGSTO1 responded to different
insecticide challenges, and the responses maybe associated with the oxidative
stress caused by insecticide treatment, which were positively correlated with
the previous studies, including that omega GSTs can be induced by insecticides
and could play a part in detoxification of insecticides in R. padi.

In
conclusion, 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 cloned
and characterized from the bird cherry-oat aphid. This study also revealed that
recombinant RpGSTO1 possesses antioxidant activity in response to
oxidative stress. The expression level of R. padi RpGSTO1 can be induced under the stresses caused by different
insecticides. Our findings provide valuable insight into the functions of the
GSTO in this serious pest. 

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