Identification,Molecular Characterization And Expression Pattern Of Glutathione S-transferase Genes In The Bird Cherry-oat Aphid Rhopalosiphum Padi

The bird cherry-oat aphid,Rhopalosiphum padi?L.?is an important pest of wheat whichwidely distributes in the world.It feeds on sap in the phloem and transmits the lot of virus,including barley yellow dwarf virus?BYDV?.Different chemical compounds are used to control this aphid.R.padi has developed resistance to various insecticides.The glutathione S-transferases?GSTs?can protect insect against oxidative damage caused by ROS?reactive oxygen species?and play role in insecticide resistance of aphids.Six subclasses?Delta,Epsilon,Omega,Sigma,Theta and Zeta?of GTSs are found in insects.Most of the previous investigations have focused on delta and epsilon GST.However,the molecular mechanisms of GSTs in insecticide resistance of aphids still need further clarification.Here,we report the identification and classification of GST genes from R.padi.The recombinant proteins of some GST gens were expressed in Escherichia coli cells.The biochemical properties of the purified recombinant GST proteins were characterized.The transcriptional patterns of some genes following exposure to various concentrations of b-cypermethrin,isoprocarb,malathion,and sulfoxaflor were analyzed.The potential roles of the GST fusion proteinS in antioxidant defense were also investigated.Our results provide valuable insight into the function of GSTs in the stress response to insecticides.1.Toxicity and GST enzyme analysis of R.padi to different synthetic chemical insecticidesWe report toxicity concentration and increased GST enzyme activity on R.padi to synthetic chemical insecticides exposure of chlorpyrifos,imidocolprid,isoprocrab,sulfoxaflor,?-cyhalothrin,?-cypermethrin and malathion insecticides.The LC₅₀0 concentration of each insecticide was used to treat the aphid,the toxicity of seven insecticides to the aphid varied.Among the seven insecticides,the?-cypermethrin had the highest LC₅₀ value.The carbamate group isoprocarb had low LC₅₀0 value.The LC₅₀ concentration of?-cypermethrin was up to 1.308mg L^-1,which was 27.25 times of that for the isoprocarb.Enzyme activity compared to the control,GST activity was decreased after 24 h of exposure to malathion and isoprocarb,while the GST activity was increased 24 h of exposure to sulfoxaflor and?-cypermethrin.After exposure to?-cypermethrin,malathion and sulfoxaflor for 12 h,the GST activity was significantly increased.This result clearly showed that GST enzyme activity can change depending on the insecticide and in timely manner.The broad spectrum of resistance and stability of resistance to insecticides in R.padi in the current study suggested that it could be a prime candidate for mass releases and compatible with most spray programs.2.Glutathione S-transferase genes identification and prokaryotic expressionIn this study,we identified 11 GST genes from R.padi.The full-length cDNA of an omega1,theta1,sigma1 and sigm2 GST genes were cloned and sequeneced from R.padi.Sequence analysis showed that these genes had the characteristics of respective gene family.Phylogenetic analysis indicated that the genes be associate with the sigma,theta and omega classes of insect GSTs.The RpGST genes contain nine?-helices and five?-sheets connected by loops.The RpGSTS1 and RpGSTS2 had 60%and 50%homology with the 3D structure of the Blattella germanica GST5.GST omega and theta genes were gene was highly expressed in transformed Escherichia coli and then protein were purified by Ni²⁺-NTA agarose gel affinity chromatographic column.The expressed recombinant RpGSTO1 protein was detected by western blot using a 6×His mouse monoclonal antibody.3.Purified recombinant GST enzyme characterization and antioxidant activityThe enzymatic properties of RpGSTO1 and RpGSTT1 were investigated.The recombinant RpGSTO1 exhibited optimum catalytic activity towards CDNB with the pH at approximately 7.0.Steady-state kinetic analysis was performed with 5 mM GSH and different CDNB concentrations at pH 7.0,and K_m and V_maxax were determined.Recombinant RpGSTO1 and RpGSTT1 showed a K_m of 0.120 and 0.129 mM and a V_maxax of 2.906 and 1.426?mol/mg/min,respectively.In addition,metal-catalyzed oxidation system was used to evaluate the ability of recombinant RpGSTT1 to protect super-coiled p UC18 plasmid DNA against oxidative stress.A disc diffusion assay showed that E.coli overexpressing Rp GSTO1 and RpGSTT1 increased resistance to cumene hydroperoxide and paraquat-induced oxidative stress.The killing zones of the E.coli expressing Rp GSTO1 and RpGSTT1 were much smaller than those around the control of the E.coli with the pET-28a vector.The defence response of RpGSTT1 resistance to cumene hydroperoxide was significantly increased compared to paraquat.The results showed that GSTs might protect against oxidative stress against cumene hydroperoxide and paraquat.4.Expression of omega,theta and sigma GST genes in different insecticide exposure of R. padiThe mRNA expression of RpGST omega,theta and sigma genes were examined to observe the effect of different insecticides on their expression levels.The relative expression level of RpGST genes were investigated after exposure to LC₂₅ and LC₅₀ concentrations of?-cypermethrin,isoprocarb,sulfoxaflor,malathion,chlorpyrifos,imidacloprid,and?-cyhalothrin.The R.padi were treated with LC₂₅ and LC₅₀ concentrations of different insecticides and the time-dependent relative expression of RpGST genes were normalized to their reference genes were quantified.Real-time quantitative PCR analysis showed that the relative expression level of RpGSTO1,RpGSTT1,RpGSTS1 and RpGSTS2 genes were different in response to different insecticides.The present work showed that the expression level of Rp GST omega and theta were changed after different exposure durations to isoprocarb,?-cypermethrin,malathion and sulfoxaflor.We found that RpGSTS1 and RpGSTS2 were highly expressed in the sulfoxaflor insecticide treatment followed by the isoprocrab treatment.This result suggesting that the GST genes could involve detoxification mechanism to insecticides in R.padi.