Identification Of Glutathione-S-transferases Genes Responding To High Temperature And Lambda-cyhalothrin In Lasioderma Serricorne

The cigarette beetle,Lasioderma serricorne(Coleoptera: Anobiidae)is a worldwide stored product pest.Glutathione-S-transferases(GSTs)belong to phase II detoxification enzymes.Studies have found that they can not only metabolize a variety of pesticides,but also resist to oxidative stress with antioxidant enzyme activities.Howerer,there are few studies on GSTs in L.serricorne.In this study,we obtaine 14 GSTs genes from the transcriptome of L.serricorne,analyze the expression patterns of these genes in response to high temperature and lambda-cyhalothrin stress,and further define the functions of two genes(Lser GSTd1 and Lser GSTe1)in resistance to insecticides and oxidative stress.The results are as follows:1.Identification and sequence analysis of GSTs gene in L.serricorneThe cDNA sequences of 14 glutathione-S-transferase(Lser GSTd1、Lser GSTd2、Lser GSTe1、Lser GSTe2、Lser GSTe3、Lser GSTo1、Lser GSTo2、Lser GSTs1、Lser GSTs2、Lser GSTs3、Lser GSTs4、Lser GSTt1、Lser GSTz1、Lser GSTu1)are obtained from the transcriptome of L.serricorne.These c DNA sequences range from 594 to 742 bp with complete ORFs,encoding proteins between 192 and 241 amino acids which are highly homologous to GSTs of other Coleoptera insects.Analysis showes that 8 Lser GSTs have conserved glutathione binding sites(G-site)at the N-terminal,11 Lser GSTs have substrate binding sites(H-site)at the C-terminal,which indicates they have glutathione transferase activity.2.Expression pattern of GSTs gene in L.serricorneThe relative expression levels of Lser GSTs are different at different developmental stages.Lser GSTd1,Lser GSTo2,Lser GSTs4 and Lser GSTu1 are highly expressed in adult stage,Lser GSTd2,Lser GSTe1 and Lser GSTe3 are highly expressed in pupal stage,and Lser GSTe2,Lser GSTo1,Lser GSTs1 and Lser GSTt1 are highly expressed in larval stage.After high temperature and lambda-cyhalothrin treatment,the contents of malondialdehyde and hydrogen peroxide in L.serricorne are increased with the enhance of activity of GST enzyme,leading to oxidative stress response in L.serricorne.The results of real-time PCR show that most Lser GSTs gene levels are significantly up-regulated.Among them,the expression levels of Lser GSTd1 and Lser GSTe1 are up-regulated to 7.65 times and 10.19 times compared to the control group at 30°C,and the expression levels of Lser GSTd1,Lser GSTe1,Lser GSTe2,Lser GSTt1 and Lser GSTz1 are significantly increased under lambda-cyhalothrin treatment.It is speculated that the proteins encoded by these genes could be involved in the oxidative stress and detoxification metabolism of L.serricorne.3.Functional analysis of Lser GSTd1 and Lser GSTe1The prokaryotic expression vectors of Lser GSTd1 and Lser GSTe1 are constructed and transformed into Escherichia coli.It is found that E.coli overexpressing Lser GSTd1 and Lser GSTe1 recombinant protein have significantly enhanced their tolerance to high temperature,paraquat and hydrogen peroxide,suggesting that the recombinant Lser GSTd1 and Lser GSTe1 protein have antioxidant enzyme activities.In addition,the catalytic activity of Lser GSTd1 and Lser GSTe1 recombinant protein is significantly inhibited by lambda-cyhalothrin,with the highest inhibition rates of 65% and 77%,indicating that Lser GSTd1 and Lser GSTe1 could interact with lambda-cyhalothrin,and may have the ability to metabolize the insecticide.In conclusion,14 GSTs are identified,the expression patterns of GSTs under high temperature and lambda-cyhalothrin stress are clarified,and the potential functions of GSTs in response to environmental stress are preliminarily analyzed.The results enrich the understanding of GSTs in L.serricorne and provide a new target for the development of environmentally-friendly control methods based on RNAi.