Identification And Functional Analysis Of Delta And Epsilon Glutathione S-transferase Genes From Cydia Pomonella

3The codling moth,Cydia pomonella(L.)(Lepidoptera: Tortricidae),is one of the most important fruittree pests in the world and an important quarantine pest in China.Studies at home and abroad have found that elevated enzyme activity of Glutathione S-transferases(GSTs)is the main mechanism of resistance to different insecticides.The Delta and Epsilon GST genes are mainly involved in the detoxification metabolism and resistance formation of chemical insecticides on insects.However,the identification of the Delta and Epsilon GST genes of C.pomonella and the study of the metabolism of chemical insecticides are not systematic and in-depth.Identification of the Delta and Epsilon GST genes and analysis of metabolic functions of the GST genes on chemical insecticides are important in C.pomonella for revealing the mechanism of GSTs-mediated metabolic resistance.In this paper,the all Delta and Epsilon GST genes were identified from the transcriptome and genomic database of C.pomonella,and their spatiotemporal expression patterns and the expression patterns after insecticide stress were studied.Some recombinant GSTs proteins were expressed in the prokaryotic expression system,and the enzyme kinetic parameters on recombinant GSTs proteins and the inhibitory activities of the insecticides on recombinant Cp GSTd3 were determined in vitro.It was confirmed by metabolic tests in vitro that Cp GSTd3 can metabolize lambda-cyhalothrin.The main research results are as follows:1.Identification of GST genes of Cydia pomonellaA total of 29 GST genes were identified by the transcripts and genomes of C.pomonella,including 26 cytosolic GST genes and 3 microsomal GST genes.The cytosolic GST genes contain 4 Delta classes,5Epsilon classes,2 Zeta classes,1 Theta classes,5 Sigma classes,3 Omega classes and 6 unclassified GST genes.The similarity of amino acid sequence of Delta and Epsilon GST genes of C.pomonella with other insects was 42.79-58.80% and 31.90-41.12%,respectively.2.Expression Patterns of Delta and Epsilon GST genes of C.pomonellaReal-time quantitative RT-PCR(RT-q PCR)were carried out to determine the expression levels of Delta and Epsilon GST genes of C.pomonella in different developmental stages,larval tissues and in larvae exposed to lambda-cyhalothrin.The results showed that the GST genes were expressed in all developmental stages of C.pomonella,and Cp GSTd3,Cp GSTe2,Cp GSTe3 and Cp GSTe4 were mainly expressed in larval stage;the expression levels of Cp GSTd2,Cp GSTd4,Cp GSTe1 and Cp GSTe5 were significantly different between male and female.The GST genes are mainly expressed in detoxification organs such as midgut,Malpighian tube and fat body,but only Cp GSTd3 is not expressed in fat bodies.After LD10 lambda-cyhalothrin treatment,the expression levels of the GST genes in larvae were increasedand then decreased under different time of treatment.The expression levels of Cp GSTd4 and Cp GSTe1 were opposite.3.Enzyme kinetic parameters of Delta and Epsilon GSTsThe recombinant proteins of Cp GSTd3,Cp GSTd4,Cp GSTe2 and Cp GSTe3 were expressed using the Escherichia coli expression system and purified.The Vmax values of the recombinant proteins were13.11±0.68 ?mol/min/mg,1.55±0.11 ?mol/min/mg,9.71±0.18 ?mol/min/mg and 12.45±0.80 ?mol/min/mg,respectively.The Km values were 0.63 ± 0.09 m M,0.67 ± 0.12 m M,0.09 ± 0.01 m M and 0.31 ± 0.06 m M,respectively.Inhibition assay in vitro results showed that lambda-cyhalothrin,bate-cyfluthrin,etofenprox,chlorpyrifos and phoxim inhibited the activity of Cp GSTd3 with IC50 values of 0.65,0.48,0.29,0.19 and0.53 m M,respectively.Metabolism in vitro results indicate that Cp GSTd3 recombinant protein can metabolize lambda-cyhalothrin.