Identification And Expression Patterns Of Glutathione-S-transferase Genes From Chilo Suppressalisand Cnaphalocrocis Medinalis

The rice striped stem borer(Chilo suppressalis)and the rice leaffolder(Cnaphalocrocis medinalis)are two of the most important rice insect pests in Asia,which causing severe economic losses to rice production in China and Southeast Asia.For a long time,the control of striped stem borer and rice leaffolder mainly relies on chemical pesticides,but on account of non-selective application of pesticides andselection pressure on these two pests caused the susceptibility reduced of these two pests to insecticides,further the control of these pests by chemical pesticides becoming increasingly difficult.At present,the study on the recuce of insecticides susceptibility to these two pests mainly focused on the increase in metabolic capabilities of detoxification enzymes as well as decrease in targe site sensitivity.Glutathione-S-transferase(GST)is one of the three major detoxification enzymes in the insects,which plays an important role on metabolicof the endogenous and exogenous toxic substances.GST catalyse the binding of harmful substances tothe thiol group of reduced glutathione(GSH)with electrophilic compounds,which make the resultant products more water soluble or excretable and reduce the toxicity of insecticides to insects.At present,reports of GSTs on Chilo suppressalis and Cnaphalocrocis medinalis have only researched that GSTs are identified in the antennal,and are not evolved with insecticide metabolicassociated tissue.Therefore,this study was conducted on the coding genes of GST from Chilo suppressalis and Cnaphalocrocis medinalis.The GSTs gene was identified from the transcriptome database of these two pests,and the expression pattern of GSTs in each tissue was clarified.And the expression pattern of GST gene under sublethal dose insecticide stress were studied.The GSTs gene of Chilo suppressalis and Cnaphalocrocis medinalis was compared by insecticidal induction experiment.The results of this study can provide a theoretical basis for exploring the molecular mechanism onthe reduction the sensitivity of insecticides and provide the prevention and control methods of other Lepidoptera pests.1.Identification and bioinformatics analysis of GSTgenes of Chilo suppressalisandCnaphalocrocis medinalisA total of 16 and 25 GSTs genes were identified by searching the midgut transcriptome,fatbody transcriptome of Chilo suppressalis and nymph transcriptome of Cnaphalocrocis medinalis.RT-quantitative PCR was conducted to determine the expression of these genes in Chilo suppressalis and Cnaphalocrocis medinalis larvae.The GSTs were classified into six known GSTs family(Delta,Epsilon,Omega,Sigama,Zeta,Theta)and unclassified families,respectively,by NCBI BLAST sequence alignment tool.Twenty-five GSTs genes were identified into five known GSTs families(Delta,Epsilon,Omega,Sigama,Zeta)and unclassified families,which have not been identified Theta classGSTs inriceleaffolder.The results of multiple alignment of theaminoacidsequenceshowed the CsGST protein and the CmGST protein both have the catalysis active site.The amino acid residues of the glutathione-binding site are highly conserved in the Delta and Epsilon families,which indicate theymay be the key to the catalytic function.The high identity of the N-terminal GSH binding site and the C-terminal substrate binding site in GSTs suggest thatGSTs function conservation of Chilo suppressalis and Cnaphalocrocis medinalis.By constructing phylogenetic trees,the relationship between GSTs of these two rice pests and other GSTs were studied.2.Tissue expression profile of GST Gene of Chilo suppressalis and its expression patterns in response to insecticide stress were quantitatively analyzed byRT-qPCR using the ?-actin gene of Chilo suppressalis as the internal control gene.The distribution patterns of GST genes in different tissues of Chilo suppressalis larvaeindicates that CsGSTd1,CsGSTe1,CsGSTo1,CsGSTo3 and CsGSTt1 were mainly expressed in the fatbody.CsGSTe3 ismainly distributed in the malpighian tubes;other genes expression profile in organizations demenstrate no significant difference.The expression of GST after sublethal dose and time induction of chlorantraniliprole was exiamined by RT-qPCR.The results showed that the expression level of CsGSTe1 was significantly higher in the Delta family and Epsilon family after 12 hours of induction,and the expression level of CsGSTe1 was significantly higher than that in CK and 6h-treated after induction of chlorpheniramine.The expression level of GSTs Omega family was significantly higher than that in CK after 12 hours of treatment.There was no significant difference in CsGSTs2 at 6h and 12h-treated with CK.CsGSTt1 showed a similar expression patternwith Delta family.After 6h treatment,the expression level of CsGSTt1 was significantly decreased,but the expression level of CsGSTt1 was significantly increased after 12 h treatment.The expression of CsGSTz1 was up-regulated compared with CK at 6h and 12h-treated,but there was no significant difference.Unclassified family GSTs were down-regulated at 6h and 12 h,the expression of CsGSTu1 was significantly decreased,and there was no significant difference in CsGSTu2.3.The mRNA expression levels of 25 GSTs genes in different tissues(midgut,mulpighian tubes,fatbody)and expression patterns in respond to insecticide of Cnaphalocrocis medinalis larvae were examind by RT-qPCR using ?-actin gene as the internal control gene.The results showed thatexpression levels of CmGSTe1,CmGSTe2 and CmGSTe5 were significantly expressed in the fatbody which higher than those in the midgut,expression level of CmGSTe1,CmGSTe2 and CmGSTe5 were significantly higher in the fatbody.After induction of chlorpyrifosby sublethal dose and time,the expression of CmGSTd2,CmGSTe6 and CmGSTe7 were significantly up-regulated.One Delta family gene(CmGSTd2)and two Epsilon family genes(CmGSTe6 and CmGSTe7)were significantly up-regulated after 24 h of induction,and the expression of CmGSTe6 was the highest one.Expression of CmGSTd1,CmGSTd3,CmGSTd4 fluctuatedin small range,which did not show a significant level.The expression patterns CmGSTe1,CmGSTe4,CmGSTe5 were consistent respectively,after 24 hours treatmentand their relative expression levels were significantly lower than CK.The relative expression of CmGSTe8 and CmGSTe9 increased compaired with CK,but there was no significant difference.The time effect of CmGSTo1 and CmGSTo2 was consistent,the relative expression levelwere down-regulated,CmGSTo3 was up-regulated,but they did not have the significant difference.The CmGSTs1,CmGSTs2,CmGSTs4 and CmGSTs5 of the Sigma family showed similar expression patterns,and the relative expression level was increased,and there was no significant difference compaired with CK.The time effects of CmGSTz1 and CmGSTu1 were similar,and the expression level of CmGSTz1 and CmGSTu1 were significantly decreased after 24 hours of induction.The expression levels of CmGSTz2 and CmGSTu2 did not change significantly compared with CK.CmGSTd2,CmGSTe6 and CmGSTe7 were significantly increased,and these three genes belonged to insect-specific Delta and Epsilon families,suggesting that these genes may play an important role in the detoxification of exogenous compounds.