Toxicity Test Of Chlorantraniliprole Against Mythimna Separata (Lepidoptera:Noctuidae) And Identification Of Glutathione S-Transferase Family

Mythimna separata(Walker)(Lepidoptera: Noctuidae),referred to as armyworm,,is an omnivorous migratory pest distributed all over the world,mainly endangering grain crops such as wheat and corn.Chlorantraniliprole(CAP)is an diamide class insecticide which target is the ryanodine receptor of insects used to control a range of pests,such as the oriental armyworm.At present,M.separata developed different degrees of resistance to insecticides such as CAP,and the mutation of target site and the enhancement of detoxification metabolism are the main reasons for drug resistance.Glutathione S-transferase(GST)is an important class of detoxifying enzymes associated with insecticide resistance.However,there are few studies on the GST superfamily of M.separata and its involvement in the metabolism of CAP.The following studies have been carried out in this thesis:1.Toxic effect of CAP on M.separata larvae.Analyzed the oral toxicity of CAP to M.separata larvae: after M.separata was fed with different doses of cap,the changes of body weight were observed,the survival curve was drawn,the LC50 was measured,and the pathological sections of midgut were made for observation.The results showed that the larvae of M.separata grew faster at sublethal concentration,and stopped growing at lethal concentration.After 24,48 and 72 hours,the LC50 was 17.615,3.127 and 1.336 mg/L respectively.The poisoned larvae contracted their somites,had abnormal midgut cells and their midgut muscles were dissociated.2.CAT,SOD and GST enzyme activities analysis.The treatment group was fed with 2 or 4 mg/L CAP,and the tissue homogenate was collected for whole insect enzyme activity determination.The activities of CAT(Catalase),SOD(Superoxide Dismutase),and GST increased at different time points after CAP stimulation.The result showed that all three enzymes showed no difference in enzyme activity between control and 2 mg/L CAP after 6 hr.However,CAT and SOD activity was increased,and GST activity was decreased when exposed to 4 mg/L CAP.After 24 hr,CAT activity had no change between control and treatments,while SOD activity was decreased when exposed to 4 mg/L CAP.There was a significant increase in GST activity after exposing to 4 mg/L CAP3.RNA sequence,gene annotation and differential expression gene analysis.Six libraries(3 control vs 3 CAP-treated)of M.separata larvae were constructed for transcriptome sequencing.A total of 43055 unigenes with an average length of 1010 bp were obtained by de novo assembly,and 22197(51.55%)unigenes were successfully annotated in GO,KEGG,COG,NR,Swiss-Prot,and Pfam databases.67 up-regulated and 692 down regulated differentially expressed genes(DEGs)were found.4.Identification and phylogenetic analysis of GST in the transcriptome of M.separata.Thirty five GST genes(MseGST)were identified from unigene.Thirty five GST genes were amplified from larval c DNA and sequenced.The phylogenetic tree(NJ method)was constructed using amino acids of insect GSTs.35 GSTs were distributed in 7 evolutionary branches except theta,including 31 cytoplasm,3 microsomes and 1 undifferentiated GST.The cytoplasmic types included 4 Delta,16 epsilon,3 Omega,7 Sigma and 1 zeta type.5.GST gene expression profile analysis.The expression profiles of GST gene in different developmental stages of armyworm,adult tissues and CAP induced larvae were analyzed by q PCR.The results showed that some GST genes were highly expressed in different developmental stages and adult tissues.For example,MseGSTd1 is mainly expressed in male and female antennae,MseGSTd2 and MseGSTd4 are highly expressed in the abdomen,and MseGSTd3 is highly expressed in the head.28 GST genes were significantly up-regulated in larvae treated with 2 mg/L cap for 6hours,while most GST genes were significantly down regulated after treated with 2 mg/L cap for 6 hours or 2 mg/L and 4 mg/L for 24 hours.6.In vitro metabolic analysis of CAP by recombinant proteins.The expression and purification of MseGSTe2 and MseGSTs6 recombinant proteins were analyzed by enzyme kinetics.In vitro metabolism experiments were carried out by high performance liquid chromatography(HPLC).The results showed that both recombinant proteins could metabolize CAP.In this thesis,the oral toxicity of CAP to M.separata was investigated,the GST superfamily of M.separata was identified,and the metabolic effect of GST recombinant protein on CAP was preliminarily explored.This study provides theoretical support for further analysis of the mechanism of GST family involved in detoxification metabolism,guiding the rational use of pesticides,and promoting the green control of armyworm.