The Clone And Expression Of Apolipoprotein D And Insecticyanins From Spodoptera Exigua And Their Binding Affinities With Insecticides

The use of insecticides is an important measurement to control crop pests.The prerequisite for insecticides to exert their insecticidal role is that they must enter the insect body and be transported to their molecular targets to disrupt the important physiological process in insects,and then lead to lethality.However,the mechanism of pesticide transport in insects remains elusive,and the mechanism for transportation of insecticide entering the haemolymph of insects remain an unsolved mystery.In this study the association of Spodoptera exigua transporting proteins and insecticide delivery will be investigated to discover the missing link in insecticide toxicology.Firstly,S.exigua apolipoprotein D(SeApoD),insecticyanin(SeIns)and lipoproteinⅢ(SeLpⅢ)genes will be cloned and the structural characteristics of the proteins encoding by these genes will be analyzed.The tissue expression profiles of these genes and the expression differences between resistant and susceptible populations of S.exigua will be analyzed.prokaryotic expression technique will will be applied to obtain the soluble protein,and the binding affinity between protein and insecticide will be analyzed.The results in detainls are dexcribed as following:1.The structural characteristics of apolipoprotein D,insecticyanin and other gene from S.exiguaThe cDNA sequences of ten genes from S.exigua were cloned,which are four apolipoprotein(ApoD)genes,one LipoproteinⅢ(LpⅢ),and five insecticyanin(Ins).The structural domains of these protein were predicted using the NCBI online tool(CDSearch),and the results showed that proteins coding by Apo dans Ins have the conserved lipocalin domain(lipocalin_apoD),suggesting the membership of lipocalin superfamily.The deduced amino acid sequences contains four conserved cysteine sites in Apod and Ins,suggesting the typical apolipoprotein D or insecticyanin characterization.The protein with the conserved domain of ApoLp-Ⅲ super family suggests the membership of apolipophorin III family.Meanwhile the phylogenetics of ApoD and Ins in Lepidoptera insects were analyzed.2.The expression profiles of apolipoprotein D.insecticyanin and other gene from S.exiguaThe relative expression levels of the genes obtained in this study were analyzed in the larvae tissues including head,epidermal,midgut,fatbody and hemolymph using RT-qPCR.The expression profiles demonstrated that SeApoDl and SeApoD2 amond four Apod genes are highly expressed in hemolymph of larvae,then in fatbody.SeApoD3 and SeApoD4 are mainly highly expressed in head of larvae.The midgut and head of larvae had relatively high expression level of SeInsl,SeIns2 is mainly expressed in fatbody and hemolymph of larvae,SeIns2 and SeIns are highly expressed only in hemolymph,and SeIns5 is highly expressed in head and then in hemolymph.The relative expression levels of these genes are also compared in the population of S.exigua susceptible strain and lambda cyhalothrin resistant to insecticide.The expression of SeApoD2,SeApoD3,SeApoD4,SeIns1,SeIns4 and SeIns5 are up-regulated in the resistant population,however,the expression of SeLpⅢ is down-regulated in the resistant population.The expression characterization of these gene in S.exigua provide theoretical basis to explore their physiological function of these genes,and the up-regulation or down-regulation of the genes in resistant population suggest the connection with insecticide resistance.3.The prokaryotic expression of apolipoprotein D,insecticyanin and other gene from S.exigua and their binding affinity analyse with insecticideThe prokaryotic expression were made using PET-30a as expression vector and BL21 as the host cell.The soluble expression of SeApoD4 and SeIns were obtained in the induction conditions of 0.5mM IPTG at 25℃ for 11h,however,other 8 proteins were expressed in inclusion bodies.Then induction conditions were modified,the soluble expression of SeLpⅢ and SeApoD5 were obtained in reduced temperature(16℃)and extended incubation time of 18 h.And then the soluble proteins of SeApoD3,SeApoD4,SeIns1 and SeLpⅢ were purified by affinity chromatography and dialysis.The binding affinity of the purified proteins with insecticides were analyzed by microscale themophoresis(MST)technique.The result showed the dissociation constant Kd for interation between lambda cyhalothrin with SeApoD4 and SeIns 1 were 0.18 μM and 9.04 μM,respectively,suggesting a high binding affinity of this chemical with two proteins respectively.SeApoD3 also showed some binding affinity with lambda cyhalothrin,its Kd is 49.81 uM,however,lipoprotein SeLpⅢ had very weak affinity with lambda cyhalothrin with a Kd value of 0.72 mM,suggesting there were no interaction bwteen this lipoprotein and this compound.the dissociation constants Kd for interaction between chlorpyrifos with SeApoD3 and SeLpIII were 10.12 μM and 4.14 μM respectively,suggest high binding affinity between them,however there were weak or no interation of chlorpyrifos with SeInsl and SeApoD4 with Kd values of 105.42 μM and 1.4 mM,respectively.These data of interaction analysis suggest ApoD and Ins proteins may be took over to deliver lambda cyhalothrin and chlorpyrifos in hemolymph of S.exigua larvae.In conclusion,the lipocalin proteins in S.exigua were unearthed comprehensively and systematically in this study and the phylogenetics and evolution of lipocalins in Lepidoptera insects were clarified.The binding affinity of lipid transportation proteins with insecticides were analyzed in insect for the first time.The discovery and identification of insecticide binding proteins answers the long sought-after question that how insecticides are delivered in hemolymph of insects.These results provide insights into the mechanism of insecticide toxicology and insecticide resistance.