Identification Of A Vemon Peptide From Neoseiulus Barkeri And Evaluation Of Its Acaricidal Activity

Tetranychus cinnabarinus is a worldwide important agricultural pest mite that can damage many agricultural products and cause serious economic losses.At present,the control of T.cinnabarinus is still mainly based on chemical control,but the long-term use of pesticides in the field and the high fertility and adaptability of this mites have led to resistance problem,so it is necessary to develop safe and efficient active substances to enrich the means of pest mite control.The natural enemies of arthropods usually have substances that have paralyzing or lethal effects on their prey,and their active ingredients are potential biological pesticides.Such substances have a higher safety profile compared to chemical pesticides because they combine effectiveness and specificity and are more easily degradable in the environment.Based on such a general background,this study intends to identify the possible lethal venom peptide in Neoseiulus barkeri,and to investigate the biological activity of this venom peptide against T.cinnabarinus.Furthermore,the interaction proteins of the venom peptide in pests were screened to clarify potential targets.The main findings were as below:1.Cloning and bioinformatics analysis of the venom peptide gene of Neoseiulus barkeriBy bioinformatic analysis,a total of two venom peptide genes were identified for N.barkeri,which were named as NbSP1 and NbSP2 according to phylogenetic tree analysis.Both genes did not have homologous in the gene pool of pest mite,which indicated that these were unique in nature enemy.They both have the typical conserved cysteine residues"C-C-CC-C-C",forming an inverse parallelβ-fold,but protein structure prediction showed that only NbSP2 formed a disulfide bond structure,i.e.ICK structure.2.Prokaryotic expression of the venom peptide gene of Neoseiulus barkeriThe recombinant proteins with pET-32a vectors were successfully constructed by homologous recombination,and the soluble recombinant proteins pET32a-NbSP1 and pET32a-NbSP2 were obtained by in situ expression.Finally,the final target proteins were purified by nickel column,and the molecular weights of the two proteins were 21.88 k Da and 21.9 k Da,which were consistent with the predicted molecular weights.3.Determination of biological activities of recombinant proteins NbSP1 and NbSP2The lethal effects of recombinant proteins NbSP1 and NbSP2 on T.cinnabarinus were analyzed by dipping and injection methods,and the results showed that the dipping method was not effective due to the hydrophobicity of the body wall of T.cinnabarinus,so the injection method was finally used for complete bioassay analysis.After injection,the mites exhibited twitching of limbs and inability to move normally,which eventually led to death.In order to expand the insecticidal spectrum of the venom peptide,the lethal effect of NbSP2 was analyzed on the 3rd instar larvae of Spodoptera litura by feeding and injection methods.It was found that the injection method worked,while the feeding method was ineffective.Two concentration gradients were set up in this experiment,and the dose of 0.05 ng/μg caused obvious mortality,which was manifested by stiffness,shortening and blackening of the body.4.Prediction of potential NbSP2 interacting proteinsThe intercalating proteins of NbSP2 were identified by affinity chromatography in T.cinnabarinus and S.litura.SDS-PAGE gel electrophoresis revealed the presence of specific banding regions following the intercalation of NbSP2 with total proteins of adult T.cinnabarinus and 3rd instar larvae of S.litura,respectively.The results suggested that the venom peptide may intercalate with proteins in the target pest mite/pest to exercise function,resulting in lethality.After LC-MS/MS analysis,a total of 29 specific proteins were identified in T.cinnabarinus and 13 specific proteins were identified in S.litura,and a total of 6 proteins were detected in both.The results of GO functional annotation showed that the interacting proteins detected in both species mainly involved"Protein binding"(26S proteasome non-ATPase regulatory subunit),"ATP binding"(Heat shock protein),"structural constituent of ribosome"(Ribosomal protein),"Dolichyl-diphosphooligosaccharide-proteinglycotransferaseactivity"(Dolichyl-diphosphooligosaccharide-protein glycosyltransferase),"DNA binding"(Histone H3),and"structural constituent of cytoskeleton"(Beta-tubulin).Among them,the 26S proteasome non-ATPase regulatory subunit may have a more important function.In addition,the ATPase proteins detected in T.cinnabarinus act in important Na~+/K~+neural pathways and may also be potential neural targets.In this study,we obtained genes with functional similarity to spider venom peptides in N.barkeri by full-length transcriptome sequencing analysis,and then identified NbSP1and NbSP2 as venom peptide genes in N.barkeri by prokaryotic expression,bioactivity assay and affinity chromatography assay,combined with homology modeling to hypothesize that NbSP2 is biologically active due to its ICK structure,and its potential interacting proteins were analyzed by LC-MS/MS analysis.The results of the study provide new ideas for the development of new biological miticides.