Identification And Ligand Binding Characteristics Of General Odorant Binding Proteins In The Diamondback Moth,Plutella Xylostella(Lepidoptera:Plutellidae)

The diamondback moth(DBM),Plutella xylostella(L.),is a serious insect pest on cruciferous plants.They are causing serious economic losses annually all over the world.The management "bottleneck" of P.xylostella is that it can rapidly develop resistance to pesticides and lack of effective natural enemies.General odorant binding protein(GOBP)is a sub-class of odorant binding proteins,the primary carriers of odor signal.They play a significant role in screening,binding and transporting hydrophobic odorant molecules to specific odor receptors of insects.Thus they are critical for survival and reproduction of insects,and correspondingly provide a new target to control DBM.Revealing the functions of P.xylostella GOBPs in olfactory recognition is indispensable to a thorough understanding of molecular mechanisms of olfaction.In this study,4 general odorant binding protein genes of P.xylostella were cloned and identified using RT-PCR technique.The recombinant proteins,PxylOBP6,PxylOBP12,PxylOBP13 and PxylOBP31 were expressed in E.coli and purified by nickel affinity chromatography column.The binding characteritics of the 4 recombinant PxylOBPs with 39 volatile odor compounds were measured by fluorescene competitive binding in vitro.Finally,the localization of the GOBPs was marked by immunofluorescence technique.The main results are as follows:1.The open reading frame of the 4 GOBPs genes PxylOBP6,PxylOBP12,PxylOBP13 and PxylOBP31 were obtained and the GenBank accession number is KT156677,KT156678,KT156679 and KT156676 respectively.All the 4 proteins contain 122?137 amino acids,molecular weight falled between 13.83?14.89KDa and pI of 4.41?8.71.Their amino acid sequences contain conserved odorant binding protein domain and six conserved cysteine residues in typical OBPs.Among them,only PxylOBP31 possessed a signal peptide in N terminal.PxylOBP6 and PxylOBP31 were clustered into one branch,PxylOBP12 and PxylOBP13 into another one by amino acid sequence homology blast and phylogenetic tree analysis,indicating that they belonged to different sub-classes of GOBP.2.Four recombinant expression vectors pET28a-PxylOBP6,pET28a-PxylOBP12,pET28a-PxylOBP13 and pET28a-PxylOBP31 were constructed and expressed in E.coli BL21(DE3).The recombinant proteins were achieved as inclusion bodies by IPTG inducing.After denaturation and refolding by dilution,4 proteins of high purity proteins were obtained by nickel affinity chromatography.The main secondary structure of the 4 GOBPs was a helical using Circular dichroism.3.The affinities of the 4 GOBPs to 39 ligand compounds were detected by fluorescence competitive binding assays.The results showed that PxylOBP6 and PxylOBP13 had a wide binding profile.They were able to bind many compounds including sex pheromones,alcohols,aldehydes,ketones,esters and olefins,and showed strong binding capacity with most of them(Ki<10 ?mol/L).2,6-butylated was the best ligand of PxylOBP6 and PxylOBP13.PxylOBP12 had strong binding capability with esters and olefin,particularly esters and olefin with long carbon chains.PxylOBP31 was able to bind cruciferous green leaf volatiles and aldehydes and ketones,but had not binding capability with pheromone and 2,5-hexanediol.4.The localization of PxylOBPs was analyzed by immunofluorescence labeling.The results showed that green fluorescence can be observed in the sections treated with anti-PxylOBP12.PxylOBP12 was mianly distributed in the distal segment of flagellum and in the joints of flagella,sparsely distributed in the body of flagella.No labeled PxylOBP12 was found in sensillum triehodeum.