Expression And Binding Specificity Analysis Of Odorant Binding Proteins In The Grain Aphid Sitobion Avenae (Fabricius)

Sitobion avenae(Fabricius)is a dominate species in the wheat growing fields of China.It cause damages in yield and quality of wheat by plunder wheat nutrition,transmitting plant virus and excreting honeydew.Olfaction plays a vital role in the process of host searching,selecting,colonization and oviposition for grain aphids.Pheromones can trigger various behavior responses in aphid.With(E)-?-farnesene(EBF)as the main or only component,aphid alarm pheromone is a crucial role in regulating interaction between plant and aphids,as well as plant-aphids-natural enemies multi trophic level.Odorant binding proteins(OBPs)is the key protein that involved in detecting peripheral odors in insect antennae,and also one of the hot topics in the study of molecular mechanisms of aphid olfaction.At present,the binding characteristic between SaveOBPs and EBF is still nuclear.Therefore,this study is focused on the identification of EBF with Save OBPs through gene cloning,constructing the prokaryotic expression vector,and expressing and purifying the recombinant proteins.Fluorescence competitive binding assays were conducted to screening the SaveOBPs that showed specific binding properties with EBF and odors.The main results were as fellows:1.Based on the antenna transcription annotation information of adult antenna in Sitobion avenae,12 genes of Sitobion avenae(SaveOBP1-10/14/15)were obtained and verified through gene cloning.2.The recombinant SaveOBPs was prokaryotically expressed in Escherichia coli and 7 SaveOBPs(SaveOBP1/3/6/7/8/9/14)were purified through ion exchange chromatography,and SaveOBP10 with histidine tags was purified by the immobilized metal nickel chelate affinity chromatography.Among of them,SaveOBP1,SaveOBP6,SaveOBP9,SaveOBP10 and SaveOBP14 were newly obtained proteins in Sitobion avenae.All of these fusion proteins were identified by LC-MS/MS(Q-TOF).3.Using N-phenyl-1-naphthylamine(1-NPN)as probe,fluorescence competitive binding ass ays were conducted to study the binding properties of SaveOBPs with components of aphid ala rm pheromones,plant volatiles of wheat,aphid sex pheromones,and EBF analogues.In the bin ding assays with EBF,for the fist time we found SaveOBP9 can bind it strongly(Ki<10?M/L),together withSaveOBP3/7/ presented the strongest binding abilities with EBF,with dissociation constants of 4.55±0.32?M,0.65±0.03?M and 0.60±0.03?M/L,respectively.We hypothesis that S aveOBP3,SaveOBP7 and SaveOBP9 are the potential target proteins that can recognize EBF.B esides,SaveOBP9 can also bound the rest components of aphid alarm pheromone(1S)-(-)-?-pine ne,(-)-(1S)-?-pinene,(+)-limonene than other SaveOBPs well,while SaveOBP7 also bound(-)-(1S)-?-pinene,(+)-limonene strongly.We found that SaveOBP9 presented strong binding abilities with plant volatile components(Z)-3-Hexen-1-ol,(Z)-3-Hexenyl acetate,Methyl salicylate,?-terpineol,It indicate that SaveOBP9 is a target protein that can recognize volatiles of wheat.For the aphid sex pheromone compon ents(4aSR,7SR,7aRS)-nepetalactone,(1RS,4aSR,7SR,7aRS)-nepetalactol,we found both Save OBP3 and SaveOBP10 showed a certain binding properity with them(Ki< 20 ?m),SaveOBP7 and SaveOBP9 displayed the strongest binding abilities with them among those proteins,We hypothesis that SaveOBP3/7/9/10 are the potential target proteins that can recognize aphid sex phe romone components.4.With high affinity,16 EBF analogues(CAU-II-09,CAU-II-11,CAU-II-15,CAU-II-19,CAU-II-20,CAU-II-21,CAU-II-28,CAU-II-29,CAU-I-B2,CAU-IV-02,CAU-C-03,CAU-A-01,CAU-A-02,CAU-A-03,CAU-A-04,CAU-A-05)were screened from 38 new EBF analogues through fluorescence competitive binding assays.Particularly,we found that SaveOBP3,SaveOBP7,SaveOBP9 displayed strong affifilities with the 16 EBF analogues among the 8 recombinated SaveOBPs.The results of this study are comparatively important in uncovering the molecular mechanisms of repelling aphids by EBF,and enriching the theory of aphid chemical ecology.In addition,it will pave the way for further developing EBF oriented repellents and provide references to green aphid control technology.