Olfaction Function Analysis Of Odorant-Binding Proteins In Aphelinus Asychis Walker

Natural enemy insects interact with hosts and plants in the long process of evolution,forming a sensitive and specific olfactory recognition system for volatiles in plant-host complex.As the first step of the olfactory system relay,odorant binding proteins can ensure the smooth and orderly operation of the olfactory system.Aphelinus asychis Walker is a kind of egg-producing aphid which is widely distributed in the world.It can parasite more than 40 kinds of aphid including Myzus persicae,Sitobion avenae and Acyrthosiphon pisum.It was found that the plant carrier system composed of Vicia faba,Acyrthosiphon pisum and Aphelinus asychis can produce and release Aphelinus asychis in large numbers,and has great potential for biological control.The study of OBPs function can not only enrich the study of plant-host-parasitic wasps in theory,but also provide reference for the study of olfactory mechanism of other natural enemy insects.At the same time,the screening of the attractive volatiles of Aphelinus asychis can be used for the subsequent development of plant synergists with attractant effect on parasitic wasps,so as to improve the control ability of parasitic wasps.In this study,we identify and functional analysis the OBPs of Aphelinus asychis,in order to elucidate the mechanism of the OBPs in olfactory recognition.The research results are as follows:1)A tissue-specific transcriptome database was constructed based on antennae and other tissues except antennae of newly emerged female and male adults of Aphelinus asychis.The OBPs of Aphelinus asychis were identified and analyzed by comparing with reported genome and antennae transcriptome data of hymenoptera.A total of 27 OBPs were identified from the antennal transcriptome of Aphelinus asychis.2)Among 27 OBPs,the expression level of 13 OBP/PBP in the head was significantly higher than that in the body,and the expression level of 12 OBPs in the body was significantly higher than that in the head.Based on transcriptome sequences,five OBPs with the highest expression in the head were amplified to obtain the full-length sequences of OBP1,OBP2,OBP3,OBP4 and OBP6.3)Three OBPs(OBP1,OBP2 and OBP4)were expressed and purified by E.coli prokaryotic expression system,and all three proteins were expressed in supernatant.Recombinant enterokinase was used to remove His-tags of recombinant proteins,and high purity OBPs without His-tags were obtained by SDS-PAGE gel electrophoresis.The binding ability of OBPs(OBP1,OBP2 and OBP4)to 95 compounds was determined by fluorescence competitive binding assay.The results that AasyOBP2 has a wide binding spectrum and strong binding ability,and can easily bind to 7 compounds(ki<10),including 3 volatile substances(β-ionone,p-anisaldehyde and methyl salicylate)and 4 other substances(folic acid,chlorogenic acid,naringin and 1-chloro-2,4-dinitrobenzene).AasyOBP1 was easily bound to3 substances(chlorogenic acid,naringin and 1-chloro-2,4-dinitrobenzene)(ki<20),while AasyOBP4 was easily bound to only one volatile substance(β-ionone)(ki<20).4)The behavioral responses of female Aphelinus asychis to 28 volatile compounds were measured using Y-type olfactory apparatus.These 28 volatiles were derived from host plant volatiles,pest induced volatiles and host volatiles of parasitic wasps.In behavioral experiments,Methyl salicylate,beta-Citronellol,cis-3-hexen-1-ol,beta-Elemene,Nonanal,2-Phenylethanol of six kinds of compounds have obvious attraction effect.