Identification And Functional Characterization Of Olfactory Relative Protein Genes In Harmonia Axyridis (Coleoptera: Coccinellidae)

In nature,with olfaction system,natural enemy insects are able to percept chemical substances released by plants,phytophagous insects and other natural enemies,thereby complete the activities including food selection,mate recognition and location of oviposition sites.Harmonia axyridis is an important natural enemy against many agricultural and forestry pests,such as aphids,mites,coccids,thrips and whiteflies.Although previous studies proved H.axyridis has significant attracting and repellent activities to a variety of volatiles,its corresponding olfactory mechanism remains unclear.Clarifying the molecular mechanism of H.axyridis olfactory can not only provide theoretical basis for the H.axyridis attractants/repellents development,but also plays virtual roles in promoting its biological control effects in farmland ecosystems.This study conducted identification and functional analysis of H.axyridis olfactory-related protein genes,aiming to clarify the mechanism of olfactory-related proteins in the process of chemical communication in the transcription and protein levels.The main findings are as follows:1.A total of 108 putative olfactory genes were identified in the adult antennae and whole body transcriptome of H.axyridis.Including candidate 19 OBPs,16 CSPs,36 ORs,13 GRs,20 IRs and 4 SNMPs were identified.OBPs all had complete open reading frames(ORFs)coding 125-141 amino acids with molecular weights ranging from 13.75–27.75 k Da.Based on the number and location of the conserved cysteines,HaxyOBPs could be divided into three groups: 11 Classic OBPs,7 Minus-C OBPs,and 1 Plus-C OBP.CSPs all had complete open reading frames(ORFs)coding 111-153 amino acids with molecular weights ranging from 12.29–17.50 k Da,having 4 conserved cysteine residues.Among the 36 ORs genes,19 had complete ORFs,encoding 352-480 amino acids,and the remaining 17 ORs genes had incomplete ORFs,encoding 109-402 amino acids.Among the 13 GRs genes,3 GRs had complete ORFs,encoding 344-440 amino acids,and 9 GRs genes had incomplete ORFs,encoding 151-338 amino acids.Among the 20 IRs genes,2 IRs had complete ORFs,encoding 918 and 923 amino acids,respectively,and 18 IRs genes were incomplete,encoding 147-1065 amino acids.None of the 4 SNMPs genes had a complete ORF,which encoded 278-551 amino acids.2.qRT-p CR was used to clarify the expression characteristics of OBPs,CSPs and ORs genes in different tissues of male and female adults of H.axyridis.HaxyOBP2,HaxyOBP3,HaxyOBP5,HaxyOBP12,HaxyOBP15,HaxyCSP4,HaxyCSP9,HaxyCSP15,Haxy ORCO,Haxy OR13,Haxy OR14 were more abundantly expressed in antennae than other body parts,including the head,thorax,abdomen,legs,and wings,which may play an important role in the olfactory recognition process of H.axyridis.3.HaxyOBP3,HaxyOBP5,HaxyOBP12,HaxyOBP15 and HaxyCSP4,HaxyCSP9,HaxyCSP15 were specifically and highly expressed in adult antennae.These genes were cloned to obtain the corresponding full-length coding sequence.The prokaryotic expression system was used to express and purify the proteins,and the binding properties of these proteins with 39 volatiles and 8 pesticides were determined by fluorescence competition binding assay.HaxyOBP15 showed a broad binding profile with 7 compounds((E)-?-farnesene,?-ionone,?-ionone,Geranyl acetate,Nonyl aldehyde,Dihydro-?-ionone,Linalyl acetate)and 3 insecticides(Deltamethyrin,Hexaflumuron,Emamectin Benzoate).HaxyOBP5 could bind Methyl salicylate,?-ionone,p-Anisaldehyde,Imidacloprid and Acetamiprid.HaxyOBP3 and HaxyOBP12 only both combined two compounds.Among 3 CSPs,HaxyCSP9 had the broadest binding spectrum,which could bind 20 compounds such as(E)-?-farnesene,?-caryophyllene,?-humulene,?-elemene;HaxyCSP9 also had good binding properties with Deltamethrin.Hasy CSP15 could bind with 8 substances including Phenylacetaldehyde,(E)-?-farnesene and Geranyl acetate.HaxyCSP4 only bound with ?-ionone,p-Anisaldehyde,Imidacloprid and Clothianidin.In addition,the binding abilities of HaxyOBP15,HaxyCSP9,HaxyCSP15 with 29 kinds of salicylic acid EBF analogues were different.HaxyOBP15 could bind with 28 kinds of analogues.HaxyOBP15 had the strongest binding ability with 14,30,10,11.The binding ability of HaxyOBP15 with 3,17,and 18 is weak.HaxyCSP15 had the strongest binding ability with 1,9,16,20,4,and 11.The binding ability of HaxyCSP15 with 8,5,14,26,18,and 23 was weak,and HaxyCSP15 did not bind with the six substances 17,24,25,27,28,and 29.HaxyCSP9 had different binding characteristics with 28 of its analogs except for 29.It had the strongest binding ability with 1,5,8,20.The binding ability with 28,18 was weak.4.The three-dimensional models of the HaxyOBP3,HaxyOBP5,HaxyOBP12,HaxyOBP15 and HaxyCSP4,HaxyCSP9,HaxyCSP15 proteins were constructed.The 7 predicted proteins were composed of 6 spirals.By using Procheck,Verify₃D,ERRAT,the quality of seven models was evaluated to be good.Using the Autodock software,molecular docking was performed to evaluate the binding ability of seven models and the related compounds.The binding of HaxyOBPs and HaxyCSPs to the compounds was mostly based on non-polar hydrophobic interaction,supplemented by “?-?” interaction and hydrogen bonding.In molecular docking,the amino acids that played important roles in OBPs were: HaxyOBP3(Y106 and I115),HaxyOBP5(V85,A90,L131,Y112),HaxyOBP12(I91),HaxyOBP15(F9,H53,L58).The amino acids that played important roles in CSPs were: HaxyCSP4(N48,A52,C60),HaxyCSP9(L48,M70,V71,L74,A85),and HaxyCSP15(H28,V68,V71,W83,F86).