Olfactory Responses Of Agrilus Mali To Hostvolatiles And Analysis Of The Binding Characteristics Of Two Chemosensory Protrins

The apple buprestid beetle,(Agrilus mali Matsumura),which belongs to regional invasive pests and quarantine objects in China,is one of the most serious pest to apple trees.The wild apple(Malus sieversii),as the rare apple germplasm worldwide and main tree species of wild fruit forests in Tianshan Mountainsn of Xinjiang,has been seriously damaged by the insect.As a destructive pest with devastating outbreaks,A.mali can seriously affect the yield and quality of apple production,and restrict the development of apple industry in Xinjiang with increasing apple cultivation.The sensitive olfactory system of insects plays an important role in their growth,development and reproduction,and affects a series of physiological activities.In this study,methods of chemical ecology and molecular biology were adopted to explore plant-derived attractants and contact pheromone attractants of A.mali,and the function of chemosensory protein was further studied,which can provide technical and theoretical support for ecological control of A.mali.The antennal responses of A.mali to volaites from three host plants(Gala,Fuping and Fushi)were studied by using GC-EAD.It was found that the antennae of the A.mali were sensitive to the stimuli of 6,6 and 4 volatiles of Fuping,Fushi and Gala respectively.The results of GC-MS and GC-EAD showed that the compounds with significant electrophysiological activities included 3-carene,(E)-4,8-Dimethylnona-1,3,7-triene,trans-?-ocimene,(Z)-Hex-3-en-1-ol,cis-?-ocimene,1,3-Cyclopentanediol,Nonanal and 6-methyl-5-heptene-2-ketone.Among them,3-carene and(Z)-Hex-3-en-1-ol could cause strong electroantennographic responses.Recombinant Amal CSP5 and Amal CSP8 proteins were successfully obtained by prokaryotic expression and protein purification.The binding characteristics of the two Amal CSPs with 20 leaf volatiles and 7 secondary metabolites were detected by fluorescence competitive binding experiments,which used 1-NPN as fluorescent probe.The binding characteristics of the two Amal CSPs to the odor ligands are obviously different.In regard to host volatiles as ligands,Amal CSP5 exhibited strong binding affinities for two esters including Hexyl benzoate(Ki = 7.18?0.09 ?M)and Hexyl hexanoate(Ki = 6.78?0.22 ?M);Nevertheless,Amal CSP8 showed strong binding to four terpenoids,?-Pinene(Ki = 9.60?0.09 ?M),3-Carene(Ki = 6.87?0.47 ?M),Myrcene(Ki = 10.15?0.07 ?M)and ?-Pinene(Ki = 9.92?0.09 ?M).For seven tested nonvolatile secondary compounds,two Amal CSPs also exhibited different binding characteristics.Amal CSP5 displayed strong binding affinities for kaempferol(Ki = 9.43?0.1 ?M)and tannin(Ki = 4.38?0.1 ?M),Amal CSP8 had better binding ability to tannin(Ki = 2.71?0.05 ?M)than other secondary compounds.The interaction between protein and ligands was further explored by homology modeling and molecular docking.We identified the three-dimensional structure model of Amal CSP5 and Amal CSP8.Ramachandran plot and profile 3D evaluated the best models of Amal CSP5 and Amal CSP8,proving that the modeles were reasonable and reliable for docking analyses.Amal CSP5 and Amal CSP8 show different structural models.The three-dimensional model of Amal CSP5 contains six ? helices,while Amal CSP8 only has five ? helices,all of which have two pairs of disulfide bonds formed by four conserved cysteine residues.The results of molecular docking show that the Leu13 residues of Amal CSP5 interacted with the oxygen in the-OH group of Hexyl benzoate and Hexyl hexanoate via H-bonding,which indicates that the Leu13 residue of Amal CSP5 plays an important role in binding with two small molecules.There were no hydrogen bonds involved in binding Amal CSP8 and four terpenes,but the four amino acids Leu27,Leu23,Val73 and Tyr77 in the binding cavity appeared in to be critical in the docking of these four terpenes,indicating that they could play an important role in the molecular recognition process.The cuticular hydrocarbons of male and female adults of A.mali were collected and identified by extraction and GC-MS.In this study,a total of 19 compounds,12 n-alkanes,6 monomethyl alkanes and 1 alcohol were identified in the cuticular hydrocarbon of the A.mali.Among them,n-C25,11-methyl-C25,n-C27,13-methyl-C27,and n-C29 are the mian components of cuticular hydrocarbon.By comparing the cuticular hydrocarbons of immature and mature male and female adults,the absolute content of hydrocarbons was found to be in dynamic change.With the increase of the adult age,the contents of n-alkanes in the cuticular hydrocarbons of male and female altered.There were significant differences in the content of hydrocarbons in the epidermis between sexually mature males and females: n-C23,n-C24,and 13-methyl-C27.