Cloning,Expression And Functional Analysis Of Genes Encoding Chemosensory Proteins In Agrilus Mali(Matsumura)

Insects can accurately identify chemical signals and avoid damage under complex environments via sophisticated and sensitive olfactory system,facilitating their prey foraging,oviposition and escaping behaviors.Chemosensory proteins(CSPs),which are dispensable to olfactory system,play a significant role in recognition with host plant volatiles and pheromones.The apple buprestid beetle,Agrilus mali Matsumura(Coleoptera:Buprestidae),is one of the most destructive trunk borers in Xinjiang wild fruit forests,and there are no effective measures in its management at present.Therefore,it makes sense to explore the recognition mechanisms of CSPs via studies of molecular biology and chemical ecology.We analyzed the sequences and physicochemical properties of the four CSP genes identified in the antennal transcriptome of A.mali.Tissue distributions and expression levels of CSPs were determined by RT-qPCR.It was found that AmalCSP1 was highly expressed in abdomens,wings and male antennae,and we detected it may have the ability to bind with sex pheromones of this pest.In addition,expression patterns of AmalCSP4 and AmalCSP5 in antennae and abdomens were higher than those of other tissues,indicating that it may have the function of binding to common odours and synthesis of sex pheromones.As AmalCSP8 is expressed mostly in antennae,it is speculated that it should have the function of identifying the of host plants.Expression vectors was successfully constructed for the four CSP genes.We found that the four CSPs were mainly expressed in the supernatant through inducing the expression of recombinant plasmids and SDS-PAGE analysis.We measured the mass concentrations of the enriched proteins,and they were 1.41,0.42,0.56,0.50mg/mL for AmalCSP1,AmalCSP4,AmalCSP5,AmalCSP8 after purification with Spin Columns,respectively.We analyzed the binding affinities of the recombinant proteins for 40 kinds of plant volatiles via fluorescent competitive binding assays.The dissociation constants(Kd)as calculated by Scatchard plots for AmalCSP4,AmalCSP5 and AmalCSP8 were 8.199,6.954 and 6.924?M,respectively.AmalCSP5 showed good binding ability with 13 odor ligands,but it did not bind with alkanes and nitriles.AmalCSP5 showed strong binding affinities to dodecanol and dodecanal,as well as C5 to C10 compounds.AmalCSP8 only bound effectively with eight ligands effectively,and exhibited week binding ability with alkanes,nitriles,and aldehydes.But it showed strong binding capacity with(Z)-?-ocimene,?-pinene and limonene.In conclusion,AmalCSP1 and AmalCSP4 could bind poorly with all the 40 host plant volatiles.We speculated that they both might bind with sex pheromones in Agrilus mali.Further assays and verification studies are necessary in the future.