Binding Characterization Of Odorant Binding Protein OBP56h?OBP69a And OBP76a In Drosophila Suzukii With Small Molecular Compounds

Drosophila suzukii is an important pest that devastates soft-skinned fruits such as strawberries,blueberries,cherries,and grapes,causing huge economic losses to the fruit industry.Compared to D.melanogaster,D.suzukii prefers to lay eggs in fresh fruits through which its olfactory sensitivity has changed.In addition,the pheromone,11-cis-vaccenyl acetate(c VA),that promotes female mating in D.melanogaster,remains loss in D.suzukii and inhibits the mating of D.suzukii.Further the odorant binding proteins(Dm OBPs),Dm OBP56 h,Dm OBP69 a and Dm OBP76 a are involved in the regulation of pheromone in D.melanogaster.However,the olfactory molecular mechanism of D.suzukii is unclear.As such,we used D.suzukii as model insects in the present study.Firstly,we screened differentially expressed DsOBPs,and purified proteins by E.coli prokaryotic expression.Then fluorescence quenching was applied to detect the binding ability of the protein to c VA,and the key binding sites were calculated using homology modeling and molecular docking techniques.Finally we analized the altered amino acid sites with virtual mutations and screened for potential ligands by fluorescent competitive binding.The main results are as follows:1.Screening of D.suzukii pheromone binding proteinsIn order to investigate DsOBPs in D.suzukii,we analyzed the transcriptome of the antennae before and after mating of D.suzukii females.After mating,1858 genes were significantly upregulated in the antennal transcriptome and 2087 genes were significantly downregulated(FDR<0.05,FC>2).After mating,nine DsOBPs were significantly up-regulated and two DsOBPs were down-regulated.The expression levels of Ds OBP69 a and Ds OBP76 a were significantly upregulated.2.Cloning,protein expression and purification of DsOBPsThe Ds OBP56 h,Ds OBP69 a,and Ds OBP76 a genes were cloned from D.suzukii,which were orthologous genes to Dm OBP56 h,Dm OBP69 a,and Dm OBP76 a,with 82.84%,72.30%,and 81.70% protein homology,respectively.The recombinant proteins p ET30a-Ds OBP56 h,p ET30a-Ds OBP69 a and p ET30a-Ds OBP76 a were expressed by the E.coli prokaryotic expression system.Ds OBP56 h protein was expressed quite a lot in the supernatant,while both Ds OBP69 a and Ds OBP76 a were mainly expressed in inclusion bodies.A large amount of pure target proteins were obtained through column,dialysis,and ultrafiltration.3.Binding mechanism of Ds OBP76 a to c VA in D.suzukiiThe fluorescence quenching experiments showed that the binding ability of Ds OBP76 a to c VA is weaker than Dm OBP76 a to c VA.We also tested the binding ability of Ds OBP56 h and Ds OBP69 a to c VA,both are weaker than the binding ability of Ds OBP76 a to c VA.Dm OBP76a(ID: 3b86.1.A)template was used to construct the Ds OBP76 a protein model.The molecular docking of Ds OBP76 a and c VA showed that the c VA molecule was surrounded by Phe6,Leu76 and Ser85,which may contribute to be the key amino acids for Ds OBP76 a binding to c VA.We also performed virtual mutations on 18 amino acid sites of Ds OBP76 a that have changed compared to Dm OBP76 a.Only Ala84 Met and Leu113 Phe mutations improved the binding capacity of Ds OBP76 a with c VA.4.Analysis of binding characteristics of DsOBPs in D.suzukiiIn order to determine the binding characteristics of DsOBPs,we screened potential ligands of DsOBPs with bis-ANS as a fluorescent probe via fluorescence competition binding experiments.The floral odor ?-ionone,bitter compounds berberine hydrochloride and coumarin,and the plant metabolite naringenin can all bind to Ds OBP56 h,Ds OBP69 a,and Ds OBP76 a,of which ?-ionone has the strongest binding ability.In addition,Ds OBP56 h can also bind with the attractant ?-cyclocitral,and Ds OBP69 a binding to the pheromone cis-7-tricosene.Overall,we purified the odorant binding proteins,Ds OBP56 h,Ds OBP69 a,and Ds OBP76 a in D.suzukii.The results showed that Ala84 and Leu113 were likely the key amino acids that led to the weak binding ability of Ds OBP76 a with c VA.Moreover,Ds OBP56 h,Ds OBP69 a and Ds OBP76 a can bind to several compounds which may play critical roles in olfactory and gustatory behaviour in D.suzukii.Our results pave the way for deeply understanding of chemical ecology in D.suzukii and establishing eco-friendly techniques for the control of the pest.