The Mechanisms Of The Interaction Between Bee Pheromone-related Proteins OBP11?CSP3 And ASP1 And The Chemical Ligands And Neonicotinoid Insecticides Of Apis Cerana Cerana

As a unique bee species in China,Apis cerana has many excellent characteristics such as tolerance to low temperature,resistance to Varroa mite,preferring swarming,and so on.In particular,A.cerana have a strong ability in search of sporadic nectar sources,and plays an important role in pollination of plants.It contacts various chemical odor substances in the external environment through water-soluble proteins such as odorant-binding proteins(OBPs)in body surface specific olfactory receptors.However,the chemical substances neonicotinoid insecticides at sub-lethal doses also have different toxicities to honeybees and other pollinating insects.The toxicity of bees and the molecular mechanism caused by this toxicity difference have not been fully analyzed.Therefore,this study has conducted in-depth research on the three existing pheromone-related proteins OBP11,CSP3 and ASP1,and also studies the mechanisms of them binding with corresponding ligands and imidacloprid.Multiplex spectroscopy was used to study the binding modes of OBP11,CSP3 and ASP1 with external odor molecules and imidacloprid.The key amino acids and major interactions of OBP11binding with various ligands and CSP3 binding with imidacloprid were determined.The effects of temperature,pH and imidacloprid on proteins binding with ligands were also studied.The main results are as follows:1.Study on protein purification,site-directed mutagenesis and ligand binding mechanism of pheromone-related protein OBP11 in A.cerana.Firstly,AcerOBP11protein preparation was performed and was checked by LC-MS/MS.The subcellular localization of antennae was performed by scanning electron microscopy and transmission electron microscopy,which showed the diversity of the antennae of worker bees and the specificity expression of AcerOBP11.Fluorescence competition experiments showed that AcerOBP11 played a dual role in the process of sensing the behavior of honeybee sex pheromone and plant volatiles.Molecular docking and site-directed mutation showed that Ile97 and Ile140 were the key amino acids of AcerOBP11 binding with ligands.The circular dichroism(CD)spectra showed that three mutant amino acid residues had an effect on the secondary structure of OBP11.Among them,Ile97 increased the?-helix content and made OBP11 protein more compact and smaller.The?-helices of OBP11-F101G and OBP11-I140G were reduced,which made the OBP11 protein spread.OBP11-F101G decreased most and protein was most stretched.2.The binding mechanism research of pheromone-related proteins OBP11binding with the neonicotinoid insecticides imidacloprid.The thermodynamic experiment and fluorescence showed the stronger binding between AcerOBP11 and imidacloprid at different temperature.The quenching mechanism of AcerOBP11 and imidacloprid was dynamic from 290K to 300K,and the mainly force was hydrophobic interaction and electrostatic force;the quenching mechanism was state from 300K to310K,and the mainly force was hydrophobic interaction.The ultraviolet absorption spectrum showed that the binding between AcerOBP11 and imidacloprid was dynamic quenching in low temperature.Circular dichroism spectrum showed that the imidacloprid decreased the?-helix of OBP11,changed the secondary structure of protein and made the struction extension.Molecular docking was used to predict the key amino acids and hydrogen bonds in the binding process between AcerOBP11 and imidacloprid.Ala138 contributed the hydrogen bond and Ile97,Ile140 and Phe101mainly contribute energy.Through the change of the fluorescence intensity of AcerOBP11 binding with HOB and 9-ODA at different temperatures and pH,we found that AcerOBP11 can not bind with 9-ODA directly.However,AcerOBP11 only at pH 4.0 had strong direct binding with HOB at 300K and 310K,the quenching mechanism was dynamic quenching.3.The purification,site-directed mutagenesis and the ligand binding mechanism research of pheromone binding proteins CSP3.The fluorescence results showed that CSP3 binding with imidacloprid was stronger than that with acetamiprid,and the binding mechanism of CSP3 with imidacloprid and acetamiprid were all static quenching.The mainly force was hydrophobic force in acetamiprid and hydrogen bond and van der Waals force was in imidacloprid.The three groups of mutants Lys100 and Phe101,Arg66,Ser6 and Tyr7 were designed and proteins were prepared.The binding mechanism of CSP3 and all mutant proteins with imidacloprid was a static quenching.All of the mutants were involved in the interaction between CSP3 and imidacloprid.The R66 and S6Y7 were significantly decreased indicating that the main structure of CSP3 was located at the N-terminus.Thermodynamic results showed that the major interactions between CSP3 wild-type and imidacloprid were hydrogen bonding and van der Waals forces.The force of mutant CSP3m-K100F101G and CSP3m-S6Y7G were not changed,but the force of mutant CSP3m-R66G was changed to hydrophobic force,which showed that the R66 mainly provided hydrogen bond.The synchronous fluorescence of CSP3 bound with imidacloprid showed that the tryptophan was the major source of CSP3 fluorescence intensity and closer to the binding site in the CSP3binding cavity.Circular dichroism spectrum showed that the?-helix of three mutants CSP3m-K100F101G,CSP3m-S6Y7G and CSP3m-R66G were more than wild type.The imidacloprid made the?-helix of three mutants decreased,showing that the imidacloprid made CSP3 spread.The binding with 8 small molecules indicated that CSP3 had stronger binding with DL-lysine and oxytetracycline,and other 5 small molecule could also bind except sulfanilamide.By studying the effect of imidacloprid on CSP3 binding with other ligands,it was found that imidacloprid promotes the binding of CSP3 to HOB and oxytetracycline at low concentrations and inhibited the binding of CSP3 to ligands at high concentrations.It's contrast to the floral material?-ionone,3,4-dimethylbenzaldehyde and the DL-lysine,K_A appeared to decrease compared with the original K_A value,which indicated that imidacloprid inhibited the binding of both with CSP3 protein.4.The protein purification and the ligand binding characteristics of ASP1.The results showed that with the increase of temperature,the K_A value gradually increased and the quenching changed from static to dynamic quenching.The main forces were hydrophobic forces and had not changed.The binding of ASP1 with HOB under different concentrations of imidacloprid showed a tendency of"W",but it was generally in a promoting state.Meanwhile,it was found that ASP1 and 9-ODA did not bind at different temperatures and pH as they both bound to HOB.With the pH was constant,the binding mechanism of ASP1with HOB was static quenching from 300K to 310K and the binding force was hydrophobic.However,at the same temperature,the lower the pH was,the larger the K_A and the stronger affinity were.