Study Binding Characterization Of Chinese Honeybee's (Apis Cerana Cerana) Odorant Binding Proteins With Ligands And Binding Mechanism Of OBP12 With Imidacloprid (Hymenoptera: Apidae)

In the long course of evolution,the bees formed a complex and sensitive olfactory system,which is a prerequisite of recogniting chemical signals in swarm and distinguishing different odor molecules in the external environment.Apis cerana cerana Fabricius is an important local and native species in China.Compared with Italian bee Apis mellifera Ligustica,it has a keen olfactory ability,foraging small and dispersed nectariferous plants,defending strongly against ectoparasitic V.destructor and chalk-brood disease,and providing tolerance for low environmental temperatures.All the advantages are associated with its developed olfactory system,of which the odorant binding protein is one of the most important protein.So it is important to study and explore the physiological mechanism of Chinese honeybee's unique olfactory behavior based on odorant-binding proteins.In this study,based on the antennae transcriptome sequencing analysis,we identified the potential odorant-binding proteins Acer OBP4,Acer OBP10,AcerOBP12,Acer OBP14,AcerOBP18,AcerOBP21 DNA sequences.We obtained the open reading frame sequences of the five genes including AcerOBP4,10,12,14,21 by using RT-PCR.And real-time fluorescent quantitative PCR was used to analyse expression profiles of AcerOBP4,10,12,14,18 and 21 in six organizations containing antennae,head,chest,abdomen,leg,wing.The recombinant AcerOBP10,12,14,21 proteins were obtained in vitro using constructed prokaryotic expression vector p ET30-AcerOBPs.By using fluorescence spectra,AcerOBPs' binding profiles can be acquired with many kinds of ligands.After researching the binding and mechanism of interaction of AcerOBP12 with imidacloprid by Multiple Spectrometry,SWISS-MODEL,molecular docking Molegro Virtual Docker and site-directed mutagenesis were be used to determine the key amino acid of the combination.Finally,we made use of polyclonal antibodies by immunization,brain anatomy and immune-electron microscopy to carry on the subcellular localization of AcerOBP12 in the bee antennae,co-localization of AcerOBP12 and acetylcholine receptors in the brain.This study obtained the main results are as follows:1.The full-length cDNA sequences of AcerOBP4,10,12,14,21 were obtained,their open reading frame are 414 bp,450 bp,453 bp,408 bp,408 bp in length,and their GenBank ID are KP717059.1,KP717060.1,KP717061.1,KP717062.1,KP717063.1,respectively.The nuclear acid sequences analysis showed Acer OBP4,10,12,14,21 encoding 137?149?150?135?135 amino acid residues.SignalP 4.0 show all of the protein have obvious N-terminal signal peptide.AcerOBP4 and 10 have six conservative cysteines,while AcerO BP12,14,21 only have four conservative cysteines.Excepted AcerOBP4,the other proteins have and contain a conservative domain that comprised based on six ?-helices by online software PSIPRED and SWISS-MODEL analysised.Molegro Virtual Docker predict they have several cavities to combine multiple ligands.Besides,Phylogentic tree showed that the kinship of AcerOBPs amino acid sequences have high similarity with other OBPs of Apis And AcerOBP10 is more similar with pheromone-binding proteins,may belong to PBP-OBP supper family.2.The expression profiling of AcerOBP4,10,12,14,18,21 in different tissues of forager was measured by real-time PCR.The results revealed that AcerOBP4,10,12,14,18,21 are different.At overall lever,AcerOBP4 was expressed at a high lever of six genes,while AcerOBP10 was expressed at a low lever.In the antennae,AcerOBP4,14,21 are high expressed,and AcerOBP4 is as 20000 times much as AcerOBP10;in the brain and chest,AcerOBP4 and AcerOBP21 are expressed at a high levers;in the abdomen and foot,all AcerOBPs expression are low compared with other organizations;in the wing,AcerOBP4 and AcerOBP14 expression in relatively high levers.3.The recombinant AcerOBP10,12,14,21 proteins were obtained using constructed prokaryo tic expression vector p ET30-AcerOBPs,and conducted its expression in the optimized conditions in vitro.The recombinant protein with biochemical activities was purified by the method of N i2+-agarose affinity chromatograph.Using 1-NPN as a fluorescence probe,the binding capability of AcerOBPs with diverse bee pheromones and many kinds of plant volatiles were measured by competitive fluorescence assay.The result showed that AcerO BPs have different binding capability with different pheromones and plant volatiles.All of the four recombinant proteins have a strong combining ability with the queen pheromone methyl p-hydroxybenzoate HOB and generally flowering plants volatile odor molecules ?-ionone.So the HOB and ?-ionone are very important to physiological function of honeybee.In addition,AcerOBP10 can be combined wit h a variety of pheromones at a high affinity,further confirmed it is a pheromone binding protein.4.Multiple fluorescent spectra,synchronous fluorescence spectra and circular dichroism(CD)spectra were combined to clarify the binding process of AcerOBP12 with imidacloprid.Basically,fluorescence intensity of AcerO BP12 could be considerably quenched by imidacloprid,indicating that a complex was formed.And the complex is more stable in lower temperature,namely the process is a static quenching.By thermodynamic analysis,the fact ?H < 0,?S > 0 indicated the hydrophobic interaction and electrostatic force were main driving force in the low temperature,and the ?H < 0,?S < 0 in high temperature indicated the vander Waals and hydrogen bond as main driving force.Moreover,synchronous fluorescence spectra showed Trp amino acid of AcerO BP12 was intensed quenched by imidacloprid and its peak caused slightly red shift.CD spectra analysis showed the decrease of ?-helix after imidacloprid addition.This indicated the interaction of AcerOBP12 and imidacloprid changed the structure of protein and interactional microenvironment closed the Trp amino acid.5.Through the SWISS-MODEL and Molecular virtual docking(MVD),the protein structure can be predicted.And the specific site Lys117,Tyr78,Arg77,Tyr46,Asp28,Phe88 and Leu121 were determined to mutate from two aspects of hydrogen-bond interaction and energy magnitude.By site-directed mutagenesis,the mutant proteins were obtained.The result showed AcerOBP12-F88 G can make the binding constant from 5.4×104 L/mol to 3.5×104 L/mol,comfirmed it is an key amino acid.But the other mutant do not obviously changed the binding constant with imidacloprid.In a word,the energy of Phe88,not hydrogen-bond,keep the combine of AcerO BP12 and imidacloprid.6.Finally,the subcellular localization of AcerOBP12 in the forager's antennae sensilla,AcerOBP12 and acetylcholine receptor in the brain were labelled by immunofluorescence using polyclonal antibodies against AcerO BP12 and acetylcholine receptor,respectively.Test results,using ELISA assay,showed high antibody titer for polyclonal antibodies against AcerOBP12 and acetylcholine receptor.And speedy and clean brain anatomy makes it is possibly to study its physiological function.In conclusion,our studies laid an important foundation for the development and application of honeybee.And it is helpful for understanding olfactory mechanism to study odorant-binding proteins model.