| In a complex natural environment,insects are sensitive to identify various chemical substances by chemical communication systems and react.There are two kinds of acidic,low molecula r weight proteins in insect chemosensory system,namely Odorant binding proteins(OBPs)and chemosensory proteins(CSPs).Apis cerana cerana Fabricius is good at collecting scattered nectar,and has a sensitive chemosensory system.Antenna is an important chemical communication organ for bee,so it has important theoretical significance to understand the mechanism of chemical communication in A.cerana cerana.In addition,the bee has attracted widespread attention because of the high toxicity of the neonic otinoid insecticides.It has been shown that the sublethal doses of neonicoti noid still interact and inhibit the function of olfaction,chemistry and learning of bees.In the early stage,CSPs and OBPs were identified in antenna of bee and were abundantly expressed.T hrough multiple fluorescence experiments and thermodynamics,we analyzed the binding properties between these proteins with natural ligands as well as neonicotinoid insecticides(imidacloprid).T he molecular docking and site directed mutagenesi s were used to identify the key amino acid residues in the binding process of CSP1 to ?-ionone and imidacloprid.T he results showed that imidacloprid at sublethal doses could inhibit the physiological function of CSP1 to ?-ionone.What's more,the gene expression analysis and site directed mutagenesis experiments were performed on a member of the OBPs family,ASP2,which specifically expressed in antenna,and the key amino acid sites were determined in the binding process between ASP2 with ?-ionone and imidacloprid.Finally,the subcellular localization of CSP1 in antennal sensilla was analy zed by immune-colloidal gold electron microscopy,which enriched the function of the antennae specific proteins in A.cerana cerana.T he main results are listed as follows:1.With the titration of ?-ionone(floral material),HOB(queen pheromones),imidacloprid and acetamiprid(neonicotinoid insecticides),the fluorescence intensity of CSP1,CSP2 and CSP4 could be considerably quenched with strong binding.T he results of synchronous fluorescence spectra showed that the fluorescence intensity of CSP1,which is specifically expressed in the antennae,was mainly derived from tryptophan.T he results of UV absorption spectra showed that the binding mode of CSP1 with ?-ionone and imidacloprid all were dynamic binding.The results of CD spectra showed that the combination of ?-ionone and CSP1 could increase the ?-helix,and the combination of imidacloprid and CSP1 could lead to the decrease of ?-helix.Molecular docking was used to simulate the 3D model of CSP1,which was combined with ?-ionone and imidacloprid.Finally,the imidacloprid at sub-lethal doses was confirmed can significantly inhibit the sensibility of the bee to ?-ionone.2.Determination of key amino acid sites and site-directed mutagenesis in CSP1 binding function.3 mutant proteins(CSP1-F44 G,CSP1-Q63 G and CSP1-F44Y26G)were analyzed by flu orescence quenching spectrum,thermodynamics and molecular docking.T he results show that the binding ability of three mutants with ?-ionone were weak,KA were decreased by 60.82 %,46.80 %,51.38 %,respectively,therefore,F44 and Q63 is the important amino acid residue,which maintain the hydrophobic interaction and electrostatic force between CSP1 and ?-ionone.In the experimental results of CSP1 with imidacloprid,the KA of CSP1-F44 G and imidacloprid decreased by 5.1 %,the KA of CSP1-Q63 G and CSP1-F44Y26 G respectively increased by 3.8 % and 20.1 % compared to wild CSP1.T he results showed that there was a great difference between the predicted results and docking,and the specific reasons need to be further confirmed.3.Verification of gene sequence va riability of odorant binding protein ASP2 in A.cerana cerana.T he ASP2 in Chinese bee was cloned into T vector,and randomly selected 10 single colonies.In the 10 colonies,the 4 sequence s(ASP2-1,4,6 and 10)was consistent with NCBI,and the bases of the other 6 sequences(ASP2-2,3,5,7,8,and 9),had changed in one or more.T he T(97)changed to C,and the G(30)changed to T,all of which appeared to be 2 times,suggesting that ASP2 might have a variety of gene phenotypes.From the comparison of protein sequence,we found that the changes of ot her bases in 9 clones did not lead to amino acid residues change,except ASP2-3.So ASP2 although had different gene phenotypes,but it did not affect the protein function.4.Determination of key amino acid sites and site-directed mutagenesis in ASP2 binding with ligands.Five mutations were identified for ASP2,including ASP2-K51,ASP2-S123,ASP2-Y10,ASP2-M55 and ASP2-K51Y10.The results of fluorescence quenching spectrum(five mutant proteins and ?-ionone)showed that the KA of five mutants were decreased from 22.69 % to 43.59 % compared to wild ASP2.The results indicated that above 4 sites all play an important role in the binding of ASP2 and ?-ionone.M55 decreased by 43.59%,indicating that it is the largest amino acid.On the other hand,in the experiment of ASP2 combined with imidacloprid,the KA of S123,Y10,and K51Y10 were decreased from 25.58 % to 48.37 %,and the KA of ASP2-K51 increased by 17.12%,compared with the wild ASP2.It was suggested that S123 and Y10 play a key role in the binding of ASP2 to imidacloprid.5.T he antennal sensilla were observed by scanning electron microscope,and were found at least seven kinds of receptors,including: sensilla placodea(Sp),sensillum ampullaceum(Sa),campaniform sensilla(Scf),sensilla basiconica(Sb),sensilla trichoidea A B CD(St A B CD),in which Sp and St were the majority.The results of immuno-electron microscopy found that CSP1 was mainly expressed in the ancillary supporting cells around the sensilla placodea,while only slightly expressed in the inner of sensilla placodea.The results showed that CSP1 and ASP2,two antennal specific proteins in A.cerana cerana had different expression patterns,suggesting the functional diversity and complexity of the antennae specific proteins. |
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