Binding Characterization Of OBP1 And OBP2 In The Scleroderma Sichuanensis Xiao And Behavior Verification

In nature,parasitic bees use olfactory information to communicate and play an important role in host positioning,finding spouses,laying eggs,and avoiding harmful substances.Parasitic bee olfactory system,there are a variety of olfactory protein,in which the combination of odor-binding protein(OBPs)and odor molecules for parasitic wasps and external odor substances in the first step.In order to study the olfactory mechanism of Scleroderma sichuanensis Xiao,it is clear that the binding ability of its odor-binding protein to host and environmental volatile odor substances.In this paper,OBP1 and OBP2 genes were expressed and purified by using the known OBP1 and OBP2 gene sequences.The recombinant plasmids were used to express and purify OBP1 and OBP2 proteins,and then the binding properties of the purified proteins were studied.The relationship between the binding properties of the two odorant binding proteins and the odor of the S.sichuanensis Xiao was analyzed.The main research results are as follows:(1)OBP1 and OBP2 proteins were successfully expressed and purified by using the primers of OBP1 and OBP2 gene sequences registered on NCBI,and high purity proteins with size of about 18 kDa and 20 kDa were obtained respectively.(2)The binding ability of OBP1,OBP2 and 18 kinds of volatile odor ligands was analyzed by fluorescence competition experiment.The results show that OBP1 and OBP2 are mainly compatible with terpene compounds,but not with alkanes and aldehydes.OBP1 and OBP2 proteins were able to bind to seven kinds of odor ligands(IC50 not more than 50 μmol/L).OBP1 binds best with ocimene by the binding constant of 12.82 μmol/L,then is a-pinene by the binding constant of 15.57 μmol/L;OBP2 binds best with(-)-a-pinene by the binding constant of 13.52 μmol/L,then are ocimene and β-pinene by the binding constant of 14.38 μmol/L and 14.51 μmol/L.(3)The Y-shaped olfactometer was used to measure the behavioral responses of 18 species of volatile odor.The results showed that no learning experience of S.sichuanensis Xiao tended to terpenes,but had no obvious trend of alkanes and aldehydes,Which tended to choose camphene、(+)-3-Carene、(+)-a-pinene、ocimene and(-)-α-pinene with the rate of 60%and have a significant difference with contrast(P<0.05).By testing the tropism choice of early emergenced S.sichuanensis Xiao which have learning experience,the result indicate that the learning experience can increase the tendency of selectance rate.(4)The two test showed that S.sichuanensis compatibility strength followed by Ocimene,(-)-α-pinene,β-pinene,camphene,(+)-3-carene and(+)-α-pinene,while benzene and β-caryophllene are only one protein in the affinity.In the choice of the behavior trend rate of 60%are ocimene,camphene,(-)-α-pinene,(+)-3-Carene and(+)-α-pinene.The results of the two tests showed that(-)-α-pinene,(+)-α-pinene,camphene,(+)-3-carene had a strong reaction,indicating that the selective binding of OBP1 and OBP2 is the basis for the behavior of the odor.