Functions And Physicochemical Basis Of Pheromone-binding Protein PBP2 And Odorant-binding Protein OBP9?11 From Ectropis Obliqua

In the process of insect reproduction,Lepidopterous bisexual insects often regulate their mating behavior through the delicate recognition of sex pheromones-that is,the newly emerged virgin female releases sex pheromone molecules in the air,which can be sensed acutely and accurately by distant male homologous insects.The recognition of this sex pheromone by males is closely related to a large number of pheromone-binding proteins(PBPs)in antennal receptor lymph,and PBPs belong to odorant-binding proteins(OBPs)family.As the main lepidoptera pest in tea garden,tea geometrid(Ectropis obliqua Prout)larvae seriously affect the yield and quality of tea.In order to study the mechanism of pheromone sensation between male and female of tea geometrid and the olfactory sensation mechanism of male moths to tea volatiles,PBP2,OBP9 and OBP11 expressed abundantly by male antennae of tea geometrid were taken as the research objects,and their physiological functions were deeply studied by biochemical and molecular biology techniques.On this basis,the physicochemical mechanism of PBP2 perceiving two main Type ? sex pheromone components(Z,Z)-3,9-6,7-epoxy-octadecadiene(Z3Z9-6,7-epo-18:Hy)and(Z,Z,Z)-3,6,9-octadecatriene(Z3Z6Z9-18:Hy)of tea geometrid was deeply analyzed.This study not only provides a theoretical basis for the mechanism of other insects' perception of Type ? sex pheromones,but also helps to provide a new idea for the interaction of multiple pheromone components in ecosystems.The results are as follows:1.Full-length cDNA sequences of EoblPBP2,EoblOBP9 and EoblOBP11 genes were cloned successfully,and their amino acid sequences,physicochemical properties,secondary and tertiary structures and homology were analyzed.It was found that they conformed to the basic characteristics of OBPs,including protein size,acidity and alkalinity,as well as six Cys.Evolutionary tree analysis revealed that EoblPBP2 belonged to the PBP protein family,while EoblOBP9 and EoblOEBP11 belonged to the OBP protein family.2.Western Blot identified the specific expression of EoblPBP2,EoblOBP9 and EoblOBP11 proteins in the antennae of tea geometrid.Immunofluorescence localization confirmed that EoblPBP2 was located in the long hair sensilla of the male tea geometrid antennae,and also distributed in scales.3.The characteristics of EoblPBP2,EoblOBP9 and EoblOBPll binding with two Type ? sex pheromone components or tea volatiles were analyzed by fluorescence competitive binding experiments.It was found that these three proteins could recognize Z3Z9-6,7-epo-18:Hy and Z3Z6Z9-18:Hy,but the main function of EoblPBP2 was to identify the sex pheromone,while EoblOBP9 was to identify tea volatiles,and EoblOBP11 was to take both into account.4.Multispectral,thermodynamic,docking and site-directed mutagenesis indicated that the major sex pheromone component Z3Z9-6,7-epo-18:Hy is more susceptible to pH-tuned than the minor component Z3Z6Z9-18:Hy,whereas Z3Z6Z9-18:Hy seems to be more susceptible to temperature and amino acid mutations than Z3Z9-6,7-epo-18:Hy.5.EoblPBP2 can recognize imidacloprid,but it is not affected by the change of pH value.In the presence of sublethal dose of imidacloprid,the binding ability of EoblPBP2 to two Type ? sex pheromone components was enhanced.