The Function Of Odorant Binding Proteins And Pheromone Receptors In The Hyphantria Cunea(Drury)

Hyphantria cunea(Drury),native to North America,is a notorious worldwide quarantine pest So far,It has become one of the most serious alien invasive species need to be cotrolled urgently in China.Insects rely on their highly sensitive olfactory sensory system to survive and reproduce.Male moths can detect sex pheromones released by female moths even miles away.According to the different chemical structure,the sex pheromones of moth insects can be divided into two categories:straight chain fatty acid derivatives of Type I and epoxy compounds of polyolefin of Type II.Sex pheromone of Hyphantria cunea a mixture of Type I and Type II.In this study,biochemical and molecular biological techniques are used to study the functions of sex pheromone binding proteins,general odorant binding proteins and pheromone receptors in Hyphantria cunea.In particular,the specific binding and accurate recognition of Type I and Type ? sex pheromones are helpful to get a better understanding of the evolutionary mechanism between pheromone structure and olfactory communication.In this study,recombinate HcunPBPs and HcunGOBPs protein were obtained by prokaryotic expression system,the binding ability of PBPs and GOBPs to sex pheromone and plant volatiles of Hyphantria cunea was determined by fluorescence competitive binding assay.The response spectrum of HcunPR3 to Hyphantria cunea sex pheromone was studied by Xenopus oocyte heterologous expression system combined with two-electrode voltage clamp technique.The main results are as follows:1.Functional Analysis of pheromone binding proteins in Hyphantria cunea.The relative expression of three HcunPBP in different tissues of adults was studied by qPCR.The results showed that the three HcunPBP were highly expressed in the antennae of female and male.Among them,the expression level of HcunPBP1 male was 6.5 times that of female,the expression of HcunPBP2 male was 1.7 times that of female,and the expression of HcunPBP3 male was 4.2 times higher than that of female.The binding ability of three PBPs to four sex pheromone components and 24 plant volatiles was determined by prokaryotic expression system,histidine label protein Ni magnetic bead purification and fluorescence competitive binding assay.The results showed that the binding ability of three PBP to Type I sex pheromone components(Ki<1.0 ?M)was significantly different from that of Type II sex pheromone components.Among them,the binding ability of HcunPBP2 to Type II sex pheromone components 1,Z3,Z6-9S,10R-epoxy-21Hy was strong,HcunPBP3 had moderate binding ability,but HcunPBP1 had no binding ability.Both HcunPBPl and HcunPBP3 had moderate binding ability to Type ? sex pheromone components Z3,Z6-9S,10R-epoxy-21Hy,but HcunPBP2 had no binding ability.In addition,among the 24 plant volatile compounds tested,3 PBP could selectively bind to some plant volatiles and had strong binding ability(Ki<10.0 ?M).Based on our results,we speculate that the three HcunPBP have obvious functional differentiation,and there is obvious preference for the sex pheromone components released by female moths.In addition to feeling the sex pheromone of female moths,PBP can also be involved in the perception of some plant odorants.2.Function Analysis of General odorant binding proteins in Hyphantria cunea.According to the antennal transcript data of Hyphantria cunea,two HcunGOBP gene sequences were cloned,and the expression profiles and odorant binding characteristics of the two GOBP genes were systematically analyzed.The results of qPCR showed that both HcunGOBP1 and HcunGOBP2 were highly expressed in the antennae of male and female moths,and the expression levels were similar between male and female moths.However,the expression of HcunGOBP2 in female antennae was 4.6 times higher than that in HcunGOBP1,and in male antennae was 5.1 times higher than that in HcunGOBP1.In addition,only very low or no expression was found in the head,chest,abdomen,foot,and wing tissues without tentacles.The results of fluorescence competitive binding test showed that HcunGOBP2 had strong binding ability to Type I pheromone components,but not to Type II sex pheromone components,and HcunGOBPl had no binding ability to the four pheromone components.Among the 24 tested plant volatiles,two HcunGOBP had different binding ability to some plant volatiles(Ki=9-50 ?M),but not to most of the selected plant volatiles.In conclusion,the two HcunGOBP have different functions,HcunGOBP1 and HcunGOBP2 correspond to different plant odorant spectra,and HcunGOBP2 may be involved in pheromone recognition.3.Functional AnaHcunlysis of pheromone receptors in Hyphantria cunea.The full-length cDNA of Hyphantria cunea typical olfactory receptor Orco and sex pheromone receptor PR3 gene was cloned by homologous cloning technique.The recognition specificity of HcunPR3/Orco for four sex pheromones in Hyphantria cunea was determined by Xenopus oocyte expression system combined with two-electrode voltage clamp technique.The results showed that HcunPR3/Orco showed obvious physiological response to one of the sex pheromone components of Type ?:1,Z3,Z6-9S,10R-epoxy-21Hy.When the concentration of 1,Z3,Z6-9S,10R-epoxy-21Hy was 10-3 M,HcunPR3/Orco produced the maximum response,EC50=l.74x-0-5 M,but did not show physiological response to Type I pheromone component and another Type II sex pheromone component.The results of this study lay a foundation for further elucidating the molecular sensory mechanism of Type ? sex pheromone,and are of great significance for the study of olfactory sensory mechanism of other moth insects with Type II sex pheromone.