Genes Cloning And Functional Identification Of Odorant Receptors In Pea Aphids,Acyrthosiphon Pisum

External environment is a library of blended and complex odors.Insects recognize external volatiles using their sensitive olfactory system and then accomplish various behavioral responses,such as,host plant and oviposition-site selection,mate recognition,and danger avoidance.Acyrthosiphon pisum is an important agricultural pest and endangers plants mainly by sucking sap and spreading plant viruses.A.pisum locates host plants by detecting plant volatiles,and also uses its sensitive olfactory system to avoid danger.When attracked by predators and parasitic wasps,A.pisum would release droplets containing alarm pheromone from the rear side of abdomen,and near individuals would detect alarm pheromone and escape or fall directly from plants.The component of alarm pheromone of many aphids is(E)-?-farnesene(EBF),including A.pisum.This thesis is a preliminary study on aphid recognition mechanism of host plant volatiles and alarm pheromone.The results would be helpful to understand how A.pisum locate host plants and avoid danger,and also lay a theoretical foundation for screening effective attractant and repellent according to odorant receptors.The main results are as follows:1.Identification,screening and functional analysis of EBF receptors from A.pisum.79 and 45 OR genes have been identified from pea aphid and cotton aphid genome,respectively.After sequences alignment and phylogenetic analysis of these genes,we chose ORs shared more than 65%of their amino acids between the two species as candidate ORs for EBF perception.In this way,15 candidate ORs were selected.Then we cloned the full length of the 15 ORs from the antennae of pea aphids.After heterologously expressed in the Xenopus oocytes,two-electrode voltage clamp was used to screen the ApisORs could be activated by EBF.The results showed that only ApisOR5 could be activated by different concentration of EBF and the EC50 was 2.12×10-7 M.The third instar nymphs were chosen to inject with dsRNA of ApisOR5,EAG and behavioral experiments were conducted at 72h after injection.The EAG results showed that the response of treated aphids to EBF decreased significantly.The behavioral experiments showed EBF perception was impaired after knocking down ApisOR5 in A.pisum.ApisOR5/Orco expressed oocytes were tested with 61 volitile compounds with two-electrode voltage clamp and the results showed that ApisOR5 could be activated by geranyl acetate,besides EBF.Behavioral experiments showed that A.pisum was strongly repelled by geranyl acetate.2.Genes cloning,expression and functional analysis of ApisOR4 and ApisOR23 from A.pisum.The full-length of ApisOR4 gene was 1161 bp,encoding 386 amino acids.The full-length of ApisOR23 gene was 1245 bp,encoding 414 amino acids.ApisOR4/Orco and ApisOR23/Orco were functional analyzed after heterologously coexpressed in the Xenopus oocytes with two-electrode voltage clamp,the results showed that ApisOR4 was tuned to 4-Ethylacetophenone,Salicylaldehyde,4-Ethylbenzaldehyde,3-Vinylbenzaldehyde,(1S)-(-)-Verbenone,(S)-cis-Verbenol,(-)-Bomeol and(1R)-(-)-Myrtenal.ApisOR23 responded to cis-2-Hexen-l-of,1-Heptanol,4-Ethylacetophenone,Hexyl acetate and trans-2-Hexen-1-al.Tissue expression pattern showed that ApisOR4 and ApisOR23 were only expressed in antennae.EAG results indicated that the ligands of ApOR4/Orco could elicit electrophysiological response of pea aphid antennae.