Funcitonal Analysis Of Chemosensory Proteins In Hobtirchia Oblita Faldermann (Coleoptera: Scarabaeida)

A thorough study of insect olfactory mechanism and a complete explanation ofolfactory signal transduction pathway are the base of exploring the specific preventionand control of pest． The scarab beetle Holotrichia oblita Faldermann（Coleoptera:Scarabaeidae） possess predominance in the underground pests in thenorthern parts of China and cause great economical loss in result of there wide hostplant and covert habitat. The researches of distinguishing outside environmentalprinciple and chemosensory protein supply a new strategy for sustainablemanagement of H. oblita．In this study,the full length cDNA library from antennae ofH. oblita were constructed by SMART (Switching Mechanism At5’end of RNATranscript) technology, and hundreds of expressed tags(ESTs)were sequenced andanalyzed. The main results are as follows:1、 Antennal cDNA library was created from the female Holotrichia oblita2、 Preliminary characterization and isolation of cDNA from H. oblita antennallibraries identified4putative chemosensory proteins(CSPs)(Gen Bankaccession number: HQ683720、HQ688991),and odorant-binding protein(OBPs)(Gen Bank accession number: HQ688992、HQ688993)3、 RecHoblCSPs were expressed using prokaryotic expression system, removedHis-tag in laboratory,and then purified by using fluorescence assays, we testedthe binding ability of the two HoblCSPs to50compounds with fluorescenceprobe1-NPN. The results showed CSP1and CSP2could widely bind the plantvolatile, but showed limited affinity to pheromone. The β-ionone was the bestligands of the two chemosensory proteins in binding assays.HoblCSP1havegood affinity to Eugenol which is a attractant to other scrabe beetle,HoblCSP2showed good and the plant volatile Methyl salicylate. Moreover,the ability of the two HoblCSPs is affected by the conformation, functional group and length of carbon chain of ligands.4、 Polyclonal Antibody of CSP1and CSP2were obtained from the serum ofrabbit and BALB/c mouse which were injected the removed His-tagHoblCSPs mix of Freund’s adjuvant. By using polyclonal antibody andelectron microscope, we observed the expression of the two HoblCSPs in thesensilla of antenna. Immunohistochemistry experiment revealed anti-CSP1and anti-CSP2could strain the section of sensilla placodeum and basiconica inboth sexes.