Immunolocalization And Ligand-binding Characterization Of Pheromone Binding Proteins From Cnaphalocrocis Medinalis

Olfactory system plays a critical role in locating host plant, mating behavior, seeking oviposition sites and other insect activities. Basing on the current study results, odorant binding protein(including pheromone binding protein) is a kind of important proteins in the olfactory reception, but the definite functions are not very explicit. The rice leaffolder, Cnaphalocrocis medinalis(Guenée)(Lepidoptera: Pyralidae) is one of the most important migratory pests of rice in China, but the olfactory mechanism of it is not clear. Here, based on gene cloning and protein expression, immolocalization of the pheromone binding proteins(PBPs) in the antennal sensillas were studied by scanning and transmission electron microscopy. And also, the binding properties of PBPs with different ligands and the correlation with the p H were analyzed. The main results are as follows:1. The prokaryotic protein expression and purification of Cmed PBP4Recombinant p ET-22b(+)-Cmed PBP4(contained a 444 bp open reading frame that encoded a protein of 147 amino acids with no signal peptide, M=16.4k D) was expressed in Escherichia coli,The supernatant was loaded into a Hi Prep TM QFF column and Hi Prep TM DEAE column and separated with a linear gradient of 0–500m M Na Cl in Tris-HCl(p H=8.0).The puri?ed protein was stored at-80 °C for preparation of antibody and the fluorescence competitive binding assays.2. Immunolocalization of Cmed PBP4 on antennal sensillasTotal proteins from male and female, and from different tissues of the adult, including abdomen, thorax, leg, and antennae, were extracted and standardized to 5mg/ml per sample. Using Western blot analysis, Cmed PBP4 were predominantly detected at the antennae only.The antiserum against Cmed PBP4 intensively labeled on triochoid sensilla, without other kinds of sensilla was labeled. In trichodea sensilla, the gold granules are very concentrated in the sensillum lymph bathing numerous dendrites. In addition, the cuticle(CW) and dendrites(D) of all sensillas are not labeled by any antiserum. The results imply that Cmed PBP4 might play an important role in the selectivity and specificity of pheromones reception.3. The fluorescence competitive binding assays of Cmed PBP4Fluorescence competitive binding assays were performed to determine the binding affinity of the Cmed PBP4 protein for for 41 chemicals(including 4 pheromones and 37 rice volatiles) at p H5.5 and p H7.4 using N-phenyl-1-naphthylamine(1-NPN) as a fluorescent probe.Competitive fluorescence binding assays showed that at p H7.4, Cmed PBP4 displayed a higher affinity for competitors than at p H5.5, at p H7.4 Cmed PBP4 bound a broad range of rice volatiles and pheromones, including 11 rice volatiles-Cyclohexanol, Nerolidol, Cedrol, Dodecanal, Ionone,(-)-α-Cedrene,(Z)-Farnesene, β-Myrcene, R-(+)-Limonene,(-)-Limonene,(+)-3-carene,and 3 pheromones-Z13-18:Ac, Z11-16:Al å'Œ Z13-18:OH,which would be the main ingredient of insect attractant.The results could provide a theoretical basis for undersanding of the function of PBPs in the olfactory and further developing of effective attractant.