Functional Study On Pheromone Binding Proteins Of Ostrinia Furnacalis (Guenée)

Asian corn borer (ACB), Ostrinia furnacalis (Guenee), which is the main corn pest in china, affects the production and quality of maize seriously. ACB is the steam borer, and it is difficult to control. Insects regulate behaviors using olfactory mechanism, therefore, the host recognition mechanism research is an important approach of ACB regulation. At present, the pheromone control with the characteristics of green, safe, high sensitivity, has became an advanced research hotspot, but the ACB olfactory mechanism was not fully studied. In order to have a deeper understanding of the olfactory mechanism of ACB, the important roles of pheromone binding proteins (PBPs) in mating, looking for host and interspecies identification have been studied, to applicate sex pheromones and plant volatiles for monitoring and managing ACB. O. furnacalis PBP1-5 were used to detect its function and application in ligand affinity, key amino acid residues, immunolocalization and electrophysiological aspects. This research will provide theoretical basis and new ideas for the prediction and prevention of ACB. The results are as following:1.5 PBPs gene coding sequences were amplified by RT-PCR, after prokaryotic expression and purification, the affinity of recombinant OfurPBP1-5 with 1-NPN and sex pheromones were measured by fluorescence competitive binding assays. Binding results revealed that (Z/E)12-14:OAc of O. furnacalis bound strongly with OfurPBP3, (Z/E)11-14:OAc of Ostrinia nubilalis (Hubner) showed strong binding ability with OfurPBP4 and OfurPBP5, but the binding affinity between OfurPBP1-5 and E11-14:OH, Z9-14:OAc were not detected. Thus, O. furnacalis PBP3 may participate in the recognition of O. furnacalis sex pheromones, while PBP4 and PBP5 for O. nubilalis sex pheromones.2. SWISS-MODEL was used to model the 3-dimensional structure. Four key amino acid residues (Phe12, Ile113, Ile52 and Leu94) were predicted through the molecular docking of OfurPBP3 to (Z/E)12-14: OAc using CDOCKER. Site-directed mutagenesis and fluorescence competitive binding assays results showed the binding affinities of mutants with (Z/E)12-14:OAc and (Z/E)11-14:OAc reduced obviously compared with the wild-type OfurPBP3, which is demonstrated Phe12, He113, Ile52 and Leu94 of OfurPBP3 are important for the efficient binding of cognate sex pheromones.3. The distribution of recombinant proteins OfurPBP1-5 in antennal sensillas were observed using immunolocalization, the results showed that OfurPBP1 and OfurPBP2 were labeled in sensilla trichodea, OfurPBP3 and OfurPBP5 were labeled in sensilla basiconca, OfurPBP4 was labeled in sensilla styloconica, respectively. Meanwhile, OfurPBP1-5 were labeled in sensillum lymph instead of the dendrite.4.6 sex pheromones and 19 corn volatiles were used for electroantennogram (EAG) to directly detect the EAG responses. The response trends were observed in different concentration gradient and indicated that 1 μg/μL was the best concentration to be tested by EAG. About 19 corn volatiles were tested under this concentration by EAG. Moreover, the responses of different ratio sex pheromones were tested in different concentration gradient. Results revealed that the most effective stimulus to male and female moth antenna was 1-Octen-3-ol, and nonanal, respectively. Male moth antenna showed strong responses to ACB sex pheromones E12-14:OAc and Z12-14:OAc, while EAGs elicited by antagonist to female moth antenna was greater in comparison with sex pheromones. For both male and female moth antenna, EAG response values increased in a dose-dependent fashion for sex pheromones and corn volatiles in the concentration of 0.01 μg/μL～1μg/μL. EAGs elicited by sex pheromone declined as the ratio of E12-14:OAc increased.