Cloning And Immunolocalization Of The Pheromone Binding Protein 2 From Atrijuglans Hetaohei Y.

As one of the major pests on walnuts, Atrijuglans hetaohei seriously affect the walnut production in China. By reason of boring behavior of larvae, huge damage for walnuts has been caused and walnuts will be early fall. At present, there is still lack of an effective control for Atrijuglans hetaohei. Sex pheromones play a critical role in mate-finding for insects, then by using those substances and their analogues can make good effect for population monitoring, mating disruption and mass trapping of pests. Through the intensive study of sex pheromone binding protein, the mechanism of interaction between sex pheromone and related protein can be reasonably illustrated, and provid theoretical foundation for insect prevention and monitoring through sex pheromone. In this study, a pheromone binding protein was cloned from Atrijuglans hetaohei. Its expression profile was studied by immunohistochemical analysis. The results are as follows:1. The open reading frame of AhetPBP2 was cloned by using RACE with the cDNA which was synthesized with total RNA extracted from antenna of 3-4 days newly emerged Atrijuglans hetaohei. The length of AhetPBP2 gene is 923 bp. The open reading frame is 504 bp, which encode a protein of 167 amino acids with the molecular weight of 19.26 ku and isoelectric point of 5.47.2. AhetPBP2 is a member of PBPs. It shows high identity with other lepidopteran PBPs. Sequence alignment indicated that the AhetPBP2 has 6 cysteine conserved site which is a typical characteristic of OBPs family.3. AhetPBP2 was expressed in E. coli BL21(DE3) and the polyclonal antibody was prepared by immunizing rabbit. By inducting of 1 mM IPTG for 10 h, soluble recombinant protein was high expressed in E. coli. The polyclonal antiserum was prepared by immunizing a male New Zealand white rabbit with the purified recombinant protein. ELISA showed that the titer of the polyclonal antibody was 1024×103.4. AhetPBP2 was found in a part of the antennal sensillas. Immunohistochemical analysis on antennae of Atrijuglans hetaohei was performed with polyclonal antibody of AhetPBP2. Processed antennae was observed under a fluorescent microscope. The result showed that AhetPBP2 was localized in a part of antennal sensillas of Atrijuglans hetaohei. We suggest that these antennal sensillas may have an important role in the perception of pheromone.