Cloning, Expression And Tissue-Localization Of The General Odorant Binding Protein â…¡ Form Plutella Xylostella

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera, Plutellidae), is one of the most destructive insect pests of crucifers, including various important oilseed and food crops. The traditional control methods of pesticides are not only an environmental hazard but also easy to make insects become resistant. Odorant binding protein (OBP) connected with odor molecules is the first reaction in the olfactory system of insects. The olfactory system of insects, a highly specific and sensitive chemical detector, can detect, recognize and transfer chemical information from the environment and triggers specific behavioral reactions, such as locating partners, food sources and oviposition and hibernation sites, and to avoid dangerous situations or unsuitable habitats and hosts. So the use of host plant odors has become one of the most ideal pest control strategies alternative to the use of insecticides. The study of the olfactory system of P. xylostella aims at investigating pesticides which would not pollute the environment.In the present study, a cDNA sequence of general odorant binding protein 2 (PxGOBP2) was cloned by RT-PCR using specific primers from the antennae of P. xylostella, Genebank accession number: GU371720. After sequence analysis, a 492 bp open reading frame encoded 163 amino acid residues, aligning under the corresponding nucleotide sequence, with a predicted MW of 18.5 KD and pI of 5.32. The deduced amino acid sequence contains 6 conservative Cystine residues, linked to form three disulfide bonds, which is the typical characteristic of odorant binding protein. According to the known sequence, prokaryotic expression vector of PxGOBP2 gene was constructed and successfully expressed in E.coli by inducing. The expressed products were consistent with the size of predicted recombinant protein by Western blot. Polyclonal antiserum, raised against the purified PxGOBPII fusion protein, was prepared by injecting a male New Zealand White rabbits. ELISA assay showed that antibodies were produced in rabbit serum 60 d after immunization and the titer of antiserum was 1:128,000.Temporal-spacial expression and tissue-specific analysis study on general odorant binding protein gene in diamondback moth was carried out by RT-PCR and Northern blot. The result shows that PxGOBP2 gene was abundantly expressed in the antennae of adult P. xylostella and there is no much difference between male and female. The transcription of PxGOBP2 gene was not detected in larvae, pupae and adults tissue except the head. Western blot analysis showed that protein encoded by PxGOBP2 gene was detected in the antennae tissue, the same as the results of RNA transcription analysis. PxGOBP2 gene transcription analysis and localization studies provide important data for further research on the function of the protein.