【摘要】：Plutella xylostella is one of the major pests to the worldwide cruciferous vegetable and oil plant. The world loses hundred millions of crops because of the P. xylostella each year. P.xylostella is the earliest founded in pesticide-resistant Lepidoptera pests and in the last decades its pesticide-resistance has significantly enhanced. Therefore, it is necessary to develop a safe and efficient new method to control the pest. Insect pheromone has been successfully applied to pest control for decades, but the molecular mechanisms for the pathway of insect pheromone has not been studied clearly. Previous studies implied that pheromone binding protein played an important role between insects and pheromone, thus, the pheromone binding proteinⅠwas cloned and its expressional profile was studied in this thesis.
A cDNA clone of P. xylostella pheromone binding proteinⅠ(PxPBPⅠ) was cloned by RT-PCR using specific primers from the antennae of P. xylostella, (submitted to GenBank ,Accession No: FJ201994.1). Sequence analysis showed that it was a 495 bp open reading frame and encoded 164 amino acid residues with a predicted MW of 18. 58 KD. The deduced amino acid sequence contains 6 conservative cystine residues and linke to form three disulfide bonds, which is the typical characteristic of odorant binding protein.
The prokaryotic expression vector of pMAL-c4E-PxPBPⅠwas constructed and PxPBPⅠsuccessfully expressed in E.coli.The expressed product confirmed by western blot was then used for preparation the polyclonal antibody. The polyclonal antiserum raised against the purified PxPBPⅠfusion protein was prepared by injecting a male New Zealand rabbits. ELISA assay confirmed that antibody was produced in rabbit serum 60d after immunization and the titer of antiserum was 1:512,000.
Temporal-spacial expression and tissue-specific analysis study on PxPBPⅠgene in vivo was carried out by RT-PCR. The result showed that PxPBPⅠgene was abundantly expressed in the head of P. xylostella male adult. The transcription of PxPBPⅠgene was not detected in female adult, larvae and pupae except for the male head. Western blot analysis showed that protein encoded by PxPBPⅠwas detected only in the male adult head tissue. This protein expression profile was consistent with the results of transcription analysis. The cloned PxPBPⅠgene and its expressional analysis layed an important basis for further study on the function of the protein.