Tissue Specific Expression Profile Of The Pheromone Binding Protein Gene In Cydia Pomonella (L.)(CpomPBP2)

Cydia pomonella (L.), causing serious economic loss to apple industry, is importantquarantine pest. Biological control, such as using of low concentration of pheromone, hasbeen used to control C. pomonella which has instead of insecticides control caused resistanceincreases of codling moth, and researches showed that control C. pomonella through using oflow concentration of pheromone is an effective method.C. pomonella adult to forage, get a partner and search spawn ground by the olfactorysystem. In insect antenna recepting mechanism, the insect exchange information with outsidethrough interaction between corresponding molecules and odorant bindingproteins(OBPs)firstly which as a "keys"to open the door from insect to the natural world. Thepheromone-binding protein2(CpomPBP2) of C. pomonella which consist in trichoidsensillaof the male antennae, is an important part of multigene family of odorant bindingprotein(OBP). In order to better understand the structure and function of CpomPBP2, and toprove C. pomonella exchanging mechanism between male and female pheromones, providetheoretical basis for its prevention and control, the method of molecular cloning andprokaryotic expression be used in experiment.We cloned a pheromone binding protein (PBP) gene from the antennal of C. pomonellaby reverse transcriptase-polymerase chain reaction (RT-PCR).The cDNA is a510bp openreading frame, which encodes a protein of169amino acids.In order to express the protein of CpomPBP2, the DNA segment was inserted into theexpression plasmid pET-28a(+) to construct a recombinant expression plasmid. TheSDS-PAGE result showed that the cloned recombinant CpomPBP2was expressed in the formof clusion bodies in E. coli BL21(DE3) with a molecular weight of20kD. The western blotshowed that the recombinant CpomPBP2had satisfied immunobiological activity. Under the1mmol/L IPTG and8h induction condition, plenty of the recombinant protein could beobtain.The polyclonal antiserum raised against the purified CpomPBP2fusion protein was prepared by injected a male New Zealand rabbits. ELISA assay confirmed that antibody wasproduced in rabbit serum after immunization and the titer of antiserum was1:512,000.Temporal-spacial expression and tissue-specific analysis study on CpomPBP2gene invivo was carried out by RT-PCR. The result showed that CpomPBP2gene was abundantlyexpressed in the head of codling moth male adult. The transcription of CpomPBP2gene wasnot detected in chest, abdomen, wing and foot, Western blot analysis showed that proteinencoded by CpomPBP2was detected only in antenna and head tissue.