Ultrastructural Observations On Adult Antennal Sensilla And Cloninq Sequence Analysis Of A Pheromone Binding Protein Gene Of The Codling Moth, Cydia Pomonella (L.)

The codling moth, Cydia pomonella(L.)is a serious invasive pest in fruit orchards, as itslarvae cause great damage in apple, pear, peach and apricot. This pest has so far spread toXinjiang, Gansu, Ningxia, Inner Mongolia and Heilongjiang Provinces, and it is a seriousthreat to China's apple industry. The olfaction system plays a critical role in the mate-finding,location of food sources and oviposition sites, and so on, and this feature can be used forpopulation monitoring, mass trapping and mating disruption of codling moth. With the insighton the molecular mechanism of the insect olfaction perception, it will be facilitated formolecular designing of effective attractant or deterrent to the key olfactory proteins.Perception of sex pheromones or other semiochemicals of insects is mediated by proteinslocated in sensory organs of the antennae, such as odorant binding proteins, odorantdegrading enzymes and olfactory receptors. Odorant binding proteins are one of the mostabundant classes of proteins in the insect sensory organs, they might represent novelinteresting targets for pest management. In this paper, the morphology and ultrastructure ofthe antennal sensilla of adult C. pomonella was studied by scanning electron microscopictechniques. And we cloned and characterized a pheromone binding protein gene (CpomPBP2)from C. pomonella. The main results are as follows:1. Eight types of sensilla were identified on the antenna of both sexes: sensilla trichodea(ST I and ST II), sensilla chaetica, sensilla coeloconica, sensilla styloconica, sensillaauricillica (SA I and SA II), sensilla basiconica, sensilla squamiformia, and B hm bristles.Sensilla trichodae were the most abundant sensilla. Sensilla squamiformia were present onthe dorsal side of the antenna, sensilla styloconica were present on the ventral side of eachflagellar subsegment, while others were found on both ventral and dorsal sides. The types andnumber of the antennal sensilla were nearly identical between male and female moths. Andthe distribution of each sensillum type was of certain regulation.2. We cloned a pheromone binding protein (PBP) gene from the antennal of C.pomonella by reverse transcriptase-polymerase chain reaction (RT-PCR), rapid amplificationof cDNA3' ends-PCR (3' RACE-PCR) and thermal asymmetric interlaced PCR (TAIL-PCR), which was named CpomPBP2. The results of sequencing and bioinformatics analysis showedthat the open reading frame (ORF) of CpomPBP2was510bp in length, encoding169aminoacids. The N-terminal initial25amino residues were predicted as a signal peptide. Thededuced amino acid sequence of CpomPBP2revealed mature proteins of144amino acidswith a molecular mass of16.45kD and an isoelectric point of5.09, and it contained sixcysteine residues in conserved positions relative to other known PBPs. The alignment of themature CpomPBP2with other lepidopteran PBPs showed high sequence identity.In order to ascertain the gene structure of CpomPBP2, a PBP gene was cloned andsequenced from genomic DNA of C.pomonella. We identified two introns within theCpomPBP2coding region. They are located between Glu22and Leu23(Intron1;1,655bp)and between Ala82and Asp83(Intron2;862bp), respectively.Using the TAIL-PCR approach, a portion of the5' upstream regulatory region ofCpomPBP2gene was isolated from the genomic DNA of C.pomonella. The promoterprediction results showed that the5' upstream region of CpomPBP2included not only thecore promoter sequences, but also several transcription factor binding sites (AML-1, BR-C-Z,CF2-II, Dfd, E74A, Ftz and Hb), predicted transcriptional start site is located at544bpupstream of initiation codon.