Molecular Cloning And Sequencing Of CDNA Encoding Odorant Binding Protein In The Antenna Of Helicoverpa Assulta And Its Expression Vector Structure In Yeast

Insect can perceive certain chemical volatiles in the environment as the chemical information sources for its behaviroral reactions such as to look for mate, food and reproduction place. Insect odorant binding proteins (OBPs) are thought to deliver odors to olfactory receptors, and thus may be the first biochemical step in odor reception capable of some level of odor discrimination. And it's important for insect to transfer odor information in the environment and give rise to certain behavior response. In this paper, the gene of general odorant binding protein 1 and pheromone binding protein in the antenna of Helicoverpa assulta was cloned and expressed in Escherichia coli and yeast.After comparing the reported OBP nucleotide sequences of insects, which was obtained from GenBank/EMBL/DDBJ, the primer was designed. The cDNA encoding the general odorant binding protein 1 (named as Hass-GOBP2) and pheromone binding protein (named as Hass-PBP) was isolated from the male and female antenna of Helicoverpa assulta by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragment (the accession numberof Hass-GOBP1 is AY864774 in GenBank, the accession numberof Hass-PBP is AY864775 in GenBank,) was further cloned into pGEM-T Easy vector, respectively. The aimed fragment was digested by two restriction enzyme BamHI/ECoRI and then was joined with pGEX-4T-1 plasmid which was digested by BamHI / ECoRI ,too. And then constructed into expression vector pGEX-4T-1 for overexpression in prokaryotic cells. Structural analysis showed that the full length of mature Hass-GOBP1 open reading frame (ORF) is 441bp, 147 amino acid residues were encoded. The predicted MW and pI are 17.2kDa and 4.71, respectively. Structural analysis showed that the full length of mature Hass-PBP open reading frame (ORF) is 405bp, 135 amino acid residues were encoded. The predicted MW and pI are 15.1kDa and 5.2, respectively. The deduced amino acid sequence showed a high identity to the reported sequence of OBP from different species of insects and shared the typical structural features with odorant binding proteins from other insects. The phylogenetic tree also showed the amino acid of Helicoverpa assulta and Helicoverpa zea have high homology.