Cloning And Expression Analysis Of Four Odorant Binding Protein Genes From Apis Cerana Cerana (Hymenoptera:Apidae)

Odorant binding proteins (OBPs) played an important role in the communication of the information from outside world. As a native and widely bred honey bee species in China, Apis cerana cerana was superior to Apis mellifera L. in characteristics such as sensitive olfactory, powerful foraging, strong resistance to cold and disease, full advantage of sporadic nectar and pollen. The creature was suited to be kept in the mountain region. Study of the OBPs from A.c. cerana may not only defined the olfactory recognition mechanism further, but also provide theoretical guidance for the scientific bee keeping and the disease control. This study aimed to clone the cDNA sequences and explore their expression profiles of AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 by molecular biology technologies, and to analysis the biochemical property and higher structure of deduced amino acid by multiple bioinformatics methods. This may provide a fundamental evidence for the future study of the physiological function of AcerOBPs. The main results were as follows:(1) The cDNA sequences of AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 were cloned from the whole body of A.c.cerana by RT-PCR. Their ID of GenBank were KT279587, KT279586, KT24648 and KT588076, ORFs were 453 bp,399 bp,408 bp and 408 bp, amino acids (AA) were 150,132, 135 and 135, respectively. Sequence analysis showed that AcerOBP12 and AcerOBP13 belong to the Classical OBP, AcerOBP14 and AcerOBP15 belong to the Minus-C OBP subfamily.(2) Bioinformatics analysis showed that AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 were all secreted proteins with a signal peptide at the N-terminus, no transmembrane structure, and a conserved domain of the PBP-GOBP superfamily. AcerOBP12, AcerOBP13 and AcerOBP15 possessed 6 a-helixs, which formed a hydrophobic binding cavity, while AcerOBP14 possessed 7 a-helixs, among which the al-a6 belong to the core helixs of classical OBPs and a7 is exposed to the surface of protein at the C-terminus.(3) The pairwise sequence alignment results of AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 showed that the homology was low. AcerOBPs were high homologous with AmelOBPs. While phylogenetic analysis revealed that AcerOBP12 was located in the branch of Classical OBP, AcerOBP14 and AcerOBP15 were grouped together with the Minus-C OBP, but AcerOBP13 was located at the root of the Minus-C group.(4) The expression levels of AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 in different tissues (antenna, head, thorax, abdomen, legs, wings) at 1,5,10,15,20,25 and 30 days were detected by Real-time PCR. The transcripts of AcerOBP12, AcerOBP13, AcerOBP14 and AcerOBP15 were differentially expressed in various tissues of the adult workers. The expression profiling at different days of AcerOBP12 were significantly higher in antenna than that in other tissues (P< 0.01), of which the highest expression level were detected in the antenna of 25-day-old workers. The expression profiles of AcerOBP13 appeared a downward trend with the age increased, the expression level in antenna, head and thorax of 1-day-old were significantly higher than in other tissues (P< 0.01); AcerOBP14 had high expression level in antenna, among which the antenna of 20-day-old workers had the highest expression level. AcerOBP15 had high expression level in antenna and legs, abdomen, which was significantly lower than in other tissues (P< 0.01); except for the head of 10-day-old workers, other tissues reached the highest expression level in forage, of which AcerOBP15 had the highest expression level in the antenna of 20-day-old.(5) In this study, AcerOBP14 was successfully cloned into prokaryotic expression vector pEASY(?)-Blunt E1. The recombined plasmid pEASY/AcerOBP14 was expressed in E.coli BL21(DE3) chemically competent cell induced by 1.0 mM IPTG. The results of SDS-PAGE showed that the expressed products were consistent with the size of predicted recombinant protein, existing with the form of inclusion body. The successful study of inducing recombinant proteins may lay a foundation for the future study of the binding property with odorant and the functional research of OBPs.