Construction And Analysis Of CDNA Libraries From The Antenna Of The Batocera Horsfieldi (Hope) And Cloning, Expression And Functional Analysis Of Olfactory Related Proteins

Insects have specialized chemosensory systems detect stimuli from the external environment, including a wide range of chemical compounds. The specialized sensilla in the chemosensory organs (antenna, maxillary palps, labial palps, legs and wings) allows the organism to recognise volatile cues that confer the capacity to detect food sources, predators, mates and oviposition sites. Two major families of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), are expressed in chemosensilla of insects and thought to carry lipophilic odorants to the olfactory receptor cells through hydrophilic surroundings and are involved in the initial biochemical step of odour reception. Clarifying the Chemical sense mechanisms of insect not only could be helpful to promote the understanding behavior essence of insects and plants, but also provide theories for controling pest. The long-horned beetle, Batocera horsfieldi (Hope)(Coleoptera:Cerambycidae), is a significant pest of Popolus. The larvae of Batocera horsfieldi cause serious economical loss in result of their eat trunk and covert habitat. In order to further understanding the olfactory mechanism of Batocera horsfieldi and control them by scientific methods, a cDNA library from antenna of B. horsfieldi was constructed and functional from hundreds of expressed sequence tags (ESTs) by patched sequencing were discovered from cDNA library. On the basis of ESTs, OBPs and CSPs of B. horsfieldi were cloned, and the transcriptional profiles in different tissues through different developmental stages were identified, also further recombinant proteins were expressed after construction of expression vectors, meanwhile, the potential function of OBP2was investigated. The main results are as follows:1The construction of cDNA library with the antenna of B. horsfieldi adultsThrough the technique of LD-PCR, ds-cDNA was synthesized, the PCR products were analysed on1%agarose gels, the bands of0.3-0.6kb and0.6-1kb were retrieved respectively and linked into the pDNR-LIB Vector. Through identification of PCR and blue-white selection, the cDNA library was identified high recombinational efficiency, reached above94.5%(hfcbwa) and93.2%(hfcbwb), respectively. And the titer of the two library (hfcbwa and hfcbwb) reached6.4×105pfu/ml and4.1×106pfu/ml.692clones from the cDNA library were selected randomly and sequenced,604high quality sequences more than100bp were produced after removing the vector sequences and contaminated sequences, And the average length of the ESTs (without vector) were386.33bp and468.62bp. Using the Phrap software,79"Contigs" and323"Singlets" were produced from the assembled ESTs, after analyzed by clustering and assemblying, get246functional gene. Compared with NCBI non-redundant protein database using BLASTx,68ESTs of Unigenes matched with olfactory related protein genes, wiped off the repeated ESTs, got4odorant binding proteins,3Chemosensory proteins,2Pheromone binding proteins and9Minus-C odorant binding proteins, which provided foundation for next research.2Molecular cloning and characterization of OBPs and CSPs from B. horsfieldi adultsFrom the B. horsfieldi antennal cDNA library, we obtained three full-length odorant-binding proteins gene by RT-PCR, from the nuclear acid sequences, all of the three BhorOBPs have6conservative cysteines, and a signal peptide which about20amino acids at N-terminus. The multiple sequences alignment of the BhorOBPs with other Coleoptera OBPs showed high sequence identity (50%) with other full-length sequences from GenBank, but the homology of these three BhorOBPs were very low, only12.68%, suggested they belong to different groups, also the results in compliance with the results of phylogenetic tree. Also three full-length cDNAs of CSPs were identified and predicted to encode small proteins (120-130amino acids). The sequences of these genes have been deposited in GenBank under the accession numbers HQ587040, HQ587041and HQ587042. Three key features of the amino acid sequences of the candidate CSP genes were consistent with the CSP family members of other insects:a predicted molecular weight of about13kDa, a hydrophobic N-terminus of about16amino acids encoding a signal peptide and the presence of a highly conserved four-cysteine motif (C-X6-C-X18-C-X2-C). The alignment results also shows the CSPs had three conserved several sequence motifs, including a commonmotif A at their N terminal YTTKYDN[V/I][N/D][L/V]DEIL, central motif B DGKELKXX[L/I]PDAL and C-terminal KYDP, in addition, aromatic residues at position53,110and123that may be functionally important are also highly conserved, along with residues located at91/92(glutamine/lysine) and129/130(lysine/tyrosine). 3Temporal and spatial expression profiles of the BhorOBPs and BhorCSPs genesUsing gene-specific BhorOBPs and BhorCSPs primers and control18s rRNA primers with cDNA prepared from different tissues, we examined the expression patterns at successive developmental stages and in various adult body parts and tissues by RT-PCR and Real time-PCR. Unlike other species, all three B. horsfieldi OBPs and CSPs transcripts were detected in antenna, labial palps, maxillary palps, forelegs, midlegs, mid abdomen and hind abdomen, but no specific products were observed in head (without antenna). On the basis of the normalized relative quantification2-ΔΔCT method for real-time Q-PCR, the transcription profiling of the tissues and developmental phases of the three BhorOBPs and BhorCSPs were obtained. The data presented the transcription levels of the three BhorOBPs were higher than those at the same periods of BhorCSPs in antenna. In general, BhorOBPs and BhorCSPs had the highest expression in antenna, and higher espression in labial palps and maxillary palps, but less in wings, legs and abdomen. The transcript levels of the BhorOBPs and BhorCSPs in all tissues of unmated or mated males were significantly higher than in unmated or mated females in most development stages except in the labial palps and maxillary palps. This is most noticeable for BhorCSPs that were generally expressed highly in the wings and legs. In the different developmental periods, BhorOBPs and BhorCSPs had two highest expression leveral periods, from larva to5day adult and around25/30day adult. These data indicated BhorCSPs maybe had other rols except olfactory sensation, also the difference transcription leveral at different developmental period and in different tissues suggest the BhorOBPs and BhorCSPs have their specifically task in detecting food sources, mates and oviposition sites.4The expression and purification of BhorOBP2and BhorCSP2proteinsThe ORF sequence of BhorOBP2and BhorCSP2were subcloned into prokaryotic vector pET32a, transferred into BL21(DE3) and expressed. The recombinant plasmids pET32a have a His-tag and a Trx-tag, the Trx-tag can increase the solubility of the protein, and facilitate forming correct disulfide bond. We deleted the Thrombin sequence of pET32a vector, then added a Thrombin sequence and a linker at the N terminal of BhorOBP2and BhorCSP2. The conditions for soluble expression was optimized by investigating different temperature (16℃), IPTG concentration (0.5mmol/L) and induction time (overnight). Then the buffer condition and chromatography methods were discussed of the proteins. Finally, through nickel affinity column, Resource Q/S ion exchange column and Superdex75gel filtration chromatography, the protein samples with high concentrations and stability were obtained.