Cloning And Analysis Of Odorant Bindingprotein Genes From Monochamus Alternatus Hope

Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle (Nematoda: Aphelenchoididae) is an important pathogen which could kill pine and cause lage loss in zoology and economy. The Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a main vector of the pine wood nematode, B. xylophilus in China. Controlling M. alternatus is the major method for controlling the pine wilt disease. While the larva of M. alternatus feeds with trunk and behaviour is hidden, it is difficult to control M. alternatus by traditional methods. Although using attractants to trap and kill M. alternatus made some progress, there were also some problems exsisting. For this reason, Clearing the olfactory receptive mechanism may provide on the pest has important reference. Insect olfactory is vitally important for insect locating hosts and looking for mates. Now widely recognized that Odorant Binding proteins (OBPs) play an important role, in binding odor molecules and transported to the olfactory receptors but its specific function is not clear. Therefore, we studied M. alternatus in this paper, cloned the OBPs genes on the basis of constructing an antennae cDNA library and measured the binding properties of OBPs with odors with a view to binding odor molecular on the basis of prokaryotic expression.the maim results blow:1. Construction of cDNA library with the antenna of M. alternatus adultsWe succeeded in canstruction of cDNA library with the antenna of M. alternatus adults and sequenced2052single clones. After matching and removing the redundancy sequense, total of1133Unigenes were obtained, consisting of857contigs and276singlets. The percentage of GC was41.8%after forecasting open reading frames. ESTs into clusters of orthologous groups, through which350ESTs were classified into21functional categories. Compared with NCBI non-redundant protein database using BLASTx, we found four OBPs and one Chemosensory Protein (CSP). It laid the foundation for studing molecular biology of M. alternatus. 2Cloning and sequence analysis of OBPs from M. alternatusIn the paper, only four OBP genes were cloned, MaltOBP2, MaltOBP3, MaltOBP4and MaltOBP5(The ID of GenBank is KF460431, KF460432, KF460433and KF460434separately), all possess complete open reading frame. The length of these sequence is402bp,408bp,423bp,378bp, coded167,164,196,141amino acids respectively, and there with a signal peptide of16,20,19and18amino acids at N-terminus respectively. The blast of MaltOBP2and MaltOBP5contain the possess four conserved cysteine sites, means the two OBPs belong to Minus-C OBPs, and MaltOBP3and MaltOBP4show six conserved cysteine sites, means the two OBPs belong to Classic OBPs. The result of blast also show that the homology is low. The phylogenetic tree showed that the classic and minus-C OBPs clustered in two distinct regions and the OBPs located different branch respectively. It agreed that the four OBPs belong different sub family and they may perform different functions in the history of M. alternatus.3The prokaryotic expression, purification and fluorescence competitive binding assays of MaltOBPsIn our research, the four MaltOBPs were successfully subcloned into prokaryotic vector pGEX-6p-1that there was a GST in the plasmids, transferred into BL21(DE3) pLysS and expressed. The fusion protein could be expressed in E. Coli. However, all of them were in inclusion body at37℃and diffcult to purify.. Last the recombinant MaltOBP3and MaltOBP5proteins were expressed in E. Coli at a low temperature. Both of the purified proteins were soluble.The recombinant protein was purified using a GST MultiTrapTM4B chromatography column, and removed the GST tag by Precission protease.MaltOBP3and MaltOBP5assessed their ligand specificity by measuring the competitive binding of fluorescent probe, N-phenyl-1-naph-thylamine, in the presence of17volatile. In our fluorescence binding assays, both MaltOBP3and MaltOBP5bound (+)-limonene oxide, p-cymene,(-)-limomene,(R)-(+)-α-pinene and (+)-β-pinene with high affinity. MaltOBP3displayed higher binding affinities for a-terpinolene,(-)-isolongifolene, β-caryophyllene, camphor and butylated hydroxytoluene than MaltOBP5, whereas MaltOBP5displayed higher binding affinities for R-(-)-limomene and (+)-α-pinene than MaltOBP3.