Cloning And Functional Analysis Of Odorant Binding Protein Genes From Monochamus Alternatus Hope

The Japanese sawyer beetle Monochamus alternatus Hope was a main vector of Bursaphelench xylophulus Nickle. Controlling M. alternatus is an effective method to manage to B.xylophulus. Odorant binding proteins(OBPs) was regarded as a medium between the odor molecules and odorant receptors and OBPs played an important role in the process of recognizing of different chemical messages insect identification information on chemical substances in the process. However, the specific function of Malt OBPs remains unclear in M. alternatus. In order to clear the function of Malt OBPs on sensory and provide the oretical basis for selecting behavior-active components of M.alternatus, we constructed the antennal transcriptome of M. alternatus, 5 Malt OBPs were selected and cloned, and the correlation between the expression profiles of these Malt OBPs and behaviors of M. alternatus were studied. And also, the binding characteritics of recombinant Malt OBPs with 18 kinds of host plant volatiles and the correlation with p H values were measured by fluorescene competitive binding. The main results are as follows:1. Construction of the antennal transcriptome of M. alternatusAntennae in male and female adults of M. alternatus were used in constructing antennal transcriptome. After sequencing, the original sequence processing, clustering,and splicing, we got a total of 85,869 contigs, the average length of 297bp; got 37,242 unigenes, the average length of 669 bp. The results of NR comment showed that 94.3% of unigenes of M. alternatus had highest similarities with unigenes of Tribolium castaneum(Herbst) and Dendroctonus ponderosae Hopkins. Unigenes of M. alternatus can be divided into 25 functional groups according to COG functional annotations. The number of unigenes were annotated for general function prediction class, replication,recombination and repair class were most, reaching 3,051 and 1,349 separately.For further selecting and blasting, We identified 25 transcripts encoding full-length odorant-binding proteins(OBPs) and 4 tanscripts encoding partial OBPs; 8 transcripts encoding full-length chemosensory proteins(CSPs) and 4 tanscripts encoding partial CSPs; 1 transcripts encoding full-length olfactory receptor(OR) and 8 tanscripts encodingpartial ORs; only one transcript encoding sensory neuron membrane protein(SNMP).2. Cloning and sequence analysis of MaltOBPs genes of M. alternatusMalt OBP9, Malt OBP10, Malt OBP19, Malt OBP21, Malt OBP24 genes(Gen Bank accession numbers are KF977562, KF977563, KF977572, KF977574, KF977577 separately.) of M. alternatus were successfully cloned by RT-PCR. The open reading frame of Malt OBP9, Malt OBP10, Malt OBP19, Malt OBP21, Malt OBP24 gene were423, 435, 615, 390, 510 bp. They encoded 140, 144, 204, 129, 169 amino acids respectively. The length of signal peptides were 19, 25, 18, 18, 0 amino acid residues respectively. Malt OBP9 and Malt OBP21 belonged to Minus-C OBPs, Malt OBP10 belonged to Classic OBPs, Malt OBP19 and Malt OBP24 belonged to Plus-C OBPs. The phylogenetic tree of OBPs showed that Classic OBPs, Minus-C OBPs and Plus-C OBPs were divided into diffferent branches obviously.3. The expression patterns of Malt OBPs genes in M. alternatusThe expression patterns of 5 MalOBPs were analyzed by Real Time Q-PCR. The results showed 5 Malt OBPs genes highly expressed in maxillary palps and labial palps in male and female adults. And 5 Malt OBPs genes also expressed in antennaes, heads, feet,abdomens and wings. The expression of OBPs genes in antennae of M. alternatus varied from age, sex and mating status.4. Expressed in prokaryotic, purified and analysized of the fluorescent binding properties of 5 Malt OBPs in M. alternatusRecombinant plasmids(p ET20b-Malt OBP9, p ET20b-Malt OBP10, p ET20 bMalt OBP19, p GEX-6P-1-Malt OBP21, p ET-17b-Malt OBP24) were successfully constructed. PGEX-6P-1-Malt OBP21 expressed in supernatant, we obtained purified Malt OBP21 through the GST Multi Trap TM 4B chromatography column and enzyme digestion of precission protease. PET20b-Malt OBP9, p ET20b-Malt OBP10, p ET20 bMalt OBP19, p ET17b-Malt OBP24 mainly expressed in inclusion bodies. After denaturation and renaturation of the inclusions, we obtained purified Malt OBPs after using DE52 and QFF affinity columns.1-NPN was selected as a fluorescent probe, competition binding properties of Malt OBP9, Malt OBP10, Malt OBP19, Malt OBP21 and Malt OBP24 with 18 kinds of host plant volatiles were measured at neutral and acidic p H environment using fluorescence competition. Malt OBP9, Malt OBP10, Malt OBP19, Malt OBP21 and Malt OBP24 generally had higher binding affinities at neutral than acidic p H environment. At neutral environment, 2 Malt OBPs belonged to Minus-C OBPs family showed high binding affinities with 4 volatiles; 2 Malt OBPs belonged to Plus-C OBPs family had a big difference on binding abilities, one Malt OBPs had relatively higher binding affinities with17 volatiles and another Malt OBPs merely showed higher binding capacities with 5volatiles. 5 Malt OBPs belonged to Minus-C OBPs, Classic OBPs and Plus-C OBPs fdid not show significant binding characteristic.This study laied an important foundation for clearing the function of Malt OBPs in M.alternatus and OBPs in insects, exploiting insect behavioral regulator.