Olfactory Genes Screening Of Two Woodwasps,sirex Noctilio Fabricius And S.nitobei Matsumura And Function Analysis Of Odorant Binding Proteins

Sirex noctilio is a major quarantine pest in the world which mainly damages Pinus.It was first discovered in China in 2013.S.nitobei is a native species in China.The two species are closely related species.The olfactory system plays an important role in insect calling,foraging,mating and oviposition.Odorant binding proteins(OBPs)is one of the important proteins of insect olfactory,which can be combined with odor molecules specificity and deliver it to Odorant receptors(ORs)and cause a series of reactions in insect olfactory system.To gain insights to the olfactory mechanism of two woodwasp species,olfactory genes were identified using antennal transcriptome analysis.Sirex OBPs which have high FPKM value in transcriptome were chose for qPCR and subsequent data analysis.At last,the crucial gene function was analyzed.The result is as follow.From our transcriptome analysis,16 OBPs,7 chemosensory proteins(CSPs),41 odorant receptors(ORs),8 gustatory receptors(GRs),13 ionotropic receptors(IRs),and one sensory neuron membrane protein(SNMP)were identified in S.noctilio,while 15 OBPs,6 CSPs,43 ORs,10 GRs,16 IRs,and 1 SNMP were identified in S.nitobei.Most of the olfactory genes identified in S.noctilio and S.nitobei were homologous and it confirmed the close relationship between the two species on genetic level.After obtaining the gene sequence of odor binding protein,the expression OBPs in several kinds of woodwasp tissues was analyzed according to qPCR.Most of OBP genes were expressed mainly in antennae of two woodwasps which further clarifying the importance of the antennae in olfactory perception.There were also some OBPs that were not expressed in the antennae.For example,SnocOBP3 was highly expressed in the genitalia of females,and SnocOBP7 and SnitOBP7 were highly expressed in the genitalia of males.Meanwhile,SnocOBP10 was specifically expressed in male heads.The expression in non-olfactory organs suggests that they may have special olfactory or non-olfactory functions.To study the function of odor binding proteins,Swiss-model was used to develop the homologous model of 4 odorant binding protein in S.noctilio antennae with high FPKM values and the model quality was evaluated.Using the Autodock software,molecular docking was performed on odorant binding proteins and important semiochemicals that had been found to attract S.noctilio.The results indicated that 4 highly expressed odorant binding protein models had high evaluation scores and high model quality.Molecular docking results showed that the best combination with pine volatiles ?-pinene and ?-pinene is SnocOBP12 with binding energy of-5.36 kJ/mol and-5.48 kJ/mol,respectively.The best combination with Z-9-nonacosene,one of female pheromone components,is SnocOBP4 with binding energy of-5.42 kJ/mol;and the best combination with male pheromone components(Z)-3-decen-1-ol and(Z)-4-decen-1-ol is SnocOBP9 with binding energy of-4.37 kJ/mol and-4.45 kJ/mol,respectively.SnocOBP6 has low binding energy with 3-carene,(Z)-7-heptacosene,and(E,E)-2,4-decadienal,and the lowest binding energy is(Z)-7-heptacosene with binding energy of-6.88 kJ/mol.SnocOBP9 has high expression and low binding energy with male volatile components.For the combined function research of SnocOBP9,the protein was expressed through E.coli expression system,and purified protein was obtained by Ni2+-NTA agarose gel column chromatography after the release of inclusion body.This study laid the foundation to eluciate the mechanism of interactions between olfactory system and specific odor molecules based on olfactory genes identification,expression,molecular docking and prokaryotic expression.Afterwards,behavior research and attachments development can be integrated to regulate the behaviors of woodwasps,to aim to control of woodwasps effectively.