Functional Analysis Of Two Odorant Binding Proteins From Wheat Aphid Sitobion Avenae(fabricius)(hemiptera;aphididae)and Identification Of Putative Chemosensory Proteins From Its Whole Body Transcriptome

The sophisticated and sensitive insect olfactory system plays a decisive role in odor perception,odor discrimination,and several important behaviors of insects,such as feeding,mating,oviposition,and avoiding natural enemies.Odorant Binding Proteins(OBPs)are considered to operate in the first step of the interaction with external odorants,to bind,and to transport the specific odorants to their specific membrane.OBPs have been reported to play the key role in detecting odors and initiating specific insect action,while the physiological mechanism of OBPs in insects is still not very clear.The wheat aphid Sitobion avenae(Fabricius)(Hemiptera: Aphididae)is responsible for causing 65% of the damage in wheat-producing areas in China,being the most dominant and destructive pest responsible for the greatest loss in wheat production.In one of the previous work,13 Save OBP and 5 Save CSP transcripts were identified,while only two studies have been performed on S.avenae in which Save OBP3 and Save OBP7 might be involved in the binding of an exclusive component of the aphid alarm pheromone.It has been reported that Save OBP9 and Save OBP10 genes were highly expressed in antennae and the striking structure with the other antennal OBPs revealed by the phylogenetic tree analysis suggests its possibly functional,behavioral role in olfactory perception of S.avenae.In this study,the Save OBP9 and Save OBP10 were cloned from S.avenae,and its olfactory functions were analyzed by fluorescence ligand binding experiments,molecular docking,RNA interference,and olfactometer(pre-and post-RNAi).Additionally,to help the better insights into the aphid olfactory system,we produced the transcriptomes of adult wingless and winged aphid.Chemosensory protein genes(CSPs)were screened and identified,and the m RNA expression level of CSP genes in wingless and winged adults were tested by q RT-PCR.The key results are outlined below;1.Functional analysis of odorant binding protein Save OBP9 from Sitobion avenaeVolatile organic compounds(VOCs)and herbivore induced plant volatiles(HIPVs)from the wheat plants were identified by GC-MS.The results showed that the amounts of 2-ethyl-1-hexanol,nonanal,tetradecane,pentadecane,butylated hydroxytoluene and hexadecane were significantly higher in healthy wheat plants as compared to infested plants after 12 hours of aphid feeding.Two HIPVs,naphthalene and decanal,were found in wheat plants infested by the aphid.After 24 hours,the contents of three compounds,hydrocarbons decanal,butylated hydroxytoluene and hexadecane,were significantly higher in healthy plants.While,the contents of three compounds,nonanal,naphthalene and tetradecane,were higher in the wheat plants infested by the aphid.The Save OBP9 was expressed successfully using a bacterial expression system,and recombinant protein were purified by Ni-ion affinity chromatography column.Fluorescence binding assay results showed that p H strongly affected the ligand binding ability to Save OBP9.Generally,the binding affinities of Save OBP9 with the ligands at p H 7.4 were higher than those at p H 5.0.In addition,Save OBP9 had broad and high(Ki < 10 ?M)binding abilities with most of the wheat volatiles(i.e.tetradecane,hexanal,octanal,decanal,?-farnesene,hexadecane,and 2-ethyl-1 hexanol)at p H 7.4.While S.avenae only showed significant behavioral preferences to four compounds,decanal,tetradecane,hexadecane and octanal.The silencing of the Save OBP9 gene by dietary RNAi resulted in significant behavioral changes and S.avenae showed non-significant preferences for tetradecane,octanal,decanal,and hexadecane.The binding sites of Save OBP9 to the volatiles were predicted well by three-dimensional docking structure modeling and molecular docking.Binding pocket residues of Save OBP9 such as Tyr77,Ile41,Ala116,Ala113,Lys38,Gln43,and Ile114 were highly overlapped and contributed to the interaction with ligands.Furthermore,we have found several covalent interactions(Pi alkayls and Sigma alkayls),van der Waals interactions,and a hydrogen bond(H-bond)between compounds and receptor proteins through a conserved binding cavity in the form of a tunnel.Save OBP9 might be involved in the chemoreception of S.avenae for wheat plants volatile organic compounds.2.Functional analysis of odorant binding protein Save OBP10 from Sitobion avenaeThe Save OBP10 was expressed successfully using a bacterial expression system,and recombinant proteins were purified by Ni-ion affinity chromatography column.Fluorescence binding assay results showed that p H strongly affected the ligand binding ability to Save OBP10.While different to Save OBP9,the binding affinities of Save OBP10 with the ligands at p H 5.0 were higher than those at p H 7.4.Save OBP10 had strong and high binding affinities(Ki ? 5 ?M)with most of wheat plant volatiles(i.e.naphthalene,trans-2-hexen-al,pentadecane,S-(-)-limonene,2-ethyl-1-hexanol,butylated hydroxytoluene,tetradecane and ? –caryophyllene)at p H 5.0.The results of Y-tube olfactometer bioassays showed that the wheat aphid S.avenae showed significant preferences to pentadecane,butylated hydroxytoluene,tetradecane and ?-caryophyllene,and repelled by naphthalene.While,they showed non-significant response to trans-2-hexen-1-al,S-(-)-limonene and 2-ethyl-1-hexanol.The dietary RNAi method had successfully reduced the m RNA transcript levels of Save OBP10,and the aphid feed with ds Save OBP10 showed significant decline in behavior response to ?-caryophyllene and non-significant behavioral preferences to tetradecane,butylated hydroxytoluene and pentadecane.The binding sites of Save OBP10 with the ligands,which had higher binding abilities(Ki ? 5 ?M),were predicted by three-dimensional docking structure simulation and molecular docking ability.Binding pocket residues of Save OBP10 such as Met 31,Met 78,Met 95,Ala 96,and Pro 97 were highly overlapped and contributed to the interaction with ligands.The docking results showed the variety of bonding between chemicals and receptor through a conserved binding cavity,which was in the form of a tunnel.We found several covalent interactions(Alkyl,Pi-alkyls,and Pi-Sigma),van der Waals interactions and a Carbon Hydrogen bond between compounds and receptor proteins.Together,these findings indicate that Save OBP10 could bind more strongly to the volatiles that are involved in S.avenae behavior regulation.3.Identification and expression analysis of putative chemosensory protein(CSP)genes based on transcriptomic analysis of english grain aphid Sitobion avenaeThe whole body transcriptomes of wingless and winged S.avenae were generated using the Illumina Nova Seq 6000 sequencing system.Among the 104,024 unigenes,in the de novo assembly,5 putative CSPs sequences were identified and complete open reading frame(ORF)of CSP1 which was identified previously with incomplete ORF.The amino acid sequence of 5 putative CSPs was distinct from all previously identified CSPs in S.avenae in another study and reflects the uniqueness.While Save CSP1 shares only 11% amino acid sequence similarity with the previously identified Save CSP1.The phylogenetic tree of the CSPs protein sequences revealed that the Save CSP1,Save CSP6,Save CSP7,Save CSP8,Save CSP9,and Save CSP10 of Sitobion avenae clustered with the CSPs of Aphis gossypii having protein identity of 100%,100%,67%,100%,99% and 99%,respectively.By molecular cloning and sequencing,all of the Save CSP sequences were validated.To analyze the expression profiles of these genes,q RT-PCR was performed.We found the 4 Save CSPs(Save CSP7,Save CSP8,Save CSP9 and Save CSP10),was markedly up regulated in the wingless morph of S.avenae.The unique features of the high abundance of CSPs in the wingless form of aphids might suggest the fundamental physiological functions in aphid physiology and ecology.