Molecular Cloning And Functional Analysis Of Chemosensory Protein Genes In Carposina Sasakii

Carposina sasakii(Matsumura)is one of the most destructive pests in orchards in north China.It causes damages in yield and quality of fruit by plunder fruit nutrition,which result in massive economic losses to fruit growers.At present,chemical pesticides are mainly used to control or reduce the pests to occur effectively in production,but this method result in more and more the serious problem of Residue,Resistance,Resurgence.It is the hotspots to control the population desity of the pest using insect behavior strategies,and sex attractants are the main products which sex pheromone is main component.Olfaction plays an important role in the process of host searching,selecting and oviposition for the peach fruit moth.Pheromone can trigger many aspect behavior responses in the peach fruit moth,while odorants from host trees modulate pheromone response.The combination of host volatiles and pheromones can improve the effect of attractant.Chemosensory proteins(CSPs)are the crucial proteins that detect environment odors in olfactory organ.It is also one of the hot topics in the study of molecular mechanisms of the peach fruit moth olfactory function.More importantly,the study can offer a theoretical basis for prevention and control C.sasakii by interfering with their olfactory system.This paper is concentrated on the identification of host volatiles with CsasCSPs by gene cloning,real-time PCR,prokaryotic expression,and purifying the recombinant proteins.Fluorescence competitive binding assay were conducted to screening the CsasCSPs that showed specific binding properties with odors.Furthermore,the stability of complexes is verified through constructing three-dimensional structure,molecular docking and molecular dynamics simulation.The main results were as follows:1.Based on the antenna transcription and body transcription annotation information in Carposina sasakii,17 genes of Carposina sasakii(CsasCSP1-17)were obtained and verified by gene cloning.The amino acid sequence of the C.sasakii CSP genes are highly homologous to other insects of Lepidoptera and that may have similar expression profiles and functions.However,the similar of 17 CSPs gene deduced amino sequence have the low identify between each other,indicating that they may have multiple functions.2.We conduct the real-time PCR by design specific primer of 17 genes of Carposina sasakii.They have different expression patterns at different tissues.The expression levels of CsasCSP7,CsasCSP9,CsasCSP14,Csas CSP15,CsasCSP16 and CsasCSP17 were highly expressed in antenna suggesting that these CsasCSPs may be involved in recognizing plant volatiles.CsasCSP4,CsasCSP5,CsasCSP 8 and Csas CSP13 were highly expressed in foot,respectively.CsasCSP4,CsasCSP5,CsasCSP9,CsasCSP16 and CsasCSP17 were highly expressed in wind.In brief,tissue expression profiles indicate that CsasCSPs serve numerous functions in olfactory system and gustation.3.The recombinant CsasCSP16 was prokaryotically expressed in Escherichia coli and purified through immobilized metal nickel chelate affinity chromatography.This was newly obtained proteins in C.sasakii.Using N-phenyl-1-naphthylamine(1-NPN)as probe,fluorescence competitive binding assays were conducted to study the binding properties of CsasCSP16 with component of plant volatiles of apple fruit.The result indicate that CsasCSP16 presented the strongest binding abilities with Pentadecane,?-Pinene,Methyl salicylate,Butyl heptanoate and 6-Methyl-5-hepten-2-one,with dissociation constants of 7.9 ±0.23 ?M,11.50± 0.03 ?M,18.69 ±0.06 ?M,19.74 ± 0.62 ?M,17.83 ± 0.58 ?M respectively.We speculate that CsasCSP16 would be the potential target proteins that can recognize host volatiles.4.The 3D structure of CsasCSP16 was based on the template of 1N8 U.Molecular docking was conducted between CsasCSP16 and five host volatiles which have strongest binding abilities.The complexes from above two confirm the stabilities by molecular dynamic simulation are within 50 ns.The result show that Pentadecane,?-Pinene,Methyl salicylate,Butyl heptanoate and 6-Methyl-5-hepten-2-one and vina Score is-5.8,-6.6,-6.1,-5.2 and-5.3,respectively and the result of molecular dynamic simulation shows that RMSD value very small,and the complexes were in equilibrium states during the 50 ns of the simulations.The residues of Ile-49,Val71·,Ile-72,Thr90 play key role in binding between CsasCSP16 and host volatiles.