Function Alanalysis Of Olfactory Genes OBP2,CSP2 And CSP3 From The Legume Pod Borer,maruca Vitrata Fabricius(Lepidoptera:Crambidae)

Maruca vitrata is a major species which damage the pulse crops throughout the tropical and subtropical.In order to prevent the damage of pests to crops,the mixture of chemical agent with sex pheromone is widely used as the means of prevention.In insect populations,the relationship between the individual and the environment is often regulated by chemical information.In insect antennae,the soluble proteins OBPs and CSPs have been proved to carry the ligand molecules through the water soluble lymph,which is involved in the electrophysiological response of insect antennae.Based on our preliminary work of our group,these experiments mainly focus on the function of olfactory genes(MvitOBP2,MvitCSP2 and MvitCSP3)from the M.vitrata during the perception of host plant volatile.Our present experimental results will provide some support and add more useful theoretical basis for prevention of M.vitrata in the field.First of all,according to our transcriptome sequencing results,the full-length of MvitOBP2,MvitCSP2 and MvitCSP3 were cloned by the polylerase chain reaction,then we analyzed the sequences of MvitOBP2,MvitCSP2 and MvitCSP3 by many bioinformatics methods such as Blast,DANMAN,Bio-edit etc.These results showed that the protein sequence of MvitOBP2,MvitCSP2 and MvitCSP3 had a high similarity with the sequence of Dichocrocis punctiferalis and Cnaphalocrocis medinalis,which were consistent with the phylogenetic tree analysis.Secondly,we detected the quantity of MvitOBP2,MvitCSP2 and MvitCSP3 in different tissues used by qRT-PCR,suggesting that MvitOBP2,MvitCSP2 and MvitCSP3 had an abundant expression in both male and female antennae,but had a little expression in the legs and wings.Besides,the competitive binding assays were conducted to detect the binding affinities of the target proteins with the seventeen synthetic ligands from floral volatile chemicals.The results showed that MvitOBP2 had a strong binding capacity to 2-methyl-3-phenylpropanal,4-ethylbenzaldehyde and butanoic acid octyl ester with Ki values of 9.50 ?M,8.17 ?M and 8.04 ?M;When the concentration of 2-methyl-3-phenylpropanal reached 9.42?M and 7.81?M,the 2-methyl-3-phenylpropanal can make the fluorescence intensity of MvitCSP2/1-NPN and MvitCSP3/1-NPN complex decreased to 50%.In addition,MvitCSP2 also has strong binding ability with limonene,and its binding constant is 8.77 ?M.According to the fluorescence competitive binding assays,we selected the compounds which have strong binding ability with these three proteins for molecular docking.The results showed that the binding energies of MvitOBP2,MvitCSP2 and MvitCSP3 to 2-methyl-3-phenylaldehyde are greater than other compounds.The binding energy of MvitOBP2 to 2-methyl-3-phenylaldehyde is-2.78.The binding energy of MvitCSP2 and MvitCSP3 to 2-methyl-3-phenylaldehyde is-4.26 and-5.51,highlighting that they have a high binding ability with these floral volatile chemicals.The key amino acid sites of MvitOBP2,MvitCSP2 and MvitCSP3 to 2-methyl-3-binding were S129,L104 and F27,respectively.In conclusion,through the functional analysis identification of MvitOBP2,MvitCSP2 and MvitCSP3,we clarified the molecular mechanism of olfactory recognition between M.vitrata and host plants.And we also explore a new way or method to interfere the olfactory behavior of insects,which is helpful to achieve the purpose of biological control to pests.