| Insects recognized the complex comical signal in the environment by their sensitive olfactory system,the comical signal include spouses released sex pheromone or volatile odor compounds of plants and so on, causing the insects'behavior of finding spouse, mating, locating host plants, looking for spawning sites and so on. In the process of insect olfaction, different carrier proteins transporting different odor compounds to their receptors located in specialized cuticular antennal olfactory sensilla or the general chemosensory systems in insects through the polarity lymph, cause olfactory nerve impulses, thereby causing a variety of behavioral responses of insects. Some studies indicated that small molecules soluble chemosensory protein play an important role in transporting hydrophobic odorants to their receptors. Use the mechanism of insect olfactory recognition, make CSPs as potential target genes, interfering with the insect host location and mating behavior and use for Integrated Pest Management has become a hot issue, was provided with theoretical and practical significance.Lucerne plant bug, Adelphocoris lineolatus (Goeze) as one of the major pests, causing serious economic loses in cotton production and other important crops. So we make the AlinCSPs of Lucerne plant bug as target, use qPCR method to study the expression pattern of AlinCSPs gene in different development stages and different tissues. We also identified, cloned, expressed and purified several new AlinCSPs by prokaryotic expression system. We used the method of homology modeling obtained the 3D structure of AlinCSPs and speculate the possible binding sites and verified by site-directed mutagenesis and fluorescent competitive binding experiments. Conduct fluorescent competitive binding experiments to select strong binding odor compounds and use these odor compounds to conduct Y-tube olfactory trails to test the behavior responds of the adults of Lucerne plant bug. The results showed as follows.1. In this study, we used prokaryotic expression system identified,expression and purificated 3 new chemoscensory proteins (AlinCSP4,AlinCSP5,AlinCSP6).We chooseβ-actin as the endogenous control to normalize the target gene expression and correct for sample-to-sample variation, choose AlinCSP4,AlinCSP5,AlinCSP6,AlinCSP7,AlinCSP8 gene as the target gene, use the method of qPCR to study the relatively expression level of AlinCSPs gene in different development stages (1st, 2nd, 3rd, 4th, 5th, 6th ) and different tissues (antenna, heads, thorax, abdomen, legs, wings ). When the amplification efficiency between the target gene and the reference gene under the same, we use the comparative 2-ΔΔCT method calculated the relative quantification of AlinCSPs directly. The result showed that AlinCSP4,AlinCSP5,AlinCSP8 and AlinCSP7 were mainly expressed at 2nd of A. lineolatus, so we speculate that AlinCSP4,AlinCSP5,AlinCSP8 and AlinCSP7 play an important role in the early development of A. lineolatus. While AlinCSP6 mainly expressed in adults of A. lineolatus. AlinCSP4 and AlinCSP6 mainly expressed in legs of adults, AlinCSP5 and AlinCSP8 mainly expressed in antenna of A. lineolatus. While AlinCSP7 mainly expressed in wings of adult A. lineolatus, so we speculate that AlinCSP7 plays an important role in the development of wings.2. The 3D result showed that AlinCSP4 consists of sixα-helices located among residues 17-22(α1),24-34(α2),40-56(α3),64-80(α4),84-92(α5) and 97-105(α6), and two pairs of disulfide bridges, Cys33-Cys40 and Cys59-Cys62. In addition, Ile78, Trp85, Ile51 and Phe52 residues may play important roles in odor ligands binding. AlinCSP5 contains a large hydrophobic pocket structure and one of the key binding sites may be Ile67. Alin-CSP6 is consists of sixα-helices located between residues 13-18(α1),20-32(α2),39-52(α3),59-76(α4),78-88(α5) and 94-102(α6), two pairs of disulfide bridges Cys29-Cys3 and Cys55-Cys58 play an important role in holding the structural stablity of the protein. We speculate that Ile7 and Phe81 may participate in binding to oleic acid amide analogues; Alin-CSP6 has a wide binding spectrum with odorant ligands which play an important role in olfaction of A. lineolatus.3. The binding properties of AlinCSP4 with 114 odorants were investigated by fluorescence competitive binding assay using a fluorescence probe 1-NPN. The AlinCSP4 has a very wide odorant binding spectrum. It was found except alkenes, all the odorant molecules including alcohols, aldehydes, ketones, ester, terpenes, heterocyclic, aromatic and aether could bind with AlinCSP4. Among them, (±) 2-undecanol, 2-butyl-1-octanol, 2-nonancne, 2-pentadecanone, nonylacetat, ethyl2-ethylbutyrate, methyl jasmonate, limonene, 2-terpinene, myrcene, 1,8-cineole, ethyl ether, 3,4-dimethyl benzaldehyde, 4-ethylbenzaldehyde and benzaldehyde showed stronger binding abilities. Two putative pheromones, butyl butanoate and 1-hexyl butyrate also have notable binding affinities to AlinCSP4. Fluorescence assays indicate that AlinCSP5'binding ability has specificity. It can specifically bind toβ-citronellol, 3-Hexanone, 2-Octanone, acetophenone, 2,3-dimethyl-benzoic acid, 3,4-Dinethylbenzaldehyde, soborneol,β-lonone and among them acetophenone showed the strongest binding ability to AlinCSP5, its IC50 data is 16.305μmol/L. The binding characterization of AlinCSP6 with 113 volatile odors was explored by a fluorescent competitive binding experiment; The result showed that AlinCSP6 has a wide binding spectrum with odorant ligands, which can bind to alcohols, aldehydes, ketones, easter, aromatic and so on. Theβ-citronellol, Trans -2 - hexene aldehyde, 6-methyl-5-Heptene -2- Ketone, 2-Undecanone, Butyl levulinate, myrcene, (1s)-(-)-β-Pinene, nonane, 3,4 - dimethyl benzaldehyde, Ethyl ether, Ocimene,Acetophenone and benzaldehyde have high binding affinities with AlinCSP6.4. Site-directed mutagenesis and fluorescence assays were used to verify the possible binding sites. Compare to the wide-type AlinCSP5, the obtained mutant can not bind toβ-Citronellol and 2,3-Dimethylbenzoic acid. The binding ability of 3-hexanone and acetophenone is not change so much. But the binding ability of 2-octanone, 3,4-Dimethylbenzaldehyde,β-lonone and Isoborneol is significant growth. We suggest that a hydrophilic amino acid at the hydrophobic pocket structure participates in initial recognition of ligands, and contributes to ligand-binding specificity of CSPs.5. In the olfactory trails, alcohol odor compound(s+-)-Isoborncol, 3-hexanol, 2-butyl-1-octanol, aldehydes compounds heptanal, ketones 2-Heptanone,β-lonone, 2-octanone, 2-hexanone, 6-methyl-5-Heptene -2-Ketone, Terpenes R-(+)-limonene,α-terpinene, carveol,α-phellandrene, Hydrocarbon compounds pentane,undecane,heterocyclicsβ-Pinene,Aromatic compounds 4-Ethylbenzaldehyde,ethers Indole have strong attract function to A. lineolatus. However, Acetophenone, Trans -2 - hexene aldehyde, oclanal have a strong repellent effect on A. lineolatus. |
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