中华蜜蜂化学感受蛋白CSP1的功能模式分析及亚细胞定位

doi:10.3864/j.issn.0578-1752.2017.15.019

Abstract

Abstract: 【Objective】The objective of this study is to research the functional mode with candidate semiochemicals and immunocytochemical localization of the recombinant chemosensory protein1 (CSP1) in Chinese honey bee, Apis cerana cerana. It has an important theoretical significance for clarifying the function of CSP1, which is specifically expressed in antennae, and enriching the chemosensory mechanism of Chinese honey bee to semiochemicals. 【Method】 CSP1 was constructed into the pET-32a (+) vector and transferred into BL21 (DE3) competent E. coli. The single positive clone was inoculated on LB medium and cultured overnight (followed by 1% (v/v) transfer to the OD₆₀₀≈0.4), then added the IPTG with a final concentration of 1 mmol·L^-1 and continued to be induced for 5 h. The recombinant CSP1 protein existing in the supernatant was purified by Ni²⁺-NTA affinity chromatography. The soluble recombinant CSP1 protein with biological activity was finally obtained after dialyzed by PBS (pH 7.4). When the excitation wavelength of the fluorescence spectrophotometer was 281 nm, the interaction between the fluorescent probe 1-NPN and CSP1 was determined. The dissociation constant was calculated by Scatchard equation, and the binding affinities of CSP1 with various candidate semiochemicals were also measured. Based on the CSPMbraA6 crystal structure (PDB entry code: 1n8v) as template, the binding model of CSP1 protein and semiochemicals was analyzed by homology modeling and molecular docking. In order to obtain the binding mode of pheromone and CSP1, according to MolDock Score, the best mechanism of the docking model was analyzed, and the hydrogen bonds between the ligand and the CSP1 residues were obtained. Finally, the polyclonal antibody of CSP1 was obtained by immunizing rabbits, and the antennae of worker bee were immobilized at low temperature, dehydrated and embedded. The subcellular distribution of CSP1 on the antennal sections samples were then immunogolded by colloidal gold and finally observed with the electron microscopy. 【Result】 After the soluble recombinant CSP1 protein was successfully induced and purified, the fluorescence quenching assay was used to obtain the dissociation constant K_1-NPN between CSP1 and 1-NPN as 2.1 μmol·L^-1. The number of binding sites n was 0.99, indicating the bind ratio of CSP1 and 1-NPN was 1﹕1. In the candidate 9 semiochemicals, CSP1 and two queen pheromone components: p-hydroxybenzoic acid methyl ester (HOB) and (E)-9-Oxodec-2-enoic acid (9-ODA), and plant volatile components, 3-carene had a strong ability to bind, the [IC₅₀] and dissociation constant K_D of the most strongly bound HOB was 10.1 and 7.68 μmol·L^-1, respectively. The results of molecular docking showed that the binding of various ligands to CSP1 was due to some specific amino acid residues (or by means of the hydrogen bonds) in the hydrophobic cavity of CSP1. Typically, the interaction between CSP1 and HOB is predicted by the contribution of eight amino acid residues, including four hydrophobic residues (Phe30, Phe44, Leu70 and Phe85), three polar neutral residues (Tyr26, Tyr27 and Ser41) and one acidic residue (Asp40). The two hydrogen bonds were produced between the oxygen atoms on the two carboxyl groups in Asp40 and the oxygen atoms on the hydroxyl group in the HOB benzene ring, respectively. The results of immuno-electron microscopy showed that CSP1 was mainly expressed in the ancillary supporting cells around the sensilla placodea, while only slightly expressed in the inner area of sensilla placodea. It was significantly different from the localization of odorant binding proteins.【Conclusion】 CSP1 of A. c. cerana has a strong ability to bind two queen pheromone components and some plant floral scents, and concentrates the function of pheromone binding protein and general odorant-binding protein. Although their functional modes are similar, there are significant differences in the antennal subcellular localization between CSP1 and odorant-binding proteins, showing the physiological characteristics variation of chemoreceptive proteins.

Key words: Apis cerana cerana, chemosensory protein, functional mode, molecular docking, immunocytochemical localization

TAN Jing, SONG XinMi, FU XiaoBin, TANG MingZhu, WU Fan, HUA QiYun, LI HongLiang. Functional Mode and Immunocytochemical Localization of Chemosensory Protein 1 (CSP1) in Apis cerana cerana[J].Scientia Agricultura Sinica, 2017, 50(15): 3052-3062.

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