苜蓿盲蝽化学感受蛋白Alin-CSP6的气味结合特性

Odorant Binding Characteristics of Chemosensory Protein Alin-CSP6 in Lucerne Plant Bug,Adelphocoris lineolatus(Goeze)

【目的】利用昆虫的嗅觉识别机制,以化学感受蛋白作为潜在的靶标,干扰昆虫的寄主定位及交配等行为,用于害虫综合治理。【方法】克隆并纯化了1个新的苜蓿盲蝽化学感受蛋白Alin-CSP6,进行了113种气味化合物对Alin-CSP6的荧光竞争性结合试验,将荧光竞争结合试验筛选出强结合力的气味标样采用"Y"型嗅觉仪验证行为反应,并采用同源建模的方法构建Alin-CSP6的3D结构。【结果】Alin-CSP6具有较广谱的结合力,能够与醇、醛、酮、酯、萜、芳香族等多种化合物结合并表现较强的结合能力。其中,β-香茅醇(β-citronellol)、反式-2-己烯醛(trans-2-hexene aldehyde)、6-甲基-5-庚烯-2-酮(6-methyl-5-heptene-2-ketone)、2-十一烷酮(2-undecanone)、乙酰丙酸丁酯(butyl levulinate)、月桂烯(myrcene)、左旋-β-蒎烯((1s)-(-)-β-pinene)、壬烷(nonane)、3,4-二甲基苯甲醛(3,4-dimethyl benzaldehyde)、乙醚(ethyl ether)、罗勒烯(ocimene)、苯乙酮(acetophenone)、苯甲醛(benzaldehyde)与Alin-CSP6有很强的结合力。"Y"型管试验结果发现,反式-2-己烯醛和苯乙酮对苜蓿盲蝽成虫表现出极显著的引诱作用,而6-甲基-5-庚烯-2-酮、左旋-β-蒎烯和4-乙基苯甲醛(4-ethylbenzaldehyde)则对苜蓿盲蝽成虫具有极显著的拒避效果。Alin-CSP6的3D结构显示该蛋白主要由6个α螺旋构成,分别是13-18(α1)、20-32(α2)、39-52(α3)、59-76(α4)、78-88(α5)和94-102(α6)。Cys29-Cys36和Cys55-Cys58两个二硫键起着稳定蛋白结构的作用。推测Alin-CSP6的Ile74和Phe81基团能够参与结合油酸酰胺或其类似物。【结论】Alin-CSP6具有较广泛的气味结合谱,在苜蓿盲蝽嗅觉过程中具有重要作用。

[Objective] The objective of this study is to use the mechanism of insect olfactory recognition, making CSPs as potential targets, and interfering with the insect host location and mating behavior for integrate pest management （IPM）. [Method] In the present study, a novel chemosensory protein Alin-CSP6 in lucerne plant bug Adelphocoris lineolatus （Goeze） was cloned and the recombinant protein was obtained. The binding characteristics of Alin-CSP6 with 113 volatile odors was explored by a fluorescent competitive binding experiment. [Result] Alin-CSP6 has a wide binding spectrum with odorant ligands, which can bind to alcohols, aldehydes, ketones, easters, aromatic and so on. The β-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 Alin-CSP6. In order to verify the behavioral influence of the odors on A. lineolatus, Y-tube olfactometer tests were conducted to observe the response of the adult to odorants which exhibited high binding abilities to Alin-CSP6. It was found that trans-2-hexene aldehyde and acetophenone showed strong attraction to lucerne plant bug, whereas 6-methyl-5-heptene-2-ketone, （1s）-（-）-β-pinene and 4-ethylbenzaldehyde displaied strong repellent action. By homologous modeling analysis the 3D structure was predicted, and it was found that Alin-CSP6 is consists of six a-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 stability of the protein. It is speculated that lle7 and Phe81 may participate in binding to oleic acid amide analogues. [Conclusion] Alin-CSP6 has a wide binding spectrum with odorant ligands which play an important role in olfaction ofA. lineolatus.