绿盲蝽气味结合蛋白AlucOBP8的结合特性分析

doi:10.16409/j.cnki.2095-039x.2015.06.002

摘要/Abstract

摘要： 气味结合蛋白(odorant binding proteins,OBPs)是参与昆虫嗅觉感知过程的一类重要蛋白,其功能之一是结合运输外界气味分子通过淋巴液到达神经元树突。本文克隆了一个绿盲蝽Apolygus lucorum(Meyer-Dür)气味结合蛋白AlucOBP8(GenBank登录号JQ675725)并进行了原核重组表达。组织表达谱分析表明,AlucOBP8基因主要在雌雄成虫触角表达。采用荧光竞争结合试验,研究了重组蛋白AlucOBP8与7种性信息素类似物和33种植物挥发物的结合能力。结果显示,7种性信息素类似物均不能与AlucOBP8有效结合,而植物挥发物中只有葎草烯(α-caryophyllene)和十二醛(dodecyl aldehyde)与AlucOBP8表现出较强的结合,结合常数分别为8.74和9.99 μmol/L。绿盲蝽雌雄成虫对这两种化合物的趋向结果表明,十二醛对绿盲蝽雄成虫具有显著的驱避作用。初步推断AlucOBP8为普通气味结合蛋白并参与寄主普通挥发气味的识别过程。

关键词: 绿盲蝽, AlucOBP8, 植物挥发物, 竞争结合试验, 趋向选择

Abstract: Odorant binding proteins (OBPs), an important class of proteins, are involved in the process of insect olfactory perception. One of OBP’s functions is to bind and transport odor molecules through the lymph to reach neuronal dendrites. In the present study, we cloned an OBP gene of Apolygus lucorum (Meyer-Dür) (GenBank No.JQ675725) and named AlucOBP8. The quantitative real-time PCR (qRT-PCR) analysis demonstrated that the AlucOBP8 was mainly expressed in the antennae of both male and female adult bugs. The fluorescence competitive binding assays were conducted to measure the binding abilities of recombinant AlucOBP8 to seven sex pheromone analogs and thirty-three plant volatiles. The results revealed that AlucOBP8 could not bind to sex pheromone analogs. Only two plant volatiles, α-caryophyllene and dodecyl aldehyde showed strong binding affinities and the dissociation constant were 8.74 and 9.99 μmol/L, respectively. We measured the taxis selection of A. lucorum to α-caryophyllene and dodecyl aldehyde by using Y tube olfactometer. The results showed that dodecyl aldehyde had significantly repellent action on A. lucorum adult males. We suspected that the AlucOBP8 might be a general odorant binding protein to play roles in odor recognition of host plant volatiles.

Key words: Apolygus lucorum, AlucOBP8, plant volatiles, binding assay, taxis selection

中图分类号:

S436.421

朱晓强, 丁玉骁, 刘航玮, 周延乐, 张永军, 郭予元. 绿盲蝽气味结合蛋白AlucOBP8的结合特性分析[J]. 中国生物防治学报, 2015, 31(6): 821-829.

ZHU Xiaoqiang, DING Yuxiao, LIU Hangwei, ZHOU Yanle, ZHANG Yongjun, GUO Yuyuan. Binding Specificity Analysis of Odorant Binding Protein AlucOBP8 of Apolygus lucorum (Meyer-Dür)[J]. journal1, 2015, 31(6): 821-829.

参考文献

[1] 陆宴辉, 吴孔明. 棉花盲蝽象及其防治[M]. 北京: 金盾出版社, 2008.
[2] Lu Y H, Qiu F, Feng H Q, et al. Species composition and seasonal abundance of pestiferous plant bugs (Hemiptera: Miridae) on Bt cotton in China[J]. Crop Protection, 2008, 27(4): 465-472.
[3] Lu Y H, Wu K M. Mirid bugs in China: pest status and management strategies[J]. Outlooks on Pest Management, 2011, 22(6): 248-252.
[4] Larsson M C, Domingos A I, Jones W D, et al. Or83b encodes a broadly expressed odorant receptor essential for Drosophila ofaction[J]. Neuron, 2004, 43(5): 703-714.
[5] Gu S H, Wang S P, Zhang X Y, et al. Identification and tissue distribution of odorant binding protein genes in the lucerne plant bug Adelphocoris lineolatus (Goeze)[J]. Insect Biochemistry and Molecular Biology, 2011, 41(4): 254-263.
[6] Leal W S. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes[J]. Annual Review of Entomology, 2013, 58: 373-391.
[7] Xu P X. A Drosophila OBP required for pheromone signaling[J]. Science, 2005, 310: 798-799.
[8] Ko K J, Lee S H, Oh E H, et al. Specificity of odorant-binding proteins: a factor influencing the sensitivity of olfactory receptor-based biosensors[J]. Bioprocess and Biosystems Engineering, 2010, 33(1): 55-62.
[9] Pelosi P, Mastrogiacomo R, Iovinella I, et al. Structure and biotechnological applications of odorant-binding proteins[J]. Applied Microbiology and Biotechnology, 2014, 98(1): 61-70.
[10] Xu P X, Atkinson R, Jones D N, et al. Drosophila OBP LUSH is required for activity of pheromone-sensitive neurons[J]. Neuron, 2005, 45(2): 193-200.
[11] 陆宴辉, 吴孔明, 蔡晓明, 等. 利用四季豆饲养盲蝽的方法[J]. 植物保护学报, 2008, 35(3): 215-219.
[12] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCtmethod[J]. Methods, 2001, 25(4): 402-408.
[13] Prestwich G D. Bacterial expression and photoaffinity labeling of a pheromone binding protein[J]. Protein Science, 1993, 151(2): 420-428.
[14] Qiao H, Tuccori E, He X, et al. Discrimination of alarm pheromone (E)-β-farnesene by aphid odorant-binding proteins[J]. Insect Biochemistry and Molecular Biology, 2009, 39(5-6): 414-419.
[15] Gu S H, Wang W X, Wang G R, et al. Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus (GOEZE)[J]. Archives of Insect Biochemistry and Physiology, 2011, 77(2): 81-99.
[16] 张涛. 绿盲蝽性信息素的提取和鉴定[D]. 北京: 中国农业科学院, 2011.
[17] 苏建伟, 陈展册, 张广珠, 等. 绿盲蝽雌虫的浸提物分析[J]. 昆虫知识, 2010, 47(6): 1113-1117.
[18] Yang C Y, Kim J, Ahn S J, et al. Identification of the female-produced sex pheromone of the plant bug Apolygus spinolae[J]. Journal of Chemical Ecology, 2014, 40: 244-249.
[19] 陈展册, 苏丽, 戈峰, 等. 绿盲蝽对性信息素类似物和植物挥发物的触角电位反应[J]. 昆虫学报, 2010, 53(1): 47-54.
[20] Aldrich J R. Chemical ecology of the Heteroptera[J]. Annual Review of Entomology, 1998, 33: 211-238.
[21] 潘洪生. 绿盲蝽与秋季蒿类寄主的化学通讯机制[D]. 北京: 中国农业科学院, 2013.
[22] Loughrin J, Manukian A, Heath R, et al. Volatiles emitted by different cotton varieties damaged by feeding beet armyworm larvae[J]. Journal of Chemical Ecology, 1995, 21(12): 1217-1227.
[23] Mccall P J, Turlings T C, Loughrin J, et al. Herbivore-induced volatile emissions from cotton (Gossypium hirsutum L.) seedlings[J]. Journal of Chemical Ecology, 1994, 20(12): 3039-3050.
[24] Whitman D W, Eller F J. Parasitic wasps orient to green leaf volatiles[J]. Chemoecology, 1990, 64(1): 69-76.
[25] Blackmer J L, Rodriguez-Saona C, Byers J A, et al. Behavioral response of Lygus hesperus to conspecifics and headspace volatiles of alfalfa in a Y-tube olfactometer[J]. Journal of Chemical Ecology, 2004, 30(12): 1547-1564.
[26] Williams L, Blackmer J L, Rodriguez-Saona C, et al. Plant volatiles influence electrophysiological and behavioral responses of Lygus hesperus[J]. Journal of Chemical Ecology, 2010, 36(5): 467-478.
[27] 于惠林, 张永军, 潘文亮, 等. 田间条件下不同诱导棉花挥发性物质的测定[J]. 应用生态学报, 2007, 18(4): 859-864.
[28] Chinta S, Dickens J C, Aldrich J R. Olfactory reception of potential pheromones and plant odors by tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae)[J]. Journal of Chemical Ecology, 1994, 20: 3251-3267.
[29] Obata T, Koh H S, Kim M, et al. Constituents of planthopper atractant in rice plant[J]. Applied Entomology and Zoology, 1983, 18: 161-169.
[30] Kallenbach M, Oh Y, Eilers E J, et al. A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments[J]. Plant Journal, 2014, 78(6): 1060-1072.
[31] 潘洪生. 棉花-棉铃虫-寄生蜂三级营养链间中红侧沟茧蜂的实时趋性反应[D]. 哈尔滨: 东北农业大学, 2010.
[32] 曹阳. 绿盲蝽对不同抗性棉花品种(系)寄主选择机制初步研究[D]. 杨凌: 西北农林科技大学, 2012.
[33] 谷少华. 苜蓿盲蝽嗅觉相关基因的发掘及功能分析[D]. 北京: 中国农业科学院, 2010.
[34] 纪萍. 绿盲蝽气味结合蛋白OBPs基因的鉴定及AlucOBP7的气味结合特性[D]. 北京: 中国农业科学院, 2013.
[35] Zhu J, Park K C, Baker T C. Identification of odors from overripe mango that attract vinegar flies, Drosophila melanogaster[J]. Journal of Chemical Ecology, 2003, 29(4): 899-909.
[36] Sun L, Gu S H, Xiao H J, et al. The preferential binding of a sensory organ specific odorant binding protein of the alfalfa plant bug Adelphocoris lineolatus AlinOBP10 to biologically active host plant volatiles[J]. Journal of Chemical Ecology, 2013, 39(11): 1221-1231.
[37] Elzen G W, Williams H J, Vinson S B. Role of diet in host selection of Heliothis virescens by parasitoid Campoletis sonorensis (Hymenoptera: Ichneumonidae)[J]. Journal of Chemical Ecology, 1984, 10(11): 1535-1541.
[38] Stensmyr M C, Dweck H K, Farhan A, et al. A conserved dedicated olfactory circuit for detecting harmful microbes in Drosophila[J]. Cell, 2012, 151(6): 1345-1357.
[39] Pelosi P, Zhou J J, Ban L P, et al. Soluble proteins in insect chemical communication[J]. Cellular and Molecular Life Sciences, 2006, 63(14): 1658-1676.
[40] Xu Y L, He P, Zhang L, et al. Large-scale identification of odorant-binding proteins and chemosensory proteins from expressed sequence tags in insects[J]. BMC Genomics, 2009, 10: 632.
[41] Ji P, Gu S H, Liu J T, et al. Identification and expression profile analysis of odorant-binding protein genes in Apolygus lucorum (Hemiptera: Miridae)[J]. Applied Entomology and Zoology, 2013, 48(3): 301-311.
[42] Hull J J, Perera O P, Snodgrass G L. Cloning and expression profiling of odorant-binding proteins in the tarnished plant bug, Lygus lineolaris[J]. Insect Molecular Biology, 2014, 23(1): 78-97.
[43] 周帅. 非生物胁迫对香樟挥发物及荧光特性的影响[D]. 杭州: 浙江农林大学, 2012.
[44] Kalberer N M, Turlings T C, Rahler M. Attraction of a leaf beetle (Oreina cacaliae) to damaged host plants[J]. Journal of Chemical Ecology, 2001, 27(4): 647-661.