两种方法测定绿盲蝽气味结合蛋白AlucOBP21 配体结合特性的结果比较

doi:10.3864/j.issn.0578-1752.2017.23.011

摘要/Abstract

摘要： 【目的】比较荧光竞争结合试验（fluorescence competitive binding assay）和微量热涌动（microscale thermophoresis，MST）技术在研究绿盲蝽（Apolygus lucorum）气味结合蛋白（odorant binding proteins，OBPs） AlucOBP21配体结合特性上的差异，探索一种新的测定昆虫OBPs结合功能的方法。【方法】提取绿盲蝽雌、雄成虫触角总RNA并进行反转录，获得绿盲蝽成虫触角cDNA。采用特异性克隆引物，以绿盲蝽成虫触角cDNA为模板进行PCR扩增，构建pET32a/AlucOBP21重组质粒。将重组质粒转化至BL21（DE3）感受态细胞进行原核表达，获得含表达标签的重组AlucOBP21蛋白。通过高亲和Ni-NTA纯化介质对重组粗蛋白进行纯化，采用重组肠激酶切掉His-tag标签，最终获得无表达标签的重组AlucOBP21蛋白。利用荧光竞争结合试验,以1-N-phenylnaphthylamine（1-NPN）为荧光探针研究重组AlucOBP21蛋白与候选配体的结合特性，其中1-NPN和气味标样均溶解在质谱纯级的甲醇中。同时，利用MST技术解析重组AlucOBP21蛋白与候选配体的结合特性，候选配体标样溶解在二甲基亚砜（dimethyl sulfoxide，DMSO）溶液中。候选配体化合物包括8种潜在的盲蝽性信息素及性信息素类似物、12种植物绿叶挥发物和绿盲蝽驱避剂主要组分二甲基二硫醚等。【结果】重组AlucOBP21蛋白在上清液和包涵体均能够表达，最终选择上清组分进行目标蛋白纯化，利用重组肠激酶在22℃切除His-tag标签得到无标签的重组AlucOBP21蛋白。荧光竞争结合试验结果显示，重组AlucOBP21与荧光探针1-NPN的解离常数为（6.88±0.31）μmol·L^-1，AlucOBP21能够与β-紫罗兰酮和β-石竹烯结合，解离常数分别为（13.74±1.93）和（13.24±2.12）μmol·L^-1，而其他候选配体均不能与重组AlucOBP21有效结合。MST测试结果显示，AlucOBP21可以与β-石竹烯、β-紫罗兰酮、β-蒎烯和柠檬烯有效结合，解离常数分别为（0.20±0.02）、（0.05±0.01）、（0.70±0.04）和（0.40±0.06）μmol·L^-1。其余候选配体化合物与AlucOBP21的热涌动不能随配体浓度变化而表现有规律的变化，故这些气味化合物不能与AlucOBP21发生结合作用。【结论】MST检测与荧光竞争结合试验相比，MST所得配体结合谱更广，不会遗漏一些结合力较弱的气味配体。

关键词: 绿盲蝽, 气味结合蛋白, AlucOBP21, 竞争结合试验, 微量热涌动测定

Abstract: 【Objective】The objective of this study is to compare two methods of fluorescence competitive binding assay and microscale thermophoresis (MST) in analysis of binding characteristics of odorant binding proteins Alucobp21 of Apolygus lucorum, and to explore a new method for determination of binding function of insect obps. 【Method】 Total RNA was extracted from antennae of both female and male A. lucorum adults by using Trizol reagent. The cDNAs were synthesized using the Superscript III Reverse Transcriptase system. AlucOBP21 was PCR-amplified using gene-specific primers. The sample cDNA of the antennae was used as the template. The PCR product was cloned into an expression vector PET-32a (+) for expression in prokaryotic BL21 (DE3) cells. The transformation of the strain with pET32a/AlucOBP21 was incubated in cultures and the crude protein with His-tag was obtained. The supernatant was obtained by sonication, purified by Ni ion affinity chromatography. Soon after the His-tag was removed with recombinant enterokinase, then the purified AlucOBP21 without His-tag was harvested. To investigate the binding abilities of AlucOBP21 to candidate ligand chemicals, fluorescence binding test was performed using 1-N-phenylnaphthylamine (1-NPN) as a fluorescence probe. 1-NPN and odor standard samples were dissolved in methanol (mass spectrometry grade). And also, the binding characteristics of recombinant AlucOBP21 to candidate ligands were explored by MST technique. In this assay, the candidate ligand samples were dissolved in dimethyl sulfoxide (DMSO) solution. Candidate ligands included 8 potential sex pheromones and sex pheromone analogues of mirids, 12 green leaf volatiles and a repellent (dimethyl disulfide) of A. lucorum.【Result】Recombinant AlucOBP21 was expressed in both supernatant and inclusion bodies. Finally, the supernatant fraction was selected to purify the target protein. Recombinant AlucOBP21 without His-tag was obtained by using recombinant entherokinase at 22℃. In fluorescence competitive binding assays, recombinant AlucOBP21 could bind with 1-NPN probe, dissociation constant was (6.88±0.31) μmol·L^-1. AlucOBP21 could bind with b-ionone and b-caryophyllene, and the dissociation constants were (13.74±1.93) and (13.24±2.12) μmol·L^-1, respectively. However, remaining candidate ligands could not effectively bind to recombinant AlucOBP21. Analysis of MST demonstrated that AlucOBP21 could bind with b-ionone, b-caryophyllene, b-pinene and limonene, and the dissociation constants were (0.20±0.02), (0.05±0.01), (0.70±0.04) and (0.40±0.06) μmol·L^-1, respectively. The thermophoresis of remaining candidate ligands with AlucOBP21 could not display regular change along with the change of ligand concentration. Therefore, these odorant chemicals could not bind with recombinant AlucOBP21.【Conclusion】In comparison with fluorescence competitive binding assay, MST detection demonstrated a broader ligand spectrum which could not miss odorant ligands with weak binding affinities.

Key words: Apolygus lucorum, odorant binding protein, AlucOBP21, competitive binding assay, microscale thermophoresis (MST)

刘航玮，张强，耿亭，董昆，安兴奎，王琪，张永军，郭予元. 两种方法测定绿盲蝽气味结合蛋白AlucOBP21 配体结合特性的结果比较[J]. 中国农业科学, 2017, 50(23): 4585-4592.

LIU HangWei, ZHANG Qiang, Geng Ting, DONG Kun, AN XingKui, WANG Qi, ZHANG YongJun, GUO YuYuan. Comparison of Two Methods in Analysis of Binding Characteristics of Odorant Binding Proteins AlucOBP21 of Apolygus lucorum[J]. Scientia Agricultura Sinica, 2017, 50(23): 4585-4592.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.23.011

https://www.chinaagrisci.com/CN/Y2017/V50/I23/4585

参考文献

[1] Kaissling K E, Keil T A, Williams J L D. Pheromone stimulation in perfused sensory hairs of the moth Antheraea polyphemus. Journal of Insect Physiology, 1991, 37(1): 71-75, 77-78.

[2] Pelosi P, Zhou J J, Ban L P, Calvello M. Soluble proteins in insect chemical communication. Cellular and molecular life sciences, 2006, 63(14): 1658-1676.

[3] Kaissling K E. Kinetics of olfactory responses might largely depend on the odorant-receptor interaction and the odorant deactivation postulated for flux detectors. Journal of Comparative Physiology A, 2013, 199(11): 879-896.

[4] Ban L, Scaloni A, D'Ambrosio C, Zhang L, Yahn Y, Pelosi P. Biochemical characterization and bacterial expression of an odorant-binding protein from Locusta migratoria. Cellular and Molecular Life Sciences, 2003, 60(2): 390-400.

[5] Jerabek-Willemsen M, Wienken C J, Braun D, Baaske P, Duhr S. Molecular interaction studies using microscale thermophoresis. Assay and Drug Development Technologies, 2011, 9(4): 342-353.

[6] Wienken C J, Baaske P, Rothbauer U, Braun D, Duhr S. Protein-binding assays in biological liquids using microscale thermophoresis. Nature Communications, 2010, 1(7): 100.

[7] Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Längst G, Baaske P. Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution. Methods in Molecular Biology, 2012, 815: 241-252.

[8] Tegler L T, Corin K, Hillger J, Wassie B, YU Y M, ZHANG S G. Cell-free expression, purification, and ligand-binding analysis of Drosophila melanogaster olfactory receptors DmOR67a, DmOR85b and DmORCO. Scientific Reports, 2015, 5: Article number 7867.

[9] Yuan H B, Ding Y X, Gu S H, Sun L, Zhu X Q, Liu H W, Khalid H D, Zhang Y J, Guo Y Y. Molecular characterization and expression profiling of odorant-binding proteins in Apolygus lucorum. PLoS ONE, 2015, 10(10): e0140562.

[10] Zhou J J, Robertson G, He X L, Dufour S, Hooper A M, Pickett J A, Keep N H, Field L M. Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components. Journal of Molecular Biology, 2009, 389(3): 529-545.

[11] Gu S H, Zhou J J, Wang G R, Zhang Y J, Guo Y Y. Sex pheromone recognition and immunolocalization of three pheromone binding proteins in the black cutworm moth Agrotis ipsilon. Insect Biochemistry and Molecular Biology, 2013, 43(3): 237-251.

[12] He X L, Tzotzos G, Woodcock C, Pickett J A, Hooper T, Field L M, Zhou J J. Binding of the general odorant binding protein of Bombyx mori, BmorGOBP2 to the moth sex pheromone components. Journal of Chemical Ecology, 2010, 36(12): 1293-1305.

[13] Duhr S, Braun D. Why molecules move along a temperature gradient. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(52): 19678-19682.

[14] 苏建伟, 陈展册, 张广珠, 戈峰. 绿盲蝽雌虫的浸提物分析. 昆虫知识, 2010, 47(6): 1113-1117.

Su J W, Chen Z C, Zhang G Z, Ge F. Identification of extracted compounds of female Lygus lucorum. Chinese Bulletin of Entomology, 2010, 47(6): 1113-1117. (in Chinese)

[15] Yang C Y, Kim J, Ahn S J, Kim D H, Cho M R. Identification of the female-produced sex pheromone of the plant bug Apolygus spinolae. Journal of Chemical Ecology, 2014, 40: 244-249.

[16] 陈展册, 苏丽, 戈峰, 苏建伟. 绿盲蝽对性信息素类似物和植物挥发物的触角电位反应. 昆虫学报, 2010, 53(1): 47-54.

Chen Z C, Su L, Ge F, Su J W. Electroantennogram responses of the green bug Apolygus lucorum (Hemiptera: Miridae), to sex pheromone analogs and plant volatiles. Acta Entomologica Sinica, 2010, 53(1): 47-54. (in Chinese)

[17] Aldrich J R. Chemical ecology of the Heteroptera. Annual Review of Entomology, 1998, 33: 211-238.

[18] 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.

Zhang T. The study of the extraction, identification and application of sex pheromone produced by Apolygus lucorum[D]. Beijing: Chinese Academy of Agricultural Sciences, 2011. (in Chinese)

[19] 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.

Pan H S. Chemical communication mechanism between Apolygus lucorum (Meyer-Dür) and its fall host plants of Artemisia spp.[D]. Beijing: Chinese Academy of Agricultural Sciences, 2013. (in Chinese)

[20] Loughrin J H, Manukian A, Heath R R, Tumlinson J H. Volatiles emitted by different cotton varieties damaged by feeding beet armyworm larvae. Journal of ChemicalEcology, 1995, 21(8): 1217-1227.

[21] McCall P J, Turlings T C, Loughrin J, Proveaux A T, Tumlinson J H. Herbivore-induced volatile emissions from cotton (Gossypium hirsutum L.) seedlings. Journal of ChemicalEcology, 1994, 20(12): 3039-3050.

[22] Blackmer J L, Rodriguez-Saona C, Byers J A, Shope K L, Smith J P. Behavioral response of Lygus hesperus to conspecifics and headspace volatiles of alfalfa in a Y-tube olfactometer. Journal of Chemical Ecology, 2004, 30(8): 1547-1564.

[23] Chinta S, Dickens J C, Aldrich J R. Olfactory reception of potential pheromones and plant odors by tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). Journal of Chemical Ecology, 1994, 20(12): 3251-3267.

[24] Obata T, Koh H S, Kim M, Fukami H. Constituents of plant hopper attractant in rice plant. Applied Entomology and Zoology, 1983, 18: 161-169.

[25] Williams L, Blackmer J L, Rodriguez-Saona C, Zhu S. Plant volatiles influence electrophysiological and behavioral responses of Lygus hesperus. Journal of Chemical Ecology, 2010, 36(5): 467-478.

[26] Loughrin J H, Manukian A, Heath R R, Turlings T C, Tumlinson J H. Diurnal cycle of emission of induced volatile terpenoids by herbivore-injured cotton plant. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(25): 11836-11840.

[27] Pan H S, Lu Y H, Wyckhuys K A, Wu K M. Preference of a polyphagous mirid bug, Apolygus lucorum (Meyer-Dur) for flowering host plants. PLoS ONE, 2013, 8(7): e68980.

[28] Qiao H L, Tuccoria E, He X L, Gazzanoc A, Fieldb L, Zhou J J, Pelosia P. Discrimination of alarm pheromone (E)-β- farnesene by aphid odorant-binding proteins. Insect Biochemistry and Molecular Biology, 2009, 39: 414-419.

[29] Gu S H, Wang W X, Wang G R, Zhang X Y, Guo Y Y, Zhang Z D, Zhou J J, Zhang Y J. Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus (Goeze). Archives of Insect Biochemistry and Physiology, 2011, 77(2): 81-99.