中红侧沟茧蜂气味结合蛋白MmedOBP18的原核表达及配体结合特性
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摘要
【目的】研究中红侧沟茧蜂Microplitis mediator气味结合蛋白MmedOBP18在触角中的表达定位,解析MmedOBP18重组蛋白的配体结合特性。【方法】原核表达中红侧沟茧蜂气味结合蛋白MmedOBP18;采用荧光免疫组织化学技术研究MmedOBP18在中红侧沟茧蜂雌蜂触角中的表达定位;通过荧光竞争结合实验分析MmedOBP18重组蛋白与99种候选配体的结合特性。【结果】在原核表达系统中成功表达MmedOBP18重组蛋白。荧光免疫定位结果显示MmedOBP18主要表达在触角锥形感器Ⅰ内的淋巴液中。荧光竞争结合实验结果表明,MmedOBP18重组蛋白能够与16种候选配体结合,与低挥发性植物挥发物2-十三酮、十二醛、十四酸和十一酸的结合能力最强,其解离常数K_i值分别为5.21, 6.42, 6.49和6.58μmol/L;而且MmedOBP18重组蛋白与非挥发性植物次生物质棕榈酸、棉酚、槲皮素及亚麻酸也有较强的结合能力,K_i值分别为3.86, 5.07, 5.08和6.51μmol/L;此外,MmedOBP18重组蛋白与鳞翅目昆虫性信息素组分顺-9-十四碳烯醛及顺-11-十六碳醛表现出较强的结合能力,K_i值分别为9.09和11.67μmol/L,提示该蛋白在寄主定位过程中可发挥重要的作用。【结论】中红侧沟茧蜂气味结合蛋白MmedOBP18能够结合长链低挥发和非挥发性化合物,推测其在嗅觉和味觉识别中发挥双重功能,主要参与对寄主和寄主生境的化学信息的近距离识别。
【Aim】 To clarify the expression of the odorant binding protein MmedOBP18 in the antenna of Microplitis mediator, and to investigate the ligand binding characteristics of the recombinant MmedOBP18. 【Methods】 The recombinant MmedOBP18 of M. mediator was expressed in the prokaryotic expression system. The localization of expression of MmedOBP18 in the antenna of female wasps of M. mediator was evaluated using fluorescence immunohistochemistry assay. The binding characteristics of the recombinant MmedOBP18 protein to 99 candidate ligands were analyzed by fluorescence competitive binding assay. 【Results】 The recombinant MmedOBP18 protein was successfully expressed in the prokaryotic expression system. The immunofluorescence localization result showed that MmedOBP18 was mainly expressed in the lymph of sensilla basiconica type I of the antenna. The recombinant MmedOBP18 could strongly bind with 16 kinds of ligands. Among the low volatile compounds, 2-tridecanone, dodecyl aldehyde, myristic acid and undecylic acid showed the strongest binding capacities to the recombinant MmedOBP18, with the dissociation constant(K_i) values of 5.21, 6.42, 6.49 and 6.58 μmol/L, respectively. The non-volatile compounds palmitic acid, gossypol, quercetin and linolenic acid also showed strong binding capabilities to the recombinant MmedOBP18, with the K_i values of 3.86, 5.07, 5.08 and 6.51 μmol/L, respectively. In addition, the lepidopteran pheromone components(Z)-9-tetradecenal and(Z)-11-hexadecenal also showed strong binding capabilities to MmedOBP18, with the K_i values of 9.09 and 11.67 μmol/L, respectively, suggesting an important role of MmedOBP18 in host localization. 【Conclusion】 The MmedOBP18 of M. mediator has strong binding capabilities to long-chain low volatile or non-volatile compounds. We speculated that MmedOBP18 may play a dual role in olfactory and taste recognition, and is mainly involved in the close range perception of chemical signals from host and host habitats.
【Aim】 To clarify the expression of the odorant binding protein MmedOBP18 in the antenna of Microplitis mediator, and to investigate the ligand binding characteristics of the recombinant MmedOBP18. 【Methods】 The recombinant MmedOBP18 of M. mediator was expressed in the prokaryotic expression system. The localization of expression of MmedOBP18 in the antenna of female wasps of M. mediator was evaluated using fluorescence immunohistochemistry assay. The binding characteristics of the recombinant MmedOBP18 protein to 99 candidate ligands were analyzed by fluorescence competitive binding assay. 【Results】 The recombinant MmedOBP18 protein was successfully expressed in the prokaryotic expression system. The immunofluorescence localization result showed that MmedOBP18 was mainly expressed in the lymph of sensilla basiconica type I of the antenna. The recombinant MmedOBP18 could strongly bind with 16 kinds of ligands. Among the low volatile compounds, 2-tridecanone, dodecyl aldehyde, myristic acid and undecylic acid showed the strongest binding capacities to the recombinant MmedOBP18, with the dissociation constant(K_i) values of 5.21, 6.42, 6.49 and 6.58 μmol/L, respectively. The non-volatile compounds palmitic acid, gossypol, quercetin and linolenic acid also showed strong binding capabilities to the recombinant MmedOBP18, with the K_i values of 3.86, 5.07, 5.08 and 6.51 μmol/L, respectively. In addition, the lepidopteran pheromone components(Z)-9-tetradecenal and(Z)-11-hexadecenal also showed strong binding capabilities to MmedOBP18, with the K_i values of 9.09 and 11.67 μmol/L, respectively, suggesting an important role of MmedOBP18 in host localization. 【Conclusion】 The MmedOBP18 of M. mediator has strong binding capabilities to long-chain low volatile or non-volatile compounds. We speculated that MmedOBP18 may play a dual role in olfactory and taste recognition, and is mainly involved in the close range perception of chemical signals from host and host habitats.
引文
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Chen L, Li HL, Zhou YX, Zhao L, Zhang LY, Ni CX, Shang HW, 2013. cDNA cloning, tissue expression and ligand binding characteristics of odorant-binding protein 2 from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Acta Entomol. Sin., 56(6): 612-621. [陈玲, 李红亮, 周宇翔, 赵磊, 张林雅, 倪翠侠, 商晗武, 2013. 桔小实蝇气味结合蛋白BdorOBP2的cDNA克隆、组织表达及配基结合特性. 昆虫学报, 56(6): 612-621]
Cui HH, Gu SH, Zhu XQ, Wei Y, Liu HW, Khalid HD, Guo YY, Zhang YJ, 2016. Odorant-binding and chemosensory proteins identified in the antennal transcriptome of Adelphocoris suturalis Jakovlev. Comp. Biochem. Physiol. Part D, 24: 139-145.
Gu SH, Wang WX, Wang GR, Zhang XY, Guo YY, Zhang Z, Zhou JJ, Zhang YJ, 2011. Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus Goeze. Arch. Insect Biochem., 77(2): 81-99.
Huang GZ, Liu JT, Zhou JJ, Wang Q, Dong JZ, Zhang YJ, Li XC, Li J, Gu SH, 2018. Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon. Insect Biochem. Molec. Biol., 98: 34-47.
Krieger J, Nickisch-Rosenegk EV, Mameli M, Pelosi P, Breer H, 1996. Binding proteins from the antennae of Bombyx mori. Insect Biochem. Molec. Biol., 26(3): 297-307.
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Li HL, Zhang LY, Zhuang SL, Ni CX, Han BY, Shang HW, 2013. Interpretation of odorant binding function and mode of general odorant binding protein ASP2 in Chinese honeybee (Apis cerana cerana). Sci. Agric. Sin., 46(1): 154-161. [李红亮, 张林雅, 庄树林, 倪翠侠, 韩宝瑜, 商晗武, 2013. 中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析. 中国农业科学, 46(1): 154-161]
Li KM, Wang SN, Zhang K, Ren LY, Ali A, Zhang YJ, Zhou JJ, Guo YY, 2014. Odorant binding characteristics of three recombinant odorant binding proteins in Microplitis mediator (Hymenoptera: Braconidae). J. Chem. Ecol., 40(6): 541-548.
Li ZQ, He P, Zhang YN, Dong SL, 2017. Molecular and functional characterization of three odorant-binding protein from Periplaneta americana. PLoS ONE, 12(1): e0170072.
Ma M, Chang MM, Lei CL, Yang FL, 2016. A garlic substance disrupts odorant-binding protein recognition of insect pheromones released from adults of the angoumois grain moth, Sitotroga cerealella (Lepidoptera: Gelechiidae). Insect Mol. Biol., 25(5): 530-540.
Pan HS, Zhao QJ, Zhao KJ, Zhang YJ, Wu KM, Guo YY, 2011. Responses of Microplitis mediator Haliday (Hymenoptera: Braconidae) to different instar larvae of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) and damaged cotton plants. Acta Entomol. Sin., 54(4): 437-442. [潘洪生, 赵秋剑, 赵奎军, 张永军, 吴孔明, 郭予元, 2011. 中红侧沟茧蜂对不同龄期棉铃虫幼虫及其为害棉株的趋性反应. 昆虫学报, 54(4): 437-442]
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Pelosi P, 1999. Odorant-binding proteins: structural aspects. Ann. NY. Acad. Sci., 855(1): 281-293.
Peng Y, Wang SN, Li KM, Liu JT, Zheng Y, Shan S, Yang YQ, Li RJ, Zhang YJ, Guo YY, 2017. Identification of odorant binding proteins and chemosensory proteins in Microplitis mediator as well as functional characterization of chemosensory protein 3. PLoS ONE, 12(7): e0180775.
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Shi QX, Luo QH, Zhao L, Zhou ZX, He GQ, Wei W, 2015. Extraction and identification of maize volatiles and cuticular volatiles of larval Helicoverpa armigera (Lepidoptera: Noctuidae) related to host-habitat location and host location of parasitic wasp Microplitis mediator (Hymenoptera: Braconidae). Acta Entomol. Sin., 58(3): 244-255. [石庆型, 罗庆怀, 赵龙, 周正湘, 何广全, 韦卫, 2015. 与中红侧沟茧蜂生境与寄主定位相关的玉米及棉铃虫幼虫体表挥发性成分的提取与鉴定. 昆虫学报, 58(3): 244-255]
Song YQ, Xie XC, Dong JF, Wu JX, 2014. cDNA cloning, expression profiling and binding properties of odorant-binding protein GmolOBP3 in the oriental fruit moth, Grapholita molesta (Lepidoptara: Tortricidae). Acta Entomol. Sin., 57(3): 274-285. [宋月芹, 解幸承, 董钧锋, 仵均祥, 2014. 梨小食心虫气味结合蛋白GmolOBP3的cDNA克隆、表达谱及结合特性分析. 昆虫学报, 57(3): 274-285]
Swarup S, Morozova TV, Sridhar S, Nokes M, Anholt RRH, 2014. Modulation of feeding behavior by odorant-binding proteins in Drosophila melanogaster. Chem. Senses, 39: 125-132.
Tang QB, Ma Y, Huang LQ, Wang CZ, 2011. Advances in mechanisms of taste perception of insects. Acta Entomol. Sin., 54(12): 1433-1444. [汤清波, 马英, 黄玲巧, 王琛柱, 2011. 昆虫味觉感受机制研究进展. 昆虫学报, 54(12): 1433-1444]
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Wan XL, Li JH, Lao C, Du YJ, 2015. Antennal lobe neurons of Spodoptera litura (Lepidoptera: Noctuidae) and their responses to plant odors and sex pheromones. Acta Entomol. Sin., 58(3): 223-236. [万新龙, 李江辉, 劳冲, 杜永均, 2015. 斜纹夜蛾触角叶神经元及其对植物气味和性信息素的反应. 昆虫学报, 58(3): 223-236]
Wang SN, Shan S, Liu JT, Li RJ, Lu ZY, Dhiloo KH, Khashaveh A, Zhang YJ, 2018. Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae). Sci. Rep., 8(1): 7649.
Wei Y, 2016. Cloning and Functional Characterization of Homologous Odorant Binding Protein 11 of Two Mirids. MSc Thesis, Chinese Academy of Agricultural Sciences, Beijing. [魏宇, 2016. 两种盲蝽同源气味结合蛋白基因OBP11的克隆和功能研究. 北京: 中国农业科学院硕士学位论文]
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Xu WA, He JH, 2006. One new species of Microplitis Foerster (Hymenoptera: Braconidae: Microgastrinae) from China. Entomotaxonomia, 28(3): 227-230. [许维岸, 何俊华, 2006. 侧沟茧蜂属一新种记述(膜翅目: 茧蜂科: 小腹茧蜂亚科). 昆虫分类学报, 28(3): 227-230]
Yan W, Liu L, Qin WQ, Luo YQ, Ma XZ, Haider N, Inayeh M, 2016. Identification and tissue expression profiling of odorant binding protein genes in the red palm weevil, Rhynchophorus ferrugineus. SpringerPlus, 5(1): 1542.
Yang YQ, Wang SN, Peng Y, Shan S, Zheng Y, Li RJ, Zhang YJ, Guo YY, 2017. Expression of the odor binding protein MmedOBP19 in the legs of Microplitis mediator (Hymenoptera: Braconidae) and its ligand binding characteristics. Acta Entomol. Sin., 60(6): 613-620. [杨叶青, 王山宁, 彭勇, 单双, 郑瑶, 李瑞军, 张永军, 郭予元, 2017. 气味结合蛋白MmedOBP19在中红侧沟茧蜂足部的表达及配体结合特征. 昆虫学报, 60(6): 613-620]
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Zhang S, Chen LZ, Gu SH, Cui JJ, Gao XW, Zhang YJ, Guo YY, 2011. Binding characterization of recombinant odorant-binding proteins from the parasitic wasp, Microplitis mediator (Hymenoptera: Braconidae). J. Chem. Ecol., 37(2): 189-194.
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Chen L, Li HL, Zhou YX, Zhao L, Zhang LY, Ni CX, Shang HW, 2013. cDNA cloning, tissue expression and ligand binding characteristics of odorant-binding protein 2 from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Acta Entomol. Sin., 56(6): 612-621. [陈玲, 李红亮, 周宇翔, 赵磊, 张林雅, 倪翠侠, 商晗武, 2013. 桔小实蝇气味结合蛋白BdorOBP2的cDNA克隆、组织表达及配基结合特性. 昆虫学报, 56(6): 612-621]
Cui HH, Gu SH, Zhu XQ, Wei Y, Liu HW, Khalid HD, Guo YY, Zhang YJ, 2016. Odorant-binding and chemosensory proteins identified in the antennal transcriptome of Adelphocoris suturalis Jakovlev. Comp. Biochem. Physiol. Part D, 24: 139-145.
Gu SH, Wang WX, Wang GR, Zhang XY, Guo YY, Zhang Z, Zhou JJ, Zhang YJ, 2011. Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus Goeze. Arch. Insect Biochem., 77(2): 81-99.
Huang GZ, Liu JT, Zhou JJ, Wang Q, Dong JZ, Zhang YJ, Li XC, Li J, Gu SH, 2018. Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon. Insect Biochem. Molec. Biol., 98: 34-47.
Krieger J, Nickisch-Rosenegk EV, Mameli M, Pelosi P, Breer H, 1996. Binding proteins from the antennae of Bombyx mori. Insect Biochem. Molec. Biol., 26(3): 297-307.
Leal WS, 2013. Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annu. Rev. Entomol., 58: 373-391.
Li HL, Zhang LY, Zhuang SL, Ni CX, Han BY, Shang HW, 2013. Interpretation of odorant binding function and mode of general odorant binding protein ASP2 in Chinese honeybee (Apis cerana cerana). Sci. Agric. Sin., 46(1): 154-161. [李红亮, 张林雅, 庄树林, 倪翠侠, 韩宝瑜, 商晗武, 2013. 中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析. 中国农业科学, 46(1): 154-161]
Li KM, Wang SN, Zhang K, Ren LY, Ali A, Zhang YJ, Zhou JJ, Guo YY, 2014. Odorant binding characteristics of three recombinant odorant binding proteins in Microplitis mediator (Hymenoptera: Braconidae). J. Chem. Ecol., 40(6): 541-548.
Li ZQ, He P, Zhang YN, Dong SL, 2017. Molecular and functional characterization of three odorant-binding protein from Periplaneta americana. PLoS ONE, 12(1): e0170072.
Ma M, Chang MM, Lei CL, Yang FL, 2016. A garlic substance disrupts odorant-binding protein recognition of insect pheromones released from adults of the angoumois grain moth, Sitotroga cerealella (Lepidoptera: Gelechiidae). Insect Mol. Biol., 25(5): 530-540.
Pan HS, Zhao QJ, Zhao KJ, Zhang YJ, Wu KM, Guo YY, 2011. Responses of Microplitis mediator Haliday (Hymenoptera: Braconidae) to different instar larvae of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) and damaged cotton plants. Acta Entomol. Sin., 54(4): 437-442. [潘洪生, 赵秋剑, 赵奎军, 张永军, 吴孔明, 郭予元, 2011. 中红侧沟茧蜂对不同龄期棉铃虫幼虫及其为害棉株的趋性反应. 昆虫学报, 54(4): 437-442]
Pelosi P, Maida R, 1995. Odorant-binding proteins in insects. Comp. Biochem. Physiol. Part B, 111(3): 503-514.
Pelosi P, 1999. Odorant-binding proteins: structural aspects. Ann. NY. Acad. Sci., 855(1): 281-293.
Peng Y, Wang SN, Li KM, Liu JT, Zheng Y, Shan S, Yang YQ, Li RJ, Zhang YJ, Guo YY, 2017. Identification of odorant binding proteins and chemosensory proteins in Microplitis mediator as well as functional characterization of chemosensory protein 3. PLoS ONE, 12(7): e0180775.
Robertson HM, Martos R, Sears CR, Todres EZ, Walden KK, Nardi JB, 1999. Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae. Insect Mol. Biol., 8(4): 501-518.
Shi QX, Luo QH, Zhao L, Zhou ZX, He GQ, Wei W, 2015. Extraction and identification of maize volatiles and cuticular volatiles of larval Helicoverpa armigera (Lepidoptera: Noctuidae) related to host-habitat location and host location of parasitic wasp Microplitis mediator (Hymenoptera: Braconidae). Acta Entomol. Sin., 58(3): 244-255. [石庆型, 罗庆怀, 赵龙, 周正湘, 何广全, 韦卫, 2015. 与中红侧沟茧蜂生境与寄主定位相关的玉米及棉铃虫幼虫体表挥发性成分的提取与鉴定. 昆虫学报, 58(3): 244-255]
Song YQ, Xie XC, Dong JF, Wu JX, 2014. cDNA cloning, expression profiling and binding properties of odorant-binding protein GmolOBP3 in the oriental fruit moth, Grapholita molesta (Lepidoptara: Tortricidae). Acta Entomol. Sin., 57(3): 274-285. [宋月芹, 解幸承, 董钧锋, 仵均祥, 2014. 梨小食心虫气味结合蛋白GmolOBP3的cDNA克隆、表达谱及结合特性分析. 昆虫学报, 57(3): 274-285]
Swarup S, Morozova TV, Sridhar S, Nokes M, Anholt RRH, 2014. Modulation of feeding behavior by odorant-binding proteins in Drosophila melanogaster. Chem. Senses, 39: 125-132.
Tang QB, Ma Y, Huang LQ, Wang CZ, 2011. Advances in mechanisms of taste perception of insects. Acta Entomol. Sin., 54(12): 1433-1444. [汤清波, 马英, 黄玲巧, 王琛柱, 2011. 昆虫味觉感受机制研究进展. 昆虫学报, 54(12): 1433-1444]
Vogt RG, Riddiford LM, 1981. Pheromone binding and inactivation by moth antennae. Nature, 293(5828): 161-163.
Vogt RG, Rybczynski R, Lerner MR, 1991. Molecular cloning and sequencing of general odorant-binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: comparisons with other insect OBPs and their signal peptides. J. Neurosci., 11(10): 2972-2984.
Wan XL, Li JH, Lao C, Du YJ, 2015. Antennal lobe neurons of Spodoptera litura (Lepidoptera: Noctuidae) and their responses to plant odors and sex pheromones. Acta Entomol. Sin., 58(3): 223-236. [万新龙, 李江辉, 劳冲, 杜永均, 2015. 斜纹夜蛾触角叶神经元及其对植物气味和性信息素的反应. 昆虫学报, 58(3): 223-236]
Wang SN, Shan S, Liu JT, Li RJ, Lu ZY, Dhiloo KH, Khashaveh A, Zhang YJ, 2018. Characterization of antennal chemosensilla and associated odorant binding as well as chemosensory proteins in the parasitoid wasp Microplitis mediator (Hymenoptera: Braconidae). Sci. Rep., 8(1): 7649.
Wei Y, 2016. Cloning and Functional Characterization of Homologous Odorant Binding Protein 11 of Two Mirids. MSc Thesis, Chinese Academy of Agricultural Sciences, Beijing. [魏宇, 2016. 两种盲蝽同源气味结合蛋白基因OBP11的克隆和功能研究. 北京: 中国农业科学院硕士学位论文]
Wu J, Zhang HH, Yang YC, Chen S, Yang JQ, Wang B, Chen JH, 2018. Molecular cloning and binding characterization of the Minus-C odorant binding protein in Bactrocera dorsalis. Acta Agric. Univ. Jiangxi., 40(2): 389-399. [吴健, 张贺贺, 杨燕川, 陈湜, 杨建全, 王波, 陈家骅, 2018. 一种桔小实蝇Minus-C气味结合蛋白的分子克隆和结合特征分析. 江西农业大学学报, 40(2): 389-399]
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