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1、光肩星天牛气味结合蛋白 AglaOBP12 的基因克隆、表达及配体结合特征 李广伟 陈秀琳 尚天翠 伊犁师范学院生物与地理科学学院 摘 要： 【目的】克隆和鉴定光肩星天牛 Anoplophora glabripennis 气味结合蛋白 (odorant binding proteins, OBPs) 基因, 明确其表达特点及与寄主植物挥发物的结合特性, 有助于阐明光肩星天牛嗅觉识别的分子机制。【方法】根据光肩星天牛雌成虫触角转录组数据, 利用 RT-PCR 克隆 OBP12 基因, 并进行生物信息学分析。通过实时定量 PCR (qRT-PCR) 测定 OBP12 在光肩星天牛成虫触角、头 (移
2、除触角) 、胸、腹、足、翅中的转录水平。利用原核表达系统和 Ni 离子亲和层析技术表达和纯化 OBP12 重组蛋白, 荧光竞争结合实验测定重组蛋白与39 种气味配体的结合能力。【结果】获得光肩星天牛气味结合蛋白基因AglaOBP12 (GenBank 登录号:KX890109) 的完整编码序列, 其开放阅读框长 414 bp, 编码 137 个氨基酸, N 末端具有 18 个氨基酸组成的信号肽序列, 蛋白序列具有 6 个保守的半胱氨酸残基, Agla OPB12 属于 Classical OBPs 亚家族基因。qRT-PCR 测定结果表明, AglaOBP12 主要在成虫触角中表达, 在其他组
3、织微量表达。在待测的 39 种寄主植物挥发物中, 重组蛋白 AglaOBP12 仅与 19 种化合物具有结合活性, 表明 AglaOBP12 对寄主植物挥发物具有明显的选择结合特性。重组蛋白 AglaOBP12 与十二烷醇、十四烷醇、法尼醇、十二醛、乙酸-顺-3-己烯酯和 -石竹烯的结合能力较强, 结合常数分别为 1.96, 0.96, 1.03, 0.82, 0.77 和 0.74mol/L。【结论】明确了 AglaOBP12 的核苷酸和氨基酸序列组成, 重组 AglaOBP12 蛋白与主链有 12 个碳原子的醇类、醛类和萜烯类挥发物有特异性的结合活性。根据 AglaOBP12 基因的表达特
4、点和重组蛋白的结合特性, 推测AglaOBP12 在光肩星天牛成虫定位补充营养寄主植物中发挥重要作用。关键词： 光肩星天牛; 气味结合蛋白; 嗅觉; 基因表达; 荧光竞争结合实验; 作者简介：李广伟, 男, 1982 年生, 甘肃会宁人, 博士, 助理研究员, 研究方向为农业害虫综合治理;E-mail:收稿日期：2017-04-08基金：新疆维吾尔自治区自然科学基金 (2015211C299) cDNA cloning, expression and ligand binding properties of the odorant binding protein AglaOBP12 in th
5、e Asian longhorned beetle, Anoplophora glabripennis (Coleoptera:Cerambycidae) LI Guang-Wei CHEN Xiu-Lin SHANG Tian-Cui College of Biology and Geography, Yili Normal University; Abstract： 【Aim】Cloning and identification of the odorant binding protein (OBP) genes and clarifying their expression featur
6、es and ligand-binding characteristics with host-plant volatiles are helpful to address the molecular mechanisms of olfaction in the Asian longhorned beetle, Anoplophora glabripennis.【Methods】Based on the antenna transcriptome data of A.glabripennis female adult, the complete coding sequence of OBP12
7、 was cloned using RT-PCR and bioinformatically analyzed.The expression levels of OBP12 in the antenna, head (without antennae) , thorax, abdomen, leg and wing were assayed by real-time quantitative PCR (qRT-PCR) .The recombinant OBP12 protein was prokaryotically expressed and then purified by Ni ion
8、 affinity chromatography.The binding affinities of the recombinant OBP12 with 39 ligands were assessed by using fluorescent competitive binding assay.【Results 】 AglaOBP12 of A.glabripennis was successfully cloned and sequenced (GenBank accession no.:KX890109) .Its ORF is414 bp in length, encoding 13
9、7 amino acids with the signal peptide of 18 amino acids at the N-terminal.The matured protein possessed six conserved cysteines and could be classified into the Classical OBP subfamily.qRT-PCR results showed that AglaOBP12 primarily expressed in the antenna and slightly expressed in other tissues.Am
10、ong the 39 chemicals tested, the recombinant AglaOBP12 had only binding activities to 19 compounds, suggesting that AglaOBP12 has obvious selective binding characteristics to host plant volatiles.The recombinant AglaOBP12 showed higher binding affinities to dodecanol, tetradecanol, farnesol, dodecan
11、al, cis-3-hexenyl acetate and -caryophyllene, with the Kivalues of1.96, 0.96, 1.03, 0.82, 0.77 and 0.74 mol/L, respectively.【Conclusion 】The nucleotide and deduced amino acid sequences of AglaOBP12 were characterized in this study.AglaOBP12 shows particularly strong binding affinities to alcohols, a
12、ldehydes and terpenes with 12 carbon atoms in the main-chain.Based on the results of qRT-PCR and fluorescent competitive binding assay, we speculated that AglaOBP12 in the Asian longhorned beetle plays an important role in locating trophic host plants.Keyword： Anoplophora glabripennis; odorant bindi
13、ng protein; olfactory; gene expression; fluorescence competitive binding assay; Received： 2017-04-08光肩星天牛 Anoplophora glabripennis (鞘翅目:天牛科) 是我国林木上一种重要的毁灭性蛀干害虫, 主要危害杨 Populus spp., 柳 Salix spp., 槭 Acer spp., 榆 Ulmus spp.和胡桃 Juglans spp.等属的多种树种 (李国宏等, 2010) 。该虫虽然寄主广泛, 但成虫对寄主植物有明显的取食和产卵偏好, 如嗜食五角枫 Acer
14、 mono 嫩梢, 更偏好在旱柳 Salix matsudana 上产卵, 灵敏的嗅觉系统在成虫定位补充营养寄主、产卵寄主植物中起着重要作用 (Yan et al., 2008) 。前人已在寄主植物挥发物鉴定、对成虫有电生理活性的挥发物组分的筛选等方面做了大量研究 (金幼菊等, 2004;Nehme et al., 2010;范丽清等, 2012, 2013;Meng et al., 2014;杜和芬等, 2016;王紫薇等, 2016;朱宁等, 2017) 。深入探究光肩星天牛嗅觉相关蛋白基因的生理功能, 阐明该虫嗅觉识别的分子机制, 将有助于推动光肩星天牛行为调控技术的开发和应用。气味结合
15、蛋白 (odorant binding proteins, OBPs) 能够选择性地结合和运输疏水性的气味分子穿过触角感器淋巴液至位于嗅觉感受神经元 (olfactory receptor neurons, ORNs) 树突膜的气味受体 (odorant receptors, ORs) 上, 激活嗅觉信号传导途径, 是昆虫感知外界环境的基础 (Justice et al., 2003;Pelosi et al., 2006) 。依据序列的同源性可将昆虫 OBPs 分为性信息素结合蛋白 (pheromone binding proteins, PBPs) 、普通气味结合蛋白 (general o
16、dorant binding proteins, GOBPs) 和触角结合蛋白 (antennal binding proteins, ABPx) 3 类 (Hekmat-Scafe et al., 2002) ;根据氨基酸序列中保守半胱氨酸数量可将 OBPs 分为 Classical OBPs, Plus-C OBPs, Dimer OBPs, Minus-C OBPs 和 Atypical OBPs 5 类 (Manoharan et al., 2013) 。随着基因组和转录组测序技术的快速发展, 已在鳞翅目、直翅目、双翅目、鞘翅目、膜翅目、半翅目等 7 个目的多种昆虫触角中鉴定到大量的 OBPs 基因, 并利用荧光竞争结合实验测定了 200 多种昆虫 OBPs 的结合功能 (Zhou et al., 2004;Gong et al., 2007;Vandermoten et al., 2011;Deng et al., 2012;Li et al., 2015, 2017;Ahmed et al.,
