茶尺蠖触角高丰度气味结合蛋白EoblOBP9、EoblOBP11的配基结合功能和模式差异研究  

Study on the Differences in Ligand-binding Function and Mode of the Antennal High-abundance Odorant-binding Proteins EoblOBP9 and EoblOBP11 of the Tea Geometrid,Ectropis obliqua Prout

作　　者：严玉婷 吴帆 张亚丽[2] 傅晓斌 崔宏春[3] 韩宝瑜 李红亮 YAN Yuting;WU Fan;ZHANG Yali;FU Xiaobin;CUI Hongchun;HAN Baoyu;LI Hongliang(College of Life Science,China Jiliang University/Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine,Hangzhou 310018,China;Hangzhou Tea Research Institute,China Coop.,Hangzhou 310016,China;Hangzhou Academy of Agricultural Sciences,Hangzhou 310024,China)

机构地区：[1]中国计量大学生命科学学院/浙江省生物计量及检验检疫技术重点实验室,浙江杭州310018 [2]中华全国供销合作总社杭州茶叶研究院,浙江杭州310016 [3]杭州市农业科学研究院,浙江杭州310024

出　　处：《茶叶科学》2021年第5期643-653,共11页Journal of Tea Science

基　　金：国家自然科学基金(31772544、32000331);浙江省自然科学基金(LQ21C030007);科技部基础资源调查专项(2018FY100405);浙江省重点计划项目(2020C02026)。

摘　　要：茶尺蠖(Ectropis obliqua Prout)是茶园重要的鳞翅目害虫,雄雌成虫能通过嗅觉系统感受和分辨外界环境的化学信息以助于其交尾和产卵地选择等行为。在此过程中,成虫触角高丰度表达的气味结合蛋白(Odorant-binding proteins,OBPs)可能发挥了重要作用。以茶尺蠖成虫触角高丰度表达的两个OBPs即EoblOBP9和EoblOBP11为对象,首先发现二者cDNA编码蛋白均含有OBPs家族的典型特征,即含有6个保守的半胱氨酸,等电点预测分别为酸性和碱性,表明二者在一级结构性质上存在差异;利用原核表达技术获得重组蛋白,并免疫小鼠制备相应的多克隆抗体,再利Western blot技术验证了二者在雄性成虫触角特异表达;通过荧光竞争结合实验分别测试二者与20种候选配基的结合力,结果表明二者均与两个性信息素成分(顺3,9-环氧-6,7-十八碳二烯与顺-3,6,9-十八碳三烯)及一种植物挥发物成分(反-2-己烯醛)呈现较强的亲和力。与此同时,二者也显示出不同的配基结合谱,如EoblOBP9与1-戊烯-3-醇、苯甲醇、苯乙酮和苯甲醛亲和力较强,结合谱较宽;而EoblOBP11与α-萜品醇亲和力较强,结合更显专一,分子对接结果也支持该结论。此外,预测二者的活性位点均位于C端,也显示出二者配基结合的共性。研究结果表明,尽管这两个茶尺蠖OBPs均在成虫触角高丰度特异表达,但无论从结构性质上还是生化结合谱上,既有保守性又存在差异性,显示出茶尺蠖触角高丰度表达OBPs功能模式的丰富度和多样性,这为解释茶尺蠖嗅觉系统在面对复杂多变的外界环境的适应机制提供了理论依据。As an important lepidopteran pest in tea gardens,the male and female adults of Ectropis obliqua Prout can perceive and distinguish the chemical information of the external environment through the olfactory system,which helps its behaviors such as mating and selection of oviposition locations.In this process,Odorant-binding proteins(OBPs)highly expressed in the antennae of adults may play an important role.In view of this,this study focused on the two OBPs high expressed in the antennae of the adult tea geometrid,namely EoblOBP9 and EoblOBP 11.First,it was found that both EoblOBP9 and EoblOBP11 contain six conserved cysteines,the typical characteristics of the OBPs family,while the isoelectric points are predicted to be acidic and basic,respectively,indicating their difference in the primary structural properties.Both recombinant proteins were obtained using prokaryotic expression technology,and the corresponding polyclonal antibodies were prepared by immunizing mice.It was verified that they were indeed expressed specifically in the antennae of male adults by Western blot.Their binding abilities with 20 candidate ligands were tested by fluorescence competitive binding experiments,and the results show that both of them were compatible with the two sex pheromone components(Z3,Z9-6,7-epo-18:Hy and Z3,Z6,Z9-18:Hy)and a plant volatile component(trans-2-hexenal)has a strong affinity.Meanwhile,two OBPs also showed different ligand binding spectra.For example,EoblOBP9 had a strong affinity with 1-penten-3-ol,benzyl alcohol,acetophenone and benzaldehyde,indicating that the binding profiles were wider;while EoblOBP11 had a strong affinity withα-terpineol with a more specific binding mode.Molecular docking analysis also supports this conclusion.In addition,it was predicted that the active sites of both proteins are located at the C-terminus,which also shows the commonality of ligand binding.In conclusion,this study shows that although both OBPs were specifically expressed in the adult tea geometrid with high abundance,they

关 键 词：茶尺蠖 触角高丰度 气味结合蛋白 原核表达 

分 类 号：S571[农业科学—作物学] S435.711