绿盲蝽论文：绿盲蝽气味结合蛋白的分子识别功能

绿盲蝽论文：绿盲蝽气味结合蛋白的分子识别功能 绿盲蝽论文:绿盲蝽气味结合蛋白的分子识别功能 【中文摘要】嗅觉在昆虫多种行为方面起着重要的作用，比如，农业害虫的寄主选择行为、择偶行为等等。昆虫的气味结合蛋白(OBPs)是一类存在于嗅觉感受器淋巴液中的可溶性的分泌蛋白，它能运输气味分子到达嗅觉神经面的受体上，是昆虫对外界气味分子的识别和化学信息素传递过程中的第一步生化反应。利用昆虫的嗅觉识别机制，以OBPs为靶标基因，干扰昆虫的寄主定位与交配等行为，将这一研究用于农业生产中的害虫治理已经成为近年来研究的热点，具有一定的理论与实践意义。绿盲蝽是一种重要的、杂食性的农业昆虫，是我国长江流域与黄河流域棉田害虫的优势种,它不仅可以为害棉花、苜蓿、枣树、向日葵、茼蒿等，同时多种果树及蔬菜都是绿盲蝽的寄主。本研究以绿盲蝽Plus-CLlucOBP1为研究目标，对其进行了原核表达，并对其进行了功能验证。本研究的主要研究结果有以下几点:1.选择出了适合Plus-C气味结合蛋白的表达菌株，经过稀有密码子与一系列的预实验，最终选择Rosetta(DE3)为Plus-C OBP1气味结合蛋白的表达菌株，避免了稀有密码子对蛋白表达的影响。2.克隆并表达了第一个绿盲蝽Plus-C气味结合蛋白--AlucOBP1，通过质谱鉴定与Western blot验证了表达蛋白，确定其带标签分子量在37KD左右，运用HisTrap亲和层析柱法纯化蛋白，发现300mM咪唑是洗脱的最适浓度。切标签后发现其分子量为25KD左右，运用 Bradford法测得最终纯化蛋白的浓度为1.462 mg/ml。3.以1-NPN为 荧光探针，通过荧光竞争结合实验，检测了50种不同种类的包括植 物挥发物及性信息素的气味分子与AlucOBP1的结合能力，结果显示， AlucOBP1能与多种气味分子结合，其中与十七烷醇、1-庚醇、丁酸 己酯、丁酸丁酯、金合欢烯、罗勒烯、2-辛酮等几种气味分子的结合 能力比较强。推测AlucOBP1可能在感受信息素以及寄主定位的过程 中发挥着重要作用。 【英文摘要】Olfaction plays a crucial role in many kinds of insect behaviors,including host selection and mating behavior. Insect odorant-bindingprotein (OBP) is one kind of soluble secreted protein existing in thelymph of the olfactory receptor which is associated with olfaction.This protein can transport, as vector, the odour molecule to thereceptors on the surface of nerve cell. OBP is thought to be the firststep in molecular recognition and the transportation of semi-chemical.And it has become a hot spot of research in recent years. In many ofthese studies, OBPs were the target genes, recognition mechanism ofinsect smell sense was applied， interference with insect’s matingand host positioning behaviors that has much theoretical andpractical application are the focus for these researches.Aygus lucorum, one species of major phytophagous agriculturalinsect pests that not only cause huge losses in crops like cotton,alfalfa, sunflower etc., but also to a variety of fruit trees (like Chinesedate) and vegetables (like artemisia). In this study, Aygus lucorumPlus-C OBP was expressed in a prokaryotic expression system, in turn its function was verified using fluorescence probe technique. Mainresults of this study are listed as the following:1. A suitable expression strain was selected for Plus-C OBP study.To avoid the rare anticodon influencing the protein expression, aseries of rare anticodon analysis were conducted; and in the end,Rosetta (DE3) was selected as the expression strain of LlucOBP1.2. A novel Plus-C OBP (AlucOBP1) in the Aygus lucorum wasexpressed and determined. A novel Plus-C OBP (AlucOBP1) in theAygus lucorum, after being cloned, expressed and purified, and then itwas verified to be AlucOBP1 by mass spectrometry appraisal andWestern blot, that determined its molecular weight about 37 KD byHisTag. After purification with HisTrap Affinity Chromatography, itwas found that both 200mM and 300mM were its optimal elutionconcentrations. Its molecular weight would become about 25 KD aftercutting down HisTag. Its protein concentration was 1.462 mg/mlmeasured by Bradford method.3. The binding efficiency of AlucOBP1 with 50 odorants of differentchemical classes was measured using a fluorescence probe 1-NPN ina fluorescence competitive binding study. Results showed that manyodorants can bind with AlucOBP1, among which, heptadecanol,1-Heptanol, ethyl butyrate, n-butyl butyrate, farnesene, ocimene,2-octanone could significantly bind with LlucOBP1. These resultssuggested that AlucOBP1 might play a key role in perception of sexpheromones and host selection for this insect species. 【关键词】绿盲蝽 气味结合蛋白 Plus-C气味结合蛋白 原核表 达 荧光竞争结合 【英文关键词】Aygus lucorum odorant-binding proteins Plus-C OBP prokaryotic expression fluorescence competitive binding 【目录】绿盲蝽气味结合蛋白的分子识别功能 英文缩略表 6-7 摘要 7-9 Abstract 9-10 1 前言 12-20 1.1 绿盲蝽的研究概况 12-14 1.1.1 绿盲蝽的 生物习性 12-13 1.1.2 绿盲蝽的为害特点 13 1.1.3 绿 盲蝽的防治 13-14 1.2 昆虫的嗅觉行为 14-16 1.2.1 昆虫触角感受器的研究 14-15 1.2.2 绿盲蝽的嗅觉感器研究 15-16 1.3 昆虫嗅觉的化学感受研究 16-18 1.3.1 气味结合蛋白的一般特征 16-17 1.3.2 气味结合蛋白的作用及功能 17 1.3.3 昆虫气味结合蛋白研究进展 17-18 1.3.4 半翅目昆虫的气味结合蛋白研究进展 18 1.4 研究依据及目的和意义 18-20 2 绿盲蝽气味结合蛋白的克隆及表达 20-36 2.1 与方法 20 2.1.1 实验材料 20 2.1.2 试剂 20 2.1.3 仪器 20 2.2 克隆与表达绿盲蝽气味结合蛋白OBP1 20-28 2.2.1 绿盲蝽气味结合蛋白OBP1的克隆 20-25 2.2.1.1 样品的准备 20 2.2.1.2 绿盲蝽触角RNA的提取 20-21 2.2.1.3 反转录合成第一条cDNA链 21 2.2.1.4 PCR扩增目的序列 21-22 2.2.1.5 目的片段胶回收 22 2.2.1.6 DNA与PGEM-T载体连接 22 2.2.1.7 转化连接产物 22-23 2.2.1.8 提取质粒 23 2.2.1.9 菌液PCR检测OBP1 23 2.2.1.10 用BamHI、Xho?酶切 OBP1 23-24 2.2.1.11 用BamHI、Xho?酶切载体PET-30(a) 24 2.2.1.12 密码子分析 24 2.2.1.13 目的片段OBP1与表达载体PET-30(a)连接 24-25 2.2.1.14 将连接后的产物转入到感受态细胞DH5α 25 2.2.1.15 提取表达质粒 25 2.2.1.16 将表达质粒转化到Rosetta(DE3)中 25 2.2.2 绿盲蝽气味结合蛋白OBP1的表达 25-28 2.2.2.1 诱导表达OBP1 25-26 2.2.2.2 SDS-PAGE电泳检测OBP1的表达情况 26 2.2.2.3 包涵体的处理 26 2.2.2.4 纯化蛋白 26-27 2.2.2.5 Western blot 27 2.2.2.6 质谱分析 27 2.2.2.7 透析 27-28 2.2.2.8 超滤 28 2.2.2.9 切除 His-tag 28 2.2.2.10 Bradford法测蛋白浓度 28 2.3 结果与分析 28-36 2.3.1 绿盲蝽气味结合蛋白OBP1的序列分析 28-30 2.3.2 绿盲蝽触角RNA 30 2.3.3 绿盲蝽气味结合蛋白OBP1的克隆 30-32 2.3.4 稀有密码子分析结果 32 2.3.5 绿盲蝽气味结合蛋白OBP1的表达 32-33 2.3.6 质谱结果 33-34 2.3.7 Western blot结果 34-35 2.3.8 切标签 35 2.3.9 制作蛋白标准曲线图并测定蛋白的浓度 35-36 3 绿盲蝽气味结合蛋白的结合特性分析 36-41 3.1 材料与方法 36 3.1.1 实验材料 36 3.1.2 所需试剂 36 3.1.3 仪器 36 3.1.4 实验原理 36 3.2 结合实验具体步骤 36-37 3.3 结果与分析 37-41 3.3.1 1-NPN和LlucOBP1的结合常数 37 3.3.2 50种气味分子与LlucOBP1的结合常数 37-41 4 全文结论 41-42 5 讨论 42-43 致谢 43-45 参考文献 45-52 攻读硕士期间论文发表情况 52