【作者】 谷少华；

【导师】 张永军；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2010， 硕士

【摘要】 昆虫个体之间以及昆虫与环境之间的关系主要靠化学通讯来调节。其中嗅觉系统对于昆虫的生存和繁衍至关重要,作为一个高度特异和灵敏的化学感应器,昆虫可以利用触角来检测、识别、转导来自环境中的化学信号并进而引发相关的行为学反应,如定位寄主植物、交配产卵、躲避有毒物质及捕食者。阐明昆虫嗅觉识别的机制可以帮助我们设计新的方法来进行害虫治理。苜蓿盲蝽是我国棉花生产上一类重要的害虫,每年都对棉花产量造成严重的损失。有证据表明盲蝽蟓可以感受特定的寄主挥发物和性信息素来进行定位寄主植物和交配产卵。嗅觉相关蛋白已被证明在昆虫嗅觉识别中是必需的,在防治苜蓿盲蝽中可作为新的靶标基因。本文通过构建苜蓿盲蝽的雌雄触角cDNA文库,通过生物信息学方法筛选到苜蓿盲蝽嗅觉相关基因,并对相关嗅觉基因在苜蓿盲蝽嗅觉识别中的功能进行了研究。主要研究结果如下：1.扫描电镜观察到苜蓿盲蝽触角呈线状,由柄节、梗节和鞭节组成。触角上存在4种感受器,分别是毛形感器、刺形感器、锥形感器和Bohm氏鬃毛。2.利用SMART技术成功构建苜蓿盲蝽雌雄触角的cDNA文库,通过生物信息学方法发掘筛选到若干嗅觉相关基因,其中包括14个气味结合蛋白基因、8个化学感受蛋白基因、2个气味受体基因、8个气味降解酶候选基因。3.利用Real-time PCR方法解析了14个苜蓿盲蝽气味结合蛋白基因(AlinOBP1-AlinOBP 14)在成虫不同组织间的表达谱。根据在各组织间的表达量差异将这14个OBP基因分成4类,ClassⅠ包括9个OBPs基因(Alin-OBP1,Alin-OBP2,Alin-OBP3,Alin-OBP4,Alin-OBP5,Alin-OBP6, Alin-OBP8,Alin-OBP12和Alin-OBP13),主要在触角中表达；ClassⅡ包括1个OBP基因(Alin-OBP11),主要在足和头部表达；ClassⅢ包括1个OBP基因(Alin-OBP14),几乎全部在头部表达；ClassⅣ包括3个OBP基因(Alin-OBP7,Alin-OBP9和Alin-OBP10),主要在雄性触角、雌性触角、头和足中表达,表达量相差不多。4.克隆、表达和纯化了一个新的苜蓿盲蝽气味结合蛋白AlinOBP 1,以1-NPN为荧光探针采用荧光竞争结合实验检测了AlinOBP 1蛋白和114种不同种类的气味分子的结合谱。结果显示AlinOBP 1与其两个性信息素类似物丁酸乙酯和丁酸反-2-己烯酯有着很强的结合能力,另外,6种棉花挥发物辛醛、壬醛、葵醛、异辛醇、β-石竹烯和β-紫罗兰酮与AlinOBP 1也有着很强的结合能力。结合实验表明AlinOBP 1能够同时结合其性信息素类似物和某些棉花挥发物,揭示AlinOBP 1在苜蓿盲蝽的嗅觉识别中可能具有多重功能。以家蚕性信息结合蛋白BmorPBP的三维结构为模板对AlinOBP 1的三维结构模型进行了预测,结果显示,AlinOBP 1蛋白的结合口袋主要由疏水性氨基酸组成,这些疏水性氨基酸可能与配体气味分子的疏水性部分能够发生相互作用。但是与此同时也有5个亲水性氨基酸(Asp19,Thr22,Asn23,Glu79和Lys102)位于结合口袋处,可能为识别配基气味分子的初始结合位点。5.克隆、表达和纯化了一个新的苜蓿盲蝽气味结合结合蛋白AlinOBP3,以1-NPN为荧光探针用荧光竞争结合实验研究了AlinOBP3蛋白与9种棉花挥发物及5种性信息素类似物的结合能力。结果表明,在9种棉花挥发物中,月桂烯、β-罗勒烯、α-水芹烯能够和AlinOBP3相结合,其中α-水芹烯和AlinOBP3的结合能力较强,结合常数为56.68μmol/L。在5种性信息素类似物中,丁酸己酯和AlinOBP3的结合能力最强,结合常数为59.53μmol/L,丁酸丁酯、丁酸反-2-己烯酯、丁酸乙酯和AlinOBP3的结合能力中等,结合常数分别为227.39μmol/L,108.77μmol/L和143.47μmol/L。据此推测,AlinOBP3可能是性信息素结合蛋白(pheromone binding protein, PBP)并在感受性信息素和植物挥发物的过程中发挥双重作用。更多还原

【Abstract】 In the insect kingdom, the relationships between individuals and the environment are often modulated by chemical communication. The olfaction system is essential for insects’survival and reproduction. As a highly specific and sensitive chemical sensor, it can detect, recognize and transfer chemical signals from the environment and trigger specific behavioral reactions, such as location host plant, mating with partners, avoidance of toxins and predators. Elucidating the mechanism of insect olfactory perception can further facilitate the design and implementation of novel intervention strategies against these pests. The lucerne plant bug, Adelphocoris lineolatus (Goeze) (Hemiptera:Miridae), is one of the most destructive pests of Bt cotton in China and cause significant yield losses every year. Evidences showed that mirid can sense particular plant volatiles and sex pheromones to locate suitable host plant and mate partners. Olfactory related proteins have been demonstrated to be required for olfaction perception in insects. In A. lineolatus, they might represent new interesting targets for the control of their population. In this paper, two high-quality cDNA libraries were constructed from the antennae of A. lineolatus. Several olfactory related genes were discovered by particular bioinformatics analysis. The functional roles of related genes in the olfaction perception of A. lineolatus are investigated. The primary results are as follows:1.The antennae and antennal sensillae of A. lineolatus were observed by scanning electron microscopy. The results showed that the antennae of of A. lineolatus was in linear shape and made up of scape, pedicel and flagellum. There are four distinct antennal sensillae, including sensilla trichodea, sensilla chaeticum, sensilla basiconica and Bohm bristles.2.By using SMART method, two high-quality cDNA libraries were constructed from the male and female antennae of A. lineolatus. Several olfactory related genes are discovered by particular bioinformatics analysis, including 14 odorant binding prorein (OBP) genes,8 chemosensory protein (CSP) genes,2 odorant receptor (Or) genes and 8 putative odorant degrading enzyme (ODE) genes.3.The expression profiles of the 14 OBPs in different adult tissues were measured by real-time qPCR. These 14 OBPs were divided into four classes based on their expression intensity in antennae,heads, legs or other tissues, respectively. ClassⅠincluded nine OBP genes (Alin-OBP1,Alin-OBP2, Alin-OBP3,Alin-OBP4,Alin-OBP5,Alin-OBP6,Alin-OBP8,Alin-OBP12 and Alin-OBP13),which are dominantly expressed in the antennae tissues. ClassⅡincluded one OBP gene (Alin-OBP11), which was mainly expressed in the legs and heads. ClassⅢincluded one OBP gene (Alin-OBP14), which was predominantly expressed in the heads. ClassⅣincluded three OBP genes (Alin-OBP7, Alin-OBP9 and Alin-OBP10), which seemed ubiquitously expressed in male antennae, female antennae, heads and legs.4. A novel OBP (AlinOBP1)in the A. lineolatus was cloned, expressed and purification in laboratory. The binding properties of AlinOBP1 with 114 odorants of different chemical classes were measured by using a fluorescence probe 1-NPN with the fluorescence competitive binding method. The results revealed that AlinOBPl exhibits high binding abilities with two major putative pheromones, Ethyl butyrate and Trans-2-Hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, Octanal, Nonanal, Decanal,2-Ethyl-l-hexanol, (3-caryophyllene andβ-ionone can also significantly bind with AlinOBPl.The binding experiments showed that the AlinOBPl can both bind with its putative pheromones and chemicals released from cotton, indicating there may exist a more multiple-function of AlinOBPl in the perireceptor events of olfaction. The predicted 3 D structure of AlinOBP1 was established with the BmorPBPl as the structural template. The AlinOBPl 3D modle demonstrated that the binding pocket is mainly organized by hydrophobic amino acids, which may be responsible for the hydrophobic interactions with the hydrocarbon part of the ligands. However, some hydrophilic residues (Asp19, Thr22, Asn23,Glu79 and Lys102) are also presented in the binding pocket, these hydrophilic residues are likely the initial recognition sites of ligands.5.A novel OBP (AlinOBP3) in the A. lineolatus was cloned, expressed and purification in laboratory. The binding properties of AlinOBP3 with nine cotton volatiles and five sex pheromone analogs were measured by fluorescence competitive binding assays with the fluorescence probe 1-NPN. The results revealed that of nine cotton volatiles, Myrcene,β-Ocimene andα-Phellandrene can bind with AlinOBP3.α-Phellandrene especially bound to AlinOBP3 with a high binding affinity, with a dissociation constant of 56.68μmol/L. Of the five sex pheromone analogs, Hexyl butyrate had the strongest binding affinity with AlinOBP3, with a dissociation constant as 59.53μmol/L. Butyl butyrate, Trans-2-Hexenyl butyrate and Ethyl butyrate had medium binding affinities with AlinOBP3,with dissociation constants of 227.39μmol/L,108.77μmol/L,143.47μmol/L, respectively. The results suggest that AlinOBP3 might be a pheromone binding protein (PBP) with a dual-function for the perception of sex pheromones and plant volatiles.更多还原