【作者】 王冰；

【导师】 曹雅忠；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 地下害虫是我国农业生产上一类重要的有害物种，具有生活隐蔽，适应性强等特点，是国内外公认的难以防治的害虫。华北大黑鳃金龟Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae)属于地下害虫重要类群之一，对农作物、果树以及林木等可以造成重大经济损失。在控制地下害虫的同时也造成了生态环境破坏等多种潜在威胁。昆虫错综复杂的嗅觉系统能够检测和识别环境中不同的挥发性小分子气味物质，这种特征在昆虫寻找寄主、交配、产卵以及逃避行为中起到了至关重要的作用。因此，对昆虫嗅觉感受机理的研究将有助于揭开昆虫感受和识别环境中的气味物质并引起相关行为反应的秘密，有助于开发新型生物制剂来干扰害虫之间的通讯，最终达到控害保益的目的，为生物防治开辟新的途径。本研究所获得的主要结果如下：（1）根据GenBank登录的华北大黑鳃金龟气味结合蛋白OBP3和OBP4的序列，设计特异性引物，获得了OBP3和OBP4基因的开放阅读框序列，并将其成功克隆到原核表达载体pET-30a（+）上，为后续气味结合蛋白功能的研究奠定了分子基础。（2）以冈比亚按蚊AgamOBP20晶体结构为模板对HoblOBP3和HoblOBP4蛋白进行同源建模。从模型上可以看出组成蛋白的α螺旋形成一个结合口袋，蛋白的C端伸入到结合口袋内，与冈比亚按蚊AgamOBP1的结构相似。对HoblOBP3和HoblOBP4以及已知的4种嗅觉相关蛋白HoblCSP1、HoblCSP2、HoblOBP1和HoblOBP2成功地进行了原核表达以及蛋白纯化。竞争结合实验分析了华北大黑鳃金龟OBP3和OBP4对植物绿叶气味以及已经鉴定的金龟子性信息素成分等42种气味分子的结合特征，研究发现HoblOBP4展现出了非常广泛的结合亲和性，尤其与6个碳的植物绿叶气味结合亲和性较高，并且能够结合L-异亮氨酸甲酯等金龟子性信息素成分。HoblOBP3只能够专一性的结合α-紫罗兰酮和-紫罗兰酮，从而显示出严谨的分子筛作用。除此之外，我们发现配体化合物官能团的种类、数量和位置，碳链的长短，碳链的开环与闭环结构，以及旋光异构等差异对蛋白结合亲和性的影响非常大。利用荧光结合实验对目前已知的6种华北大黑鳃金龟嗅觉相关蛋白之间的相互关系以及是否存在二聚体进行了探讨，我们推测HoblOBP2能够分别和HoblOBP1、HoblOBP4形成异源二聚体。（3）本研究根据免疫组织化学法利用胶体金双标记技术在亚细胞水平上检测了二元蛋白混合物在华北大黑鳃金龟触角感器中的共定位情况。实验结果显示，HoblOBP1/HoblOBP2和HoblOBP2/HoblOBP4均在华北大黑鳃金龟板型感器和锥形感器中共表达，这一实验结果是对OBPs之间能够形成二聚体以感受外界气味分子以及信息素这一理论的验证和支持，为疏水性通道理论提供了强有力的证据。（4）本研究利用DUALsystem Biotech公司文库构建试剂盒，构建了华北大黑鳃金龟触角酵母双杂交系统均一化的cDNA文库，并将其克隆到酵母双杂交系统的表达载体pPR3-N上，为下一步利用酵母双杂交技术研究蛋白互作搭建了技术平台。构建好的均一化cDNA文库经质量检测发现，原始文库的库容量为1.1×106，平均插入片段大小约为1.2kb，达到了高质量文库的标准，保证了文库的覆盖性。（5）本实验利用DUALsystem Biotech公司DUALhunter starter kits酵母双杂交筛选试剂盒，构建了华北大黑鳃金龟气味结合蛋白OBP1和OBP2诱饵载体，筛选了华北大黑鳃金龟触角酵母双杂交系统均一化的cDNA文库，经鉴定以及在GenBank中进行Blast比对分析，以OBP1为诱饵鉴定出6个互作物，以OBP2为诱饵鉴定出8个互作物。通过-半乳糖苷酶活性检测，以OBP1为诱饵，最终确定的阳性互作物为receptor for activated protein kinase C-like，以OBP2为诱饵最终确定的阳性互作物为proclotting enzyme，我们推测这两种蛋白可能是华北大黑鳃金龟在嗅觉识别过程中的相关蛋白。更多还原

【Abstract】 Underground pests are a harmful class group in agricultural industry in China, and their coverthabitats and strong adaptability result in difficulties in the prevention and control of pests. The scarabbeetle, Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae), belongs to such a class and hascaused serious economic damage to crops, fruit trees and forest trees in China. The chemical methods ofcontroling underground pests could lead to many potential threats. Therefor, an environmentallyfriendly method for controlling of H. oblita is needed. The sophisticated insect olfactory system candetect and discriminate between different kinds of odorants, which are volatile small organic moleculesin the environment. This characteristic property plays a crucial role in insect behaviors, such as hostseeking, mating, ovipositing, as well as escape behaviors. Therefore, the secret that arousing a series ofinsect behavior reactions by perceiving and distinguishing amounts and kinds of odorants will beunveiled by investigating olfactory receptive mechanism, which contributes to exploit the newbiological agent to mess up communications among pests. This kind of new conception will providenew way for biological control, and gain our ends to protect crops from damage by pests. Ourconclusions are listed as follow.(1) According to registered OBP3and OBP4sequences from H. oblita in GenBank, we designedspecific primers and obtained ORF sequences of OBP3and OBP4from H. oblita. These two sequenceswere cloned into expression vector pET-30a respectively, which provided molecular basis for proteinfunctional analysis.(2) The crystal structure of Anopholes gambiae OBP20(AgamOBP20) was chosen as a templatefor both HoblOBP3and HoblOBP4to predict three-dimensional structures. The three-dimensionalmodel of HoblOBP3and HoblOBP4presented a large binding pocket, and the C-termini extended intothe binding pocket, which was similar to AgamOBP1. Six olfactory associated proteins of H. oblita,HoblOBP3, HoblOBP4, HoblCSP1, HoblCSP2, HoblOBP1and HoblOBP2, were expressed in E.coliand purified using Ni ion affinity chromatography. We selected42potential organic compounds on thebasis of competitive binding assays with proteins of HoblOBP3and HoblOBP4, which includedcompounds from volatile green plants and putative sex pheromones of some beetle species. The resultsshowed that HoblOBP4exhibited a broader spectrum of activity and well bound aliphatic compoundsconsisting of6carbon atoms, and putative sex pheromone compounds of some beetle species, such asL-isoleucine methyl ester. However, HoblOBP3only appeared high affinity to α-ionone and-ionone.In addition, the differences of position, variety and amount of functional groups, length difference ofcarbon chain, ring-shaped or ring-open structure of carbon chain, and optical isomerism in compoundswere the main impact factors that affected binding affinity. The interrelations and formation of dimerbetween olfactory associated proteins were discussed using competitive binding assays. We speculatedthat HoblOBP2could form dimer with HoblOBP1and HoblOBP4respectively.(3) We used the technique of colloidal gold post-embedding immunocytochemistry to detect colocalization of binary OBPs at the subcellsular level. The presented results demonstrated that thebinary mixtures of HoblOBP1/HoblOBP2and HoblOBP2/HoblOBP4were both coexpressed in sensillaplacodea and sensilla basiconica. This conclusion supported one assumption that OBPs could formdimer in order to perceive plant odors and sex pheromones, which provided evidence for thehydrophobic tunnel hypothesis.(4) The construction kits of DUALsystem Biotech were used to construct the normalized cDNAlibrary from H. oblita antenna in order to screen by yeast two-hybrid assay. Then, the completed librarywas cloned into vector pPR3-N expressing in yeast, which put up the platform for screening by yeasttwo-hybrid assay. Subsequently, the quality of the normalized cDNA library was tested. The resultsshowed that the number of independent transformants in the original library was1.1×106, and theaverage insert size was greater than1.2kb, which was up to the standard of the high-quality andhigh-spreadability cDNA library.(5) DUALhunter starter kits of DUALsystem Biotech were used to construct the bait vector ofHoblOBP1and HoblOBP2respectively. Then, the H. oblita antenna normalized cDNA library wasscreened. The screened results showed that six interactors screened with HoblOBP1bait vector andeight interactors screened by HoblOBP2bait vector were identified using PCR technique and analyzedusing Blast in GenBank. In the end, by assaying for detection of-galactosidase activity, the strongestpositive interactor, receptor for activated protein kinase C-like, was identified by HoblOBP1bait, andthe proclotting enzyme was viewed as the strongest positive interactor identified by HoblOBP2bait.Therefore, we speculated that the two positive interactors might work in the process of olfactoryrecognition.更多还原