【作者】 钟涛；

【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， 2012， 博士

【摘要】 昆虫借助灵敏的嗅觉可对环境中各类信息线索进行有效识别，从而准确获悉食物和配偶的方位并躲避捕食者的伤害在嗅觉感知过程中，气味结合蛋白是最先发挥功能的一类蛋白依据豌豆蚜已知基因序列对麦长管蚜气味结合蛋白基因同源克隆，获得7种气味结合蛋白基因（SaveOBP2，JN165749；SaveOBP3，JN165750；SaveOBP4，JN165751；SaveOBP5，JN165752；SaveOBP7，GQ847859；SaveOBP8，GQ888708；SaveOBP9，GQ847860）完整编码序列，都已在GenBank登录对序列进行分析，发现只有SaveOBP5序列中不含气味结合蛋白家族保守结构域从构建的系统发育树中可以看出麦长管蚜气味结合蛋白基因序列分别与麦无网长管蚜和豌豆蚜的直系同源基因有比较一致的进化趋势，可能暗示基因序列与寄主生境的选择压力有某种特殊的联系对其中6种气味结合蛋白构建了重组表达质粒，并在大肠杆菌中诱导表达借助金属镍亲和层析技术对包含His标签的融合蛋白成功纯化，Western blot结果证实融合蛋白得到完整表达经重组牛肠激酶（rEK）切除His标签，获得接近天然状态的气味结合蛋白利用荧光竞争结合实验研究SaveOBPs与小麦挥发物组份和告警信息素的结合特性，以1-NPN为探针，证实6种气味结合蛋白对C6–C8的小分子气味表现出广谱的结合特性其中，SaveOBP2能够在微摩尔浓度水平上（竞争解离常数在10μM左右）对北京837小麦的挥发物组份较强结合，对其中绿叶气味（如反-2-己烯醛反-2-己烯-1-醇顺-3-己烯-1-醇）以及苯甲醛都表现出较高的亲和性同时，研究发现SaveOBP7能够强烈地结合反-β-法呢烯气味（竞争解离常数为1.3μM），表明SaveOBP7很可能是识别蚜虫告警信息素的一类重要功能蛋白对不同蛋白的结合能力进行比较后发现SaveOBP2SaveOBP3SaveOBP4SaveOBP5和SaveOBP8对C6–C8绿叶气味的结合能力也是存在明显差别的，SaveOBP2在结合小分子醇醛和酯类气味中表现最佳，该蛋白可能在识别寄主挥发物过程中发挥重要作用利用实时荧光定量技术对其中3个基因（SaveOBP3SaveOBP7和SaveOBP8）进行组织中表达的定位研究，发现其在触角组织中都有表达，推测它们可能参与麦长管蚜的嗅觉识别过程SaveOBP7在足和翅中都未检测到表达，而主要表达于触角组织中SaveOBP3和SaveOBP8均能周身表达，而且在足中表达量较高，可能暗示着它们发挥特殊的嗅觉功能在分析麦长管蚜气味结合蛋白基因在不同发育期的表达，这3个基因在麦长管蚜若蚜的前3个龄期的表达量总体上差别不大，而在若蚜4龄期表达量升高并出现一个表达量高峰，成蚜期又下降并恢复到之前的水平；SaveOBP3和SaveOBP8在有翅成蚜中的表达量均高于无翅成蚜，SaveOBP7在有翅成蚜和无翅成蚜的表达量相当，这种表达差异可能反映出这3种蛋白在麦长管蚜有翅蚜的寄主搜寻过程中存在重要作用更多还原

【Abstract】 Acute olfaction allows insects to orientate food and mates, and hide from predators quite accuratelyby discriminating semiochemical cues in natural environment. Odorant binding proteins (OBPs) are thefirst functional proteins which involved in the olfactory perception. According to the known pea aphidsequence, seven OBPs genes with complete coding sequence were obtained from Sitobion avenae byhomologous gene cloning, and they were deposited in GenBank (SaveOBP2, JN165749; SaveOBP3,JN165750; SaveOBP4, JN165751; SaveOBP5, JN165752; SaveOBP7, GQ847859; SaveOBP8,GQ888708; SaveOBP9, GQ847860). All the SaveOBPs were predicted to contain a highly conserveddomain of the PBP and GOBP superfamily separately except SaveOBP5. Two consistent evolutionarytrends of orthologous genes were observed from the phylogenetic trees which constructed by aphidOBPs available, one was S. avenae and Metopolophium dirhodum, the other was S. avenae andAcyrthosiphon pisum, which indicated that some particular relationships might exist between OBPgenes and the selective pressure from the host surroundings.Six expression plasmids contained separate SaveOBPs were reconstructed and they were expressedin Escherichia coli host cells by inducing. Fusion proteins with histidine tags were easily purified by theimmobilized metal nickel chelate affinity chromatography, and western blot confirmed that fusionproteins were totally expressed. The histidine tags were digested by the recombinant enterokinase (rEK),and the rest part of fusion proteins were quite close to the natural state of SaveOBPs. Fluorescencecompetition binding assays were applied to explore the binding characters of SaveOBPs for wheatvolatiles and aphid alarm pheromone. The six SaveOBPs showed a broad binding character for the smallodorants with C6–C8when they competed1-NPN. SaveOBP2was able to bind several components ofhost plant volatiles which were released from the ‘Beijing837’ wheat crops at micromolar level (thedissociation constants of inhibitors were at about10μM), among which benzaldehyde and someodorants belonging to green leaf volatiles like trans-2-hexenal, trans-2-hexen-1-ol and cis-3-hexen-1-olexhibited higher affinities. Meanwhile, SaveOBP7could bind trans-β-farnesene very strongly (thedissociation constant of inhibitor was at1.3μM), which indicated that SaveOBP7was an importantfunctional protein in recognizing aphid alarm pheromone. After comparing the binding abilities amongSaveOBP2, SaveOBP3, SaveOBP4, SaveOBP5and SaveOBP8, I found that they had obviousdiscrepancy in discriminating green leaf volatiles of C6–C8. SaveOBP2was pretty excellent in bindingalcohols, aldehydes and esters with small molecular weight, which suggested that SaveOBP2might playan important role in sensing host plant volatiles.The expression and localization in tissues of three genes (SaveOBP3, SaveOBP7and SaveOBP8)were investigated by real-time fluorescent quantitative PCR. These genes were thought to be involved inolfactory recognition, because they all expressed in the antennae. SaveOBP7was detected in neither legnor wing, but was predominantly expressed in the antennae. Both SaveOBP3and SaveOBP8wereexpressed in the whole body, and were much higher in the leg tissues, which implied special olfactoryfunctions they might have. Additionally, in the study of expression among different developmental stages, SaveOBP3, SaveOBP7and SaveOBP8were expressed in the first three nymphal periods withrather small differences, but the expression increased and reached a peak in the4th nymphal period, andthen it dropped back to the former amount in the adult period; SaveOBP3and SaveOBP8wereexpressed higher in the alate adults than the apterous ones, but SaveOBP7was equally expressed, theirdifferential expressions suggested that important roles might exist in the host-seeking of the alate adults.更多还原