麦蚜嗅觉关联基因鉴定及其对EBF及类似物特异识别机制研究

(E)-β-farnesene is the key (or only) component of aphid alarm pheromone of most species, can repel aphids and attract natural enemies, with a good potential use for control aphids infested effectively. Many documents show that the odorant binding proteins (OBPs) in insect antennae selectively bind with EBP and other semichemicals. Therefore, to discriminate the particular OBPs for perception odorants or pheromones, can improve the repellent exploitation and development. In the project, wheat aphids (eg. Sitobion avenae) chose to be test targets, the aphid olfactory gene repertoire are to identify from antennal transcriptomes, through unigenes generation and prediction, genes identification and functional annotation by the bioinformatic methods and quantitative real-time PCR technique. The antennal specific expressed OBPs are cloned and sequenced, and the recombinant proteins are prepared through expression in E. coli cell and complex purification procedures , then the specific antisera prepared by the recombinant proteins . The genes and proteins of OBPs location in antennal sensilla are detected by both in situ hybridization (in mRNA level) and immnocytochemistry method (in protein level). The binding affinity between OBPs with ligands (EBP and its structure analogues, and other semichemicals ), are checked by fluorescence binding assays, and a real-time specific binding with surface plasmon resonance(SPR) technique, and the aphid behavioral responses to ligands investigated by Y-tube or Four-Armed olfactometer, to the end, the candidate OBPs of good binding affinity with EBF and plant odorants and of bioassay reaction , are obtained. The function of the candidate OBPs gene are checked by RNAi technique. Through feeding aphid on the artificial diet with synthetized dsRNA of target OBPs, the effect of RNAi is checked in the olfactory behavior by Y-tube test and target gene expression by qRT-PCR. 3D structural modeling method including homology modeling, molecular docking, molecular dynamics were applied to set up the binding model between EBF analogues and aphid OBP targets, to clarify the interactional mechanism, find OBPs binding and functional domains, and finally obtain the selective OBP components for EBF and its analogues, and repelling odorants. The expected results of project have a significant value on environmental friendly and green ecological pest management techniques R&D, based on novel aphid repellents, and also enrich the theory of aphid chemical ecology.

(E)-β-法呢烯（EBF）作为蚜虫报警激素主要或唯一组分，可以驱避蚜虫、吸引其天敌。据报道昆虫触角中气味结合蛋白（OBPs）能选择性结合EBF和其他气味分子，因此发掘与EBF、其他气味分子特异识别的OBP种类，能加速蚜虫驱避剂的研发。本项目以麦长管蚜为对象，通过触角转录组测序与生物信息学预测及qRT-PCR分析，获得嗅觉相关几类基因；针对OBPs基因进行分离克隆、原核表达及触角感器中定位，经荧光结合及SPR技术实时结合监测，嗅觉行为测定，发现与配体结合能力强的OBPs；采用RNAi技术，采用饲喂蚜虫，检测蚜虫选择行为变化及靶标基因抑制效果；通过同源模建与分子对接，揭示OBP与配体互作机制，进而找到与EBF（类似物）及其他气味分子的特异结合OBPs蛋白，揭示其对麦蚜驱避调控的分子机理。研究结果对蚜虫绿色防控技术研发，及蚜虫化学生态学理论的丰富具有重要意义。

摘要：.蚜虫报警信息素(E)-β-法呢烯（EBF）可以驱避蚜虫，但麦蚜对EBF嗅觉机制尚不清楚。在本基金项目资助下,以麦蚜优势种（麦长管蚜和禾谷缢管蚜）为试虫，取得以下主要研究进展：（1）麦长管蚜触角感器超微结构观察结果表明，成蚜触角感器包括原生感觉圈、次生感觉圈、毛形感器、钟形感器和单个腔锥形感器等5种感器。原生感觉圈由板形感器和腔锥形感器组成，具有感知植物挥发物及蚜虫报警信息素的功能。若蚜与成蚜感器基本相同，但没有次生感觉圈和具橛感器。从触角超微结构得出，麦蚜若蚜与成蚜均有感知EBF生理基础。（2）以麦长管蚜为对象，通过触角转录组测序、生物信息学预测及qRT-PCR分析，注释并鉴定了12个典型气味结合蛋白（OBPs），1个Plus-C OBP和5个化学感受蛋白（CSPs），其中6个OBPs和3个CSPs为麦长管蚜中首次发现。（3）以2种麦蚜为对象，对OBPs基因进行分离克隆、原核表达及荧光结合研究，发现RapdOBP3和RpadOBP7均能与EBF结合，RapdOBP7倾向于特异结合EBF，而RpadOBP3与多个配体均结合而非特异结合。发现麦长管蚜的三种OBPs即SaveOBP3、SaveOBP7、SaveOBP9均与EBF强烈结合，推测SaveOBP3、SaveOBP7、SaveOBP9是识别EBF的目标蛋白。同源建模及模拟分子对接的结果与配体结合结果一致。（4）从麦长管蚜中克隆出嗅觉受体伴侣蛋白SaveOrco基因，运用RNAi技术沉默SaveOrco后，蚜虫对EBF、绿叶气味、蚜虫诱导植物挥发物的EAG及行为反应均显著减弱；蚜虫在间断式、高剂量的EBF胁迫处理下响应降低、有翅蚜比率大幅下降；EBF处理的母代和初产若蚜，均可提高其有翅蚜率；RNAi干扰后，上述诱导效应消失。研究证实蚜虫SaveOrco蛋白参与感知EBF信号转导途径，可能在蚜虫嗅觉响应以及若蚜翅型发育调控中发挥作用。（5）以EBF为分子模型，OBP为靶标，设计并合成了38个新型EBF类化合物，通过荧光竞争结合测定筛选出16个与麦蚜OBPs的结合活性EBF类似物，为进一步开发以EBF为导向的趋避剂奠定了理论基础。.上述研究结果为揭示EBF对麦蚜驱避调控的分子机理，对蚜虫绿色防控技术研发，及蚜虫化学生态学理论的丰富具有重要意义。