Determine the primary responses of target parasites to xenobiotic compounds utilising the model organism C. elegans

生物体有机制来解毒暴露于许多外来的和可能对他们有害的化合物（异种生物）。了解这一机制对于确定植物产品被害虫和食草动物摄取之间的相互作用以及合理设计杀虫剂至关重要。这是第二个研究领域，是当前工业合作伙伴奖提案的重点。该项目侧重于具有不同作用模式的特定化合物，并试图确定代谢每种化合物的途径。每条途径都可能以层次结构的方式运作，其中几个关键基因（称为转录因子）在暴露于一种或多种外源药物后被激活。然后，这些基因将依次开启那些编码代谢异种生物的产物的基因。我们已经确定了这个项目中一些转录因子的可能身份。我们将在使用小的，自由生活的线虫秀丽隐杆线虫的实验中建立特定转录因子的参与。我们将敲除转录因子的功能，并确定这是否会增强特定外源性药物的毒性。这将证明消融基因的功能在协调该化学物质的代谢中起作用。然后，我们将用微阵列技术筛选蠕虫体内的所有基因，以识别那些受上游转录因子调节的基因。这部分工作得到了先正达的大力支持。我们将生成实时响应特定外源的秀丽隐杆线虫报告系，以评估响应的时间过程。这种线虫具有很高的潜力，可用于铅化合物及其合成变体的快速通量筛选。转录因子必须以某种方式感知它们所响应的外来物。这种相互作用可能是直接的，文献中有这样的例子。我们将克隆我们的转录因子基因，并在细菌中产生它们的蛋白产物，然后确定转录因子蛋白是否与异种生物结合。我们还将研究与我们的重点化学品相关的更多样化的化合物。这些化合物将由先正达合成，具有关键的化学基团取代。这将使我们能够确定引起特定代谢反应的化学结构的性质。这项工作以秀丽隐杆线虫为中心。它是一种有用的模式生物，因为它积累了详细的遗传和其他知识。出于这些原因，先正达公司在寻找用于农药开发的先导化合物时使用了它。它在这项工作中的使用将确保快速进展，但我们也将对英国农业具有高经济影响的害虫进行转化研究。我们选择了苍白球线虫，因为马铃薯囊肿线虫是英国农业的主要害虫，估计每年给我们的马铃薯产业造成5000万英镑的损失。杀线虫剂是英国马铃薯种植者最大的可变生产成本。鉴于线虫每年给全球农业造成的损失估计为1250亿美元，它们潜在的全球市场是可观的。目前的杀线虫剂既对环境有害，也可能对人类健康有害，由于这些原因，一些杀线虫剂已经从市场上撤下。有效但对环境无害的化合物有很大的市场机会。这项工作是及时的，因为它将直接与bbsrc资助的苍白藻测序项目相关联，该项目由本提案的PI与桑格中心、RES和SCRI合作领导。考虑到秀丽隐杆线虫在产品开发中的核心作用，这项工作将为杀线虫剂以及杀虫剂的设计和评估提供有价值的信息。这些新产品将有助于实现粮食安全和保护环境。

Organisms have mechanisms to detoxify exposure to many compounds that are foreign and might be detrimental to them (xenobiotics). Understanding this mechanism is central to defining interactions between plant products ingested by pests and herbivores and also to the rational design pesticides. It is the second research field that is the focus of the current proposal for an industrial partner award. The project focuses on specific compounds with different modes of action and seeks to determine the pathways that metabolise each example. Each pathway is likely to operate in a hierarchical nature with a few key genes, termed transcription factors, being turned on after exposure to one or more xenobiotics. These genes will then in turn switch on those that encode products that metabolise the xenobiotic. We have already determined the likely identity of some of these transcription factors for this project. We will establish the involvement of particular transcription factors in assays using the small, free-living nematode Caenorhabditis elegans. We will knock-out the function of the transcription factors and determine if this enhances toxicity of particular xenobiotics. This will demonstrate that the ablated gene's function had a role in coordinating the metabolism of that chemical. We will then screen all the genes in the worm with microarray technology to identify those that are regulated by these upstream, transcription factors. This part of the work has substantial support from Syngenta. We will generate reporter lines of C. elegans that respond to specific xenobiotic(s) in real time to evaluate the time course of responses. Such nematodes have high potential for use later in rapid throughput screens of lead compounds and synthesised variants of them. The transcription factors must somehow perceive the xenobiotics to which they respond. This interaction could be direct and there are examples of this in the literature. We will clone our transcription factor genes and generate their protein products in bacteria before determining if the transcription factor protein does binds to the xenobiotic. We will also look at a greater diversity of compounds that are related to our focus chemicals. These compounds will by synthesised by Syngenta to have key chemical group substitutions. This will allow us to determine the nature of the chemical structure that elicits a particular metabolic response. The work centres on C. elegans. It is a useful model organism because of detailed genetic and other knowledge accumulated about it. It is used for these reasons by Syngenta in its search for lead compounds for pesticide development. Its use in this work will assure rapid progress but we will also carry out translational research to a pest with high economic impact in UK agriculture. We have chosen Globodera pallida as potato cyst nematodes are the major nematode pest of UK agriculture costing our potato industry an estimated £50 m/year. Nematicides are the largest variable cost of production for UK potato growers. Their potential global market is appreciable given nematodes cause estimated annual losses to global agriculture of $125b. Current nematicides are harmful both to the environment and possibly human health and several have already been removed from the market for these reasons. There is a large market opportunity for effective but environmentally benign compounds. The work is timely as it will be linked directly to the BBSRC-funded G. pallida sequencing project led by the PI of this proposal in collaboration with Sanger Centre, RES and SCRI. The work will provide information that will be valuable in the design and evaluation of not just nematicides but also insecticides given the central role of C. elegans in product development. These new products will help deliver food security and safeguard the environment.