Understanding the evolution and function of xenobiotic detoxification enzymes in a global crop pest

了解全球农作物害虫中外源解毒酶的进化和功能

• 批准号: BB/X010058/1
• 负责人: Bartlomiej Troczka
• 金额: $ 50.28万
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX010058%2F1
• 关键词: Understanding; evolution; function; xenobiotic; detoxification

Detoxification enzymes play an important role in the ability of insects to neutralise threats caused by foreign chemicals (xenobiotics), such as plant secondary metabolites and synthetic pesticides. Indeed, insect pests, such as the aphid Myzus persicae, frequently adapt to novel host plants or develop resistance to synthetic insecticides via quantitative or qualitative changes in these enzymes. One of the most important superfamilies of enzymes involved in the evolution of insect resistance to plant-derived and human-made toxins are cytochrome P450s (or CYPs). M. persicae has over 60 individual genes encoding P450 enzymes (the CYPome), however, only a handful of these have been functionally characterised to date. This is a reflection of the time and effort required to produce functional protein for analysis in the lab. As a consequence, while we have a good understanding of the type and number of P450 genes in insects we know very little about what they actually do. In the context of pest species, key questions remain on which P450s have the capacity to detoxify insecticides, the extent to which genetic variation in these P450s in insect populations can influence insecticide metabolism and which of the building blocks (amino acids) that make up P450 proteins are particularly important in interacting with insecticides and breaking them down (structure-function determinants). In this project I aim to exploit a unique set of genomic resources recently created for M. persicae, in combination with recent advances in synthetic biology and modelling of protein structure, to understand how global diversity in an entire gene superfamily impacts the ability of M. persicae to adapt to natural and synthetic xenobiotics.In the first objective of the project, I will investigate where and when all the individual P450s in M. persicae are present through the insect body and life cycle in order to gain insight into their potential function. I will then use state of the art modelling tools to create 3D structures of each protein to gain an intimate understanding of how they interact with small molecules such as toxins and predict which P450s break down insecticides. These computational predictions will be tested in the laboratory utilizing recent advances in protein production to individually express the entire complement of P450 enzymes in M. persicae and examine their ability to metabolise insecticides and other model substrates using biochemical approaches. This will provide the first ever functional dataset on the complete suite of P450s in an insect, and unprecedented insights into the biological function of each member of the CYPome including those that are key actors in xenobiotic metabolism.In the second objective of the project, I will leverage a global sample of >110 re-sequenced clones of M. persicae, in combination with 3D modelling and functional expression to understand the potential for standing genetic variation in CYP genes in aphid populations to be co-opted in the evolution of resistance. The data obtained from these analyses, together with that derived from Objective 1, will allow me to identify key structure-function determinants of P450-mediated insecticide metabolism. The results obtained from the first two objectives of this project will provide fundamental insights into the evolution and function of an important group of enzymes that are found in all kingdoms of life, however, they also have the potential for significant applied impact. To realise this impact I will translate the knowledge gained into a set of molecular tools that can be used to rapidly screen compounds against aphid pest P450s and so identify resistance-breaking chemistry. In summary, the knowledge and tools generated in this study will provide i) a holistic understanding of the function and diversity of the insect CYPome and its relevance for the evolution of insecticide resistance; ii) a toolkit to study and combat P450-mediated resistance.

解毒酶在昆虫中和外来化学物质（例如植物次生代谢物和合成农药）造成的威胁的能力中发挥着重要作用。事实上，害虫，例如桃蚜，常常通过这些酶的量或质变化来适应新的寄主植物或对合成杀虫剂产生抗性。细胞色素 P450（或 CYP）是参与昆虫对植物源性和人造毒素抗性进化的最重要的酶超家族之一。 M. persicae 有超过 60 个编码 P450 酶（CYPome）的单独基因，然而，迄今为止，只有少数基因得到了功能表征。这反映了生产用于实验室分析的功能性蛋白质所需的时间和精力。因此，虽然我们对昆虫 P450 基因的类型和数量有了很好的了解，但我们对它们的实际作用却知之甚少。就害虫物种而言，关键问题仍然是哪些 P450 具有杀虫剂解毒能力、昆虫种群中这些 P450 的遗传变异在多大程度上可以影响杀虫剂代谢以及构成 P450 的组成部分（氨基酸）蛋白质在与杀虫剂相互作用并分解它们（结构-功能决定因素）方面特别重要。在这个项目中，我的目标是利用最近为桃李子创建的一组独特的基因组资源，结合合成生物学和蛋白质结构建模的最新进展，以了解整个基因超家族的全局多样性如何影响桃李子的能力。在该项目的第一个目标中，我将调查 M. persicae 中所有单个 P450 在昆虫身体和生命周期中何时何地存在，以便深入了解它们的潜在功能。然后，我将使用最先进的建模工具创建每种蛋白质的 3D 结构，以深入了解它们如何与毒素等小分子相互作用，并预测哪些 P450 分解杀虫剂。这些计算预测将在实验室中进行测试，利用蛋白质生产的最新进展，在桃李子中单独表达 P450 酶的全部补体，并使用生化方法检查它们代谢杀虫剂和其他模型底物的能力。这将提供第一个关于昆虫中全套 P450 的功能数据集，并对 CYPome 每个成员的生物学功能（包括那些在外源代谢中起关键作用的成员）提供前所未有的见解。 在该项目的第二个目标中，我将利用超过 110 个桃蚜重新测序克隆的全球样本，结合 3D 建模和功能表达，了解蚜虫种群中 CYP 基因的长期遗传变异的潜力共同选择了抵抗的演变。从这些分析中获得的数据以及从目标 1 中获得的数据将使我能够确定 P450 介导的杀虫剂代谢的关键结构-功能决定因素。该项目前两个目标获得的结果将为所有生命王国中发现的一组重要酶的进化和功能提供基本见解，然而，它们也有可能产生重大的应用影响。为了实现这一影响，我将把获得的知识转化为一套分子工具，可用于快速筛选针对蚜虫 P450 的化合物，从而识别突破抗性的化学物质。总之，本研究产生的知识和工具将提供 i) 对昆虫 CYPome 的功能和多样性及其与杀虫剂抗性进化的相关性的整体理解； ii) 研究和对抗 P450 介导的耐药性的工具包。