A genomic approach to understanding insecticide resistance in crop pests

农作物的害虫通常用化学杀虫剂来控制。不幸的是，随着时间的推移，许多害虫对用于控制的杀虫剂产生了抗药性。昆虫主要通过两种方式产生抗药性。首先是由于与杀虫剂结合的蛋白质发生了变化，这意味着它对杀虫剂的毒性作用不再那么敏感；其次是由于分解或结合杀虫剂并使其无效的酶的产生增加。本研究旨在研究两种重要作物害虫——桃薯蚜和褐飞虱的抗药性。桃蚜是英国和欧洲一系列作物的主要害虫，而褐飞虱是亚洲水稻作物的主要害虫，两者都通过直接取食和传播病毒对植物造成损害，造成巨大的经济损失。这两种作物害虫都对用于控制它们的许多杀虫剂产生了抗药性，目前使用的主要化学类别是新烟碱类。然而，最近有对这类杀虫剂产生抗药性的报告。生化研究表明，这种抗性很可能是由于分解杀虫剂的酶的产生增加，特别是一组称为细胞色素P450单加氧酶（P450）的酶。P450是一类具有多种功能的酶，包括毒素分解，昆虫被发现有46-143个P450基因，每个基因产生一种不同的酶。害虫可以通过增加它们产生的一种或多种P450酶的数量来对杀虫剂产生抗性。在这个项目中，我们的目标是研究桃蚜和绿螨对新烟碱的抗性是否由p450的过量产生引起，并确定哪些p450参与其中以及它们过量产生的原因。研究与代谢抗性有关的大基因家族并不容易，但基因组学（研究基因及其功能）和新相关技术的最新进展意味着现在更可行。本研究将利用这些新资源来鉴定目标害虫的P450基因。这些包括许多昆虫物种（包括蚜虫）的基因组（生物体的整个DNA内容）序列，表达序列标签或ESTs（通过对表达基因的一端或两端进行测序而产生的通常为200至500个核苷酸长的DNA片段）和可负担得起的高通量测序技术，这些技术可以在几小时内确定数亿个碱基（DNA单位）的序列。鉴定出的P450基因将使用新的分子方法进行研究，这种方法可以确定基因在RNA和蛋白质中的表达水平。RNA干扰技术（将双链RNA引入细胞以抑制基因的表达）将用于沉默P450基因，从而研究它们在耐药性中的作用。最后，P450基因将被克隆并表达为蛋白质，以观察它们是否会分解或与杀虫剂结合。当这些作物害虫中涉及抗性的特定p450被确定后，我们将开发诊断工具来监测昆虫种群的抗性。这些是旨在减缓或防止耐药性发展的耐药性管理战略的基本要求。通过控制抗药性来延长杀虫剂的使用寿命是至关重要的，因为可供控制的杀虫剂数量有限，而且欧洲议会提出的关于杀虫剂的新立法将大大减少用于农业的杀虫剂的可用性。该项目将与农用化学品公司和杀虫剂抗性行动小组的合作伙伴合作开展，以确保这项研究的结果能够迅速得到利用。

Insect pests of crops are often controlled using chemical insecticides. Unfortunately over time many pests have evolved resistance to the insecticides used for control. Insects have been shown to develop resistance in two main ways. Firstly by changes in the protein that the insecticide binds to which means that it is no longer as sensitive to the toxic effect of the insecticide and secondly by increased production of enzymes that break down or bind to the insecticide and render it ineffective. In this proposal we aim to study insecticide resistance in two important crop pests the peach potato aphid (Myzus persicae) and the brown planthopper (Nilaparvata lugens). M. persicae is a major pest on a range of crops in the UK and Europe and N. lugens is a major pest of rice crops in Asia, both cause damage to plants through direct feeding and the transmission of viruses resulting in high economic losses. Both of these crop pests have evolved resistance to many of the insecticides used for their control and the main chemical class currently being used is the neonicotinoids. However reports of resistance to this insecticide class have recently been described. Biochemical studies have shown that this resistance is likely to be caused by increased production of enzymes that break down the insecticide, in particular a group of enzymes called cytochrome P450 monooxgenases (P450s). P450s are a class of enzymes with many functions including the breakdown of toxins and insects have been found to have between 46-143 P450 genes, each producing a different enzyme. Insect pests can become resistant to insecticides by increasing the amount of one or more of the P450 enzymes they produce. In this project we aim to examine if resistance in M. persicae and N. lugens to neonicotinoids is caused by over-production of P450s and determine which P450s are involved and why they are over-produced. It is not easy to study the large gene families involved in metabolic resistance however recent advances in the field of genomics (the study of genes and their function) and new associated technologies means that it is now more feasible. This study will exploit these new resources to identify P450 genes in the target pest species. These include the genome (the entire DNA content of an organisms) sequences of a number of insect species (including an aphid), expressed sequence tags or ESTs (small pieces of DNA sequence usually 200 to 500 nucleotides long that are generated by sequencing either one or both ends of an expressed gene) and affordable high-throughput sequencing technologies that allow many hundreds of millions of bases (a unit of DNA) of sequence to be determined in a matter of hours. The identified P450 genes will then be studied using new molecular methods that allow determination of the levels of expression of genes into RNA and protein. The technique RNA interference (the introduction of double-stranded RNA into a cell to inhibit the expression of a gene) will be used to silence P450 genes and therefore examine their role in resistance. Finally the P450 genes will be cloned and expressed as protein to see if they break down or bind to insecticide. When the specific P450s involved in resistance in these crop pests have been identified we will develop diagnostic tools to monitor insect populations for resistance. These are an essential requirement of resistance management strategies which aim to slow or prevent the development of resistance. Prolonging the life of insecticides by managing resistance is vital as there are only a limited number of insecticides available for control and proposed new legislation on pesticides from the European Parliament will dramatically cut the availability of insecticides for use in agriculture. This project will be carried out in collaboration with partners in agrochemical companies and the Insecticide Resistance Action Group to ensure the findings of this study can be rapidly exploited.