The genetic basis for resistance to bioinsecticides in diamondback moth Plutella xylostella

近年来，已经开发了几种生物杀虫剂，它们的作用模式通常非常特异，因此可能比传统的化学杀虫剂更环保。甚至有人提出，由于这些杀虫剂的生物来源，它们可能避免目标害虫物种产生抗药性。毫不奇怪，这并没有被证明是这样的，这两种杀虫剂Bt和多杀菌素的农业使用增加，导致了小菜蛾抗药性的进化，小菜蛾是十字花科作物最具破坏性的全球害虫。了解耐药性的作用方式和机制对于长期、可持续地使用这些化合物至关重要。此外，虽然转基因Bt作物在英国还没有商业用途，但了解对现有Bt作物喷雾剂的抗性基础对于评估未来转基因作物抗性的可能性至关重要。令人惊讶的是，尽管Bt毒素在叶面喷洒和转基因作物中具有全球重要性，但其靶点还远未明确。在这里，我们的目标是在田间获得对这些杀虫剂的抗性的小菜蛾种群中确定抗性的遗传基础。已知Bt（Cry 1Ac毒素）和多杀菌素抗性都涉及单个主基因，在这两种情况下，我们已经确定了连锁的分子标记。我们将用匿名的分子标记来定位这些抗性基因周围的基因组区域。然后，通过大插入BAC文库，我们将完全测序两个抗性基因周围的区域。为了鉴定抗性基因，我们将测试从该区域鉴定的候选基因，以确定它们是否在幼虫和适当的组织（如中肠）中表达，以及抗性和易感群体之间的表达是否存在差异。此外，我们将使用RNAi敲低实验来确定是否存在降低候选基因的表达对易感品系中的抗性水平的影响。我们还将检测候选基因座的基因转录本中抗性和敏感品系之间的序列差异（如果有的话）。这将允许开发用于检测田间捕获样品中的抗性等位基因的基于PCR的快速方法，这将促进抗性等位基因传播的田间监测。鉴定具有新作用模式的杀虫剂可以在防止农业害虫群体中交叉抗性的演变中发挥重要作用，并且新型生物杀虫剂如Bt和多杀菌素在补充和/或取代旧的化学杀虫剂方面发挥了重要作用。然而，抗性的演变是不可避免的，在目标害虫，除非采取措施来对抗它。更好地了解抗性的机制，因此，这些化合物的作用机制将是至关重要的，在开发新的毒素具有不同的作用模式，并在制定战略，在该领域的抵抗力。

Recent years have seen the development of several bio-insecticides, which are often very specific in their mode of action and therefore potentially more environmentally friendly than traditional chemical insecticides. It was even suggested that, due to their biological origin, these insecticides might avoid the evolution of resistance in targeted pest species. Unsurprisingly this has not proven to be the case, and increased agricultural use of two of these insecticides, Bt and Spinosad, has led to the evolution of resistance in the diamondback moth, the most destructive global pest of cruciferous crops. Understanding the mode of action and mechanisms of resistance is crucial for long term, sustainable use of these compounds. Furthermore, although transgenic Bt crops are not available for commercial use in the UK, an understanding of the basis for resistance to existing Bt crop sprays will be vital in assessing the likelihood of resistance to transgenic crops in the future. Surprisingly, despite its global importance in both foliar sprays and transgenic crops, the targets for Bt toxin are far from clear. Here, we aim to characterise the genetic basis of resistance in populations of diamondback moth that have acquired resistance to these insecticides in the field. Both Bt (Cry1Ac toxin) and spinosad resistance are known to involve a single major gene and in both cases we have already identified linked molecular markers. We will target the regions of the genome surrounding these resistance genes with anonymous molecular markers. Then, by means of a large-insert BAC library, we will fully sequence the region surrounding the two resistance genes. In order to identify the resistance gene we will then test candidate genes identified from this region to see whether they are expressed in the larvae and in appropriate tissues such as the midgut, and also whether there are differences in expression between resistant and susceptible populations. Additionally, we will use RNAi knockdown experiments to determine whether there is an effect of reducing the expression of the candidate gene on levels of resistance in susceptible lines. We will also characterise sequence differences, if any, between the resistant and susceptible lines in the gene transcript of the candidate locus. This will allow development of rapid PCR-based methods for detecting resistant alleles in field caught samples that will facilitate field monitoring of the spread of resistance alleles. Identification of insecticides with novel modes of action can play an important role in preventing the evolution of cross resistance in populations of agricultural pests, and novel biopesticides such as Bt and spinosad have played an important role in complementing and/or replacing older chemical insecticides. However the evolution of resistance is inevitable in targeted pests unless measures are taken to combat it. A better understanding of the mechanisms of resistance, and hence the mechanism of action of these compounds will be crucially important in developing novel toxins with distinct modes of action, and in developing strategies for combating resistance in the field.