Developing a Gene Silencing Technology for Insect Vectors of Disease

昆虫基因功能的研究为理解这一非常多样化和成功的动物群体的生理、行为、免疫学和疾病传播过程提供了至关重要的一步。有了这些知识，我们就可以开发对抗疾病的模型和控制害虫种群的策略。然而，获得这些知识并不像想象的那么简单。在这个建议中，我们将开发一种新技术，使昆虫基因的研究更容易。第一个完整基因组序列的发表是历史上一个决定性的时刻。然而，仅仅知道一个基因的序列并不足以解释它的功能。幸运的是，人们发现了一种强大的技术，可以在单个基因的基础上询问基因功能。该工具利用了一种古老的细胞抗病毒防御反应：RNA干扰（RNAi）。当一个细胞遇到双链（ds）形式的RNA（如病毒感染）时，它处理RNA并用它来“关闭”或沉默宿主细胞中与dsRNA序列互补的特定基因。通过人工合成具有已知序列的dsRNA并将其引入目标细胞，可以通过观察其活性丧失的后果来了解特定基因的作用。因此，RNAi和其他所谓的反向基因技术正在彻底改变生物科学。选择用于基因组测序的许多生物代表的物种要么造成痛苦（例如冈比亚按蚊），要么为人类生理学和疾病提供了惊人的模型（例如果蝇黑胃肠）。事实上，dsRNA技术被迅速应用于按蚊，正是RNAi技术在昆虫中的应用为我们的项目提供了重点。昆虫的dsRNA传递方法有点复杂。昆虫通常被注射dsRNA，或者偶尔被喂食。虽然到目前为止，绝大多数昆虫都能接受dsRNA介导的RNAi，但小于一定大小的昆虫由于注射损伤和麻醉（必须注射大量的药物）而死亡率很高，而大型昆虫需要大量昂贵的dsRNA来合成。其他因素，如沉默效果的持续时间相对较短（这可能不适合长寿的昆虫），意味着目前的技术不适合许多昆虫物种。如果我们要充分利用新出现的昆虫遗传信息，RNAi方法必须进化以适应更广泛的物种。本项目将开发一种新的RNAi技术，该技术依赖于转基因共生肠道细菌在体内合成dsRNA并被昆虫宿主摄取。dsRNA将直接针对昆虫的基因，导致敲低效应，从而揭示目标基因的作用。我们的模式昆虫将是长角Rhodnius prolixus；一种大型、长寿的吸血虫，与罗得红球菌进化成共生关系。新孵化的昆虫没有共生细菌，必须通过摄入其他昆虫污染的粪便来获得它们。这意味着表达dsRNA的细菌有可能在昆虫群体中自然传播。这项技术将减少昆虫的处理和相关的死亡率，并提高成本效益。我们面临的障碍是确保细菌和dsRNA表达的充分和稳定的转化，保持它们的共生特征和适应性，它们重新繁殖昆虫的能力，以及保持它们的昆虫宿主的适应性。我们设想，这项新技术不仅可以改善昆虫的反向遗传学研究，扩大可以研究的物种范围，而且可能最终形成一种新的和高度特异性的害虫控制策略的基础，该策略将针对昆虫生存或繁殖所必需的基因。

The study of insect gene function provides a crucial step towards understanding physiology, behaviour, immunology and disease transmission processes in this very diverse and successful group of animals. Armed with this knowledge we can develop models to fight disease and strategies to control pest insect populations. Obtaining this knowledge, however, is not as simple as it could be. In this proposal we will develop a new technology to make the study of insect genes easier. The publication of the first complete genome sequence was a defining moment in history. However, simply knowing the sequence of a gene is not enough to explain its function. Luckily a powerful technique was found to interrogate gene function on an individual gene basis. This tool exploits an ancient cellular antiviral defence response: RNA interference (RNAi). When a cell encounters RNA in a double-stranded (ds) form (as in viral infections), it processes the RNA and uses it to 'switch off' or silence a specific gene in the host cell whose sequence complements that of the dsRNA. By artificially synthesising dsRNA with a known sequence and introducing it to target cells, it is possible to understand the role of a specific gene by observing the consequences of its loss of activity. RNAi and other so-called reverse genetics techniques are thus revolutionizing biological sciences. Many of the organisms selected for genome sequencing represent species that either inflict suffering (e.g. the mosquito Anopheles gambiae) or that provide spectacular models for human physiology and disease (e.g. the fruitfly Drosophila melanogaster). Indeed the dsRNA technique was rapidly adapted for use in the Anopheles mosquito, and it is the application of RNAi technologies in insects that provides the focus for our project. The dsRNA delivery method for insects is a little complicated. Insects are most commonly injected with, or occasionally fed, dsRNA. While the great majority of insects so far addressed are amenable to dsRNA-mediated RNAi, insects below a certain size suffer high mortality associated with injection injury and anaesthesia (and one must inject an awful lot of them), whereas large insects require expensive quantities of dsRNA to be synthesised. Other factors, such as the relatively short duration of the silencing effect (that may not suit long-lived insects) means that the technology in its current state is inappropriate for many insect species. If we are to make the most of emerging insect genetic information, RNAi methods must evolve to accommodate a wider variety of species. This project will develop a new RNAi technique that relies on the in vivo synthesis of dsRNA by transgenic symbiotic gut bacteria, and its ingestion by the insect host. The dsRNA will be directed against genes of the insect, leading to a knockdown effect that will reveal the role of the target gene. Our model insect will be Rhodnius prolixus; a large, long-lived blood-sucking bug that has evolved a symbiotic relationship with Rhodococcus rhodnii bacteria. Newly-hatched insects are free from symbiotic bacteria and must acquire them through ingestion of R. rhodnii - contaminated faeces from other insects. This means that dsRNA expressing bacteria have the potential to spread naturally through a colony of insects. The technique should reduce insect handling and associated mortality, and boost cost-efficiency. The hurdles we face are in ensuring adequate and stable transformation of the bacteria and the expression of dsRNA, the retention of their symbiotic characteristics and fitness, their ability to repopulate insects, and in maintaining the fitness of their insect hosts. We envisage that this new technique would not only improve reverse genetics studies in insects and widen the range of species that can be studied, but also that it may eventually form the basis of a novel and highly specific pest control strategy that will target genes essential to insect survival or reproduction.