Developing a Gene Silencing Technology for Insect Vectors of Disease

对昆虫基因功能的研究为了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关键的一步。有了这些知识，我们就可以开发抗击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它可能的那样简单。在这项提案中，我们将开发一种新技术，使昆虫基因的研究变得更容易。第一个完整基因组序列的公布是历史上的一个决定性时刻。然而，仅仅知道基因的序列并不足以解释它的功能。幸运的是，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。这个工具利用了一种古老的细胞抗病毒防御反应：RNA干扰(RNAi)。当细胞遇到双链(Ds)形式的RNA(如在病毒感染中)时，它会处理RNA，并使用它来关闭或沉默宿主细胞中的特定基因，该基因的序列与dsRNA的序列互补。通过人工合成具有已知序列的dsRNA并将其引入靶细胞，可以通过观察特定基因失去活性的后果来了解其作用。因此，RNAi和其他所谓的反向遗传学技术正在给生物科学带来革命性的变化。许多被选择进行基因组测序的生物代表了要么造成痛苦的物种(例如冈比亚按蚊)，要么是为人类生理和疾病提供壮观模型的物种(例如果蝇黑腹果蝇)。事实上，dsRNA技术很快就被应用于按蚊，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。昆虫最常见的是注射或偶尔喂食dsRNA。虽然到目前为止所讨论的绝大多数昆虫都能受到dsRNA介导的RNAi的影响，但一定大小的昆虫由于注射损伤和麻醉而遭受高死亡率(并且必须注射大量的dsRNA)，而大型昆虫需要合成昂贵的dsRNA。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。如果我们要最大限度地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。该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，并将其摄入昆虫宿主。DsRNA将针对昆虫的基因，导致一种击倒效应，将揭示目标基因的作用。我们的模型昆虫将是Rhodnius prolixus；一种大型、长寿的吸血昆虫，它已经进化出与罗德尼红球菌细菌的共生关系。新孵化的昆虫没有共生菌，必须从其他昆虫那里摄取被罗得尼氏菌污染的粪便才能获得共生菌。这意味着表达dsRNA的细菌有可能通过昆虫群体自然传播。这项技术应该会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的充分和稳定的转化和dsRNA的表达，保持它们的共生特性和适应性，它们重新繁殖昆虫的能力，以及维持它们的昆虫宿主的适应性。我们预计，这项新技术不仅将改善昆虫的反向遗传学研究，扩大可研究的物种范围，而且最终可能形成一种新的、高度特异的害虫控制策略，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.