Functional Genomics by Sense-RNAi: A Forward Genetic Approach for Cell-Type-Targeted Mutagenesis and for Polyploids

该项目的目标是开发一种基于被称为RNA干扰（RNAi）的基因沉默现象的功能基因组学新工具。一种特殊形式的RNAi，被称为Sense-RNAi，将被优化为一种专注于基因组学的工具，在作物植物中有特殊的应用。这种方法将产生一系列的单株植物，每个单株中有一个基因被沉默。通过筛选Sense-RNAi沉默的植物群体，研究人员将能够识别出有助于研究者特别感兴趣的有价值性状的基因。通过使用Sense-RNAi而不是标准的RNAi方法，预计沉默转基因构建体将非常有效地产生。这将允许沉默集中在作物植物中选定的基因子集上，极大地提高识别影响特定性状的基因的总体效率。这项新技术将在一种模式植物中进行开发、测试和优化，然后应用于农作物，首先从番茄开始。基因沉默方法将集中在毛状体中表达的基因上，毛状体是叶子表面的微小毛发，有助于保护植物免受昆虫和微生物的侵害。该资助项目的预期结果主要是优化沉默一组重点基因的方法，包括必要的植物转化载体和将该方法成功应用于作物植物的实验方案。结果将通过出版物和用户手册传播，该手册将在网上公布，并视需要增订。向量将通过公共存储库不受限制地提供给用户，我们预计这将是ABRC。

The goal of this project is to develop a new tool for functional genomics based on the gene silencing phenomenon known as RNA interference (RNAi). A particular form of RNAi, known as Sense-RNAi, will be optimized for use as a focused genomics tool with special application in crop plants. The method will produce a series of individual plants, in each of which a single gene has been silenced. By screening a population of Sense-RNAi silenced plants, investigators will be able to identify genes that contribute to valuable traits of particular interest to the investigator. By using Sense-RNAi rather than standard RNAi approaches, it is expected that silencing transgene constructs will be produced very efficiently. This will allow silencing to be focused on a select subset of the genes in a crop plant, increasing dramatically the overall efficiency of identifying genes that affect a given trait. This new technology will be developed, tested and optimized in a model plant species and then applied to crop plants, beginning with tomato. The gene silencing method will be focused on genes expressed in trichomes, which are tiny hairs on the surface of a leaf that help protect the plant from insects and microbes. The expected outcome of the funded project is primarily the optimized method for silencing of a focused set of genes, including the necessary plant transformation vectors and experimental protocols for successful application of this method to crop plants. Results will be disseminated through publications and via a user's manual that will be posted on the web and updated as needed. Vectors will be provided to users without restriction via a public repository, which we anticipate will be ABRC.