EAGER: RNAi Gene Discovery Tool to Randomly Generate Dominant Mutant Pools in Plants

近10年来，植物小RNA介导的基因沉默及其相关技术的发展取得了巨大的进展。迄今为止，已经在植物中发现了两种常见的小RNA：siRNA和miRNA。 siRNA可以进一步细分为三种不同类型：反式siRNA（tasiRNA）、天然反义siRNA和重复相关siRNA。 虽然所有这些小RNA都可以用来沉默植物中的基因，但tasiRNA是一种独特但有趣的小RNA群体，仅在各种植物物种中非常保守。在植物中产生tasiRNA需要miRNA和RNA干扰途径，这使得tasiRNA的基因沉默成为基因功能研究的潜在强大系统。与其他类型的小RNA需要预结构化的双链RNA（dsRNA）作为其生产的前体不同，tasiRNA可以使用单链RNA前体通过RNA依赖性RNA聚合酶在细胞中产生dsRNA。这种独特的品质使得通过基因工程在tasiRNA前体内部引入随机短序列成为可能。EAGER项目将探索植物tasiRNA途径在模式植物拟南芥中产生植物突变体库的新应用，作为概念验证。除了研究作物基因功能的新工具的广泛影响外，该项目还将为1名博士后研究员和9名本科生和高中生提供研究培训机会。 代表性不足的少数民族和妇女，以及来自肯塔基州东部农村的学生，将特别成为目标和招募。所有关于开发的方法和产生的突变体的信息将通过出版物和拟南芥信息资源（TAIR）获得。 所有载体和转基因种子均可从PI和拟南芥生物资源中心（ABRC）获得。

Tremendous progress has been made over the last 10 years in small RNA-directed gene silencing and the development of related technologies in plants. To date, two general kinds of small RNAs have been discovered in plants: siRNAs and miRNAs. siRNAs can be further subdivided into three different types: trans-active siRNAs (tasiRNAs), natural antisense siRNAs, and repeat-associated siRNAs. Although all of these small RNAs can be used to silence genes in plants, tasiRNAs are a unique but intriguing population of small RNAs that are well conserved exclusively in various plant species. Production of tasiRNAs in plants requires both the miRNA and the RNA interference pathways, which makes gene silencing by tasiRNAs a potentially powerful system for gene function studies. Unlike other kinds of small RNAs that need pre-structured double-stranded RNAs (dsRNAs) as precursors for their production, tasiRNAs can use single-stranded RNA precursors to produce dsRNA through RNA-dependent RNA polymerase in cells. This unique quality makes it possible to introduce a randomized short sequence inside the tasiRNA precursors through genetic engineering. This EAGER project will explore the novel application of the plant tasiRNA pathway for generating plant mutant pools in the model plant Arabidopsis as proof-of-concept.In addition to the broader impacts of a new tool for investigating gene function in crop plants, the project will provide research training opportunities for one postdoctoral fellow and nine undergraduate and high school students. Underrepresented minorities and women, as well as students from rural eastern Kentucky, will be especially targeted and recruited. All information regarding the method(s) developed and mutants generated will be available through publication and through the Arabidopsis Information Resource (TAIR). All vectors and transgenic seed will be available from the PI and from the Arabidopsis Biological Resource Center (ABRC).