SGER: EVALUATING RIBONUCLEASE P AND RNAi AS TOOLS FOR TARGETED RNA DEGRADATION IN PLANTS

随着基因组序列的获取，一个新出现的挑战是需要有效的工具来特定和可调控地干扰基因表达，以建立基因功能。这个SGER项目旨在使用一种策略，以拟南芥为测试有机体，对植物中的任何细胞RNA进行定向和严格控制的降解。该方法依赖于使用RNaseP，一种参与tRNA生物发生的普遍存在的酶，作为一种试剂，特异性地切割内源性目标RNA并破坏其可译性或其他生物功能(S)。这种RNaseP介导的切割依赖于另一种名为外部引导序列(EGS)的小RNA的表达，当它与目标mRNA结合时，生成的复合体是RNaseP的底物。通过比较基于RNaseP和EGS的方法与RNAi在干扰记者(荧光素酶、绿色荧光蛋白)和组织特异性转录因子(狼人和GLABROUS1)的目标mRNAs表达方面的有效性、序列特异性、诱导性和细胞自主性，将阐明这些潜在的基因功能发现工具的优缺点。除了作为植物生物学家的功能基因组学工具外，EGS方法还可以通过工程抗病或改变代谢/生理性状来提高农业生产力。拟议研究的更广泛影响。拟议的研究是基于在单子叶和双子叶植物上取得的有希望的初步结果的坚实基础。该项目将使(I)对处于职业生涯不同阶段的研究人员进行最先进的多学科培训，(Ii)建立学术研究合作网络，并加强一个开创性的土地授予机构的研究基础设施，以及(Iii)开发一种在农业生物技术方面具有重大回报的令人兴奋的新技术。预计两年后，所有必要的实验方案都将可用，以帮助其他研究人员使用EGS方法设计他们自己的研究，以定向干扰基因表达。为了确保这项研究的结果在研究界产生最大影响，应要求将立即提供EGS战略所需的试剂和工具。

An emerging challenge pursuant to acquisition of genomic sequences is the need for efficient tools for specific and regulatable disruption of gene expression to establish gene function. This SGER project aims to use a strategy for targeted and tightly controlled degradation of any cellular RNA in plants, using Arabidopsis as a test organism. The method depends on using RNase P, a ubiquitous enzyme involved in tRNA biogenesis, as a reagent that specifically cleaves an endogenous target RNA and disrupts either its translatability or other biological function(s). This RNase P-mediated cleavage depends on the expression of another small RNA called an external guide sequence (EGS), which when bound to the target mRNA renders the resulting complex a substrate for RNase P. By comparing the efficacy, sequence specificity, inducibility and cell autonomy of the RNase P- and EGS-based method versus RNAi in disrupting the expression of target mRNAs of reporters (luciferase, green fluorescent protein) and tissue-specific transcription factors (WEREWOLF and GLABROUS1), the pros and cons of these potential gene-function discovery tools will be elucidated. In addition to serving as a functional genomics tool for plant biologists, the EGS method could be used to enhance agricultural productivity by engineering disease resistance or altering metabolic/physiological traits. Broader impacts of the proposed research . The proposed studies are based on a solid foundation of promising preliminary results in a monocot and a dicot. The project will enable (i) multi-disciplinary state-of-the-art training for researchers at various stages in their careers, (ii) a network of academic research collaborations and enhancement of the research infrastructure at a pioneering land-grant institution, and (iii) the development of a new, exciting technology with significant payoffs in agricultural biotechnology. It is anticipated that after two years all the necessary experimental protocols to help other researchers design their own studies for targeted disruption of gene expression using the EGS method will become available. To ensure that the results of this study will have maximum impact in the research community, reagents and tools needed for the EGS strategy will be provided immediately upon request.