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

获取基因组序列的一个新挑战是需要有效的工具来特异性和可调控地破坏基因表达以建立基因功能。该SGER项目旨在使用一种策略，以拟南芥为试验生物，对植物中任何细胞RNA进行靶向和严格控制的降解。该方法依赖于使用RNase P，一种参与tRNA生物合成的普遍存在的酶，作为特异性切割内源性靶RNA并破坏其可翻译性或其他生物功能的试剂。这种RNase P介导的切割依赖于另一种称为外部指导序列（EGS）的小RNA的表达，当其与靶mRNA结合时，使所得复合物成为RNase P的底物。通过比较基于RNase P和EGS的方法与RNAi在破坏报告基因的靶mRNA表达方面的功效、序列特异性、诱导性和细胞自主性，（荧光素酶，绿色荧光蛋白）和组织特异性转录因子（WEREWOLF和GLABROUS 1），这些潜在的基因功能发现工具的优点和缺点将被阐明。除了作为植物生物学家的功能基因组学工具外，EGS方法还可用于通过工程抗病性或改变代谢/生理性状来提高农业生产力。拟议研究的更广泛影响。 建议的研究是基于一个坚实的基础上有前途的初步结果，在单子叶植物和双子叶植物。该项目将使（i）在其职业生涯的各个阶段的研究人员的多学科国家的最先进的培训，（ii）学术研究合作网络和研究基础设施的增强在一个开拓性的土地赠款机构，和（iii）一个新的，令人兴奋的技术与农业生物技术的显着回报的发展。 预计两年后，所有必要的实验方案，以帮助其他研究人员设计自己的研究，有针对性地破坏基因表达使用EGS方法将成为可用。为确保本研究的结果在研究界产生最大影响，将根据要求立即提供EGS策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.