SGER: Functional Genomics Tools Based on Double Standard RNA-mediated Transcriptional and Post-transcriptional Gene Silencing

该研究的主要目标是最大限度地提高RNA干扰（RNAi）作为植物功能基因组学工具的效率和有效性，特别是多倍体。目前，RNAi技术受到几个限制，这些限制严重限制了其用于基因功能的全基因组测定以及针对由基因组编码的基因的特定子集的更集中的反向遗传研究的有用性。通过RNAi的高通量诱变是不可能的，因为在沉默构建体中插入相同序列的两个拷贝的必要性要求单独制备每个构建体，这是费力且昂贵的。本文提出的方法利用了正义共抑制，一种用靶序列的单拷贝诱导RNA沉默的方法。这允许在库中同时构建大量基因的沉默构建体（即，以大规模平行方式产生沉默构建体）。在SGER下，这个小型探索性项目的理由是，它为以高通量方式在作物植物，特别是多倍体中产生突变的问题带来了一种新的方法。此外，我们将评估dsRNA介导的转录沉默的相对有效性相比，转录后沉默用于集中的反向遗传实验，这是植物功能基因组学项目的共同组成部分，针对特定的相关基因家族。结果和方法将通过植物染色质数据库（ChromDB：http：//chromdb.biosci.arizona.edu/）的RNAi工具部分，通过在适当的科学会议上的演讲，以及通过在科学期刊上的出版物公开提供。新的载体将通过拟南芥生物资源中心（ABRC：http：//www.biosci.ohiostate.edu/plantbio/Facilities/abrc/abrchome.htm）等公共资源库免费提供。更广泛的影响：该项目将资助一名代表性不足的少数民族高中生，他将参加拟议的研究，并通过亚利桑那大学荣誉学院和上级高中生卓越暑期计划获得独立的学习学分。

The principal goal of the proposed research is to maximize the efficiency and the effectiveness of RNA interference (RNAi) as a tool for functional genomics in plants, especially polyploids. Currently, RNAi technology suffers from several limitations that severely limit its usefulness for genome-wide determination of gene functions, as well as for more focused reverse genetic investigations directed at specific subsets of the genes encoded by a genome. High-throughput mutagenesis by RNAi is not possible because the necessity of inserting two copies of the same sequence in silencing constructs requires that each construct be made individually, which is laborious and expensive. The approach proposed here exploits sense co-suppression, a means of inducing RNA silencing with a single copy of the target sequence. This allows the construction of silencing constructs for a large number of genes simultaneously in pools (i.e., production of silencing constructs in a in massively parallel manner). The justification under SGER for this small, exploratory project is that it brings a new approach to the problem of generating mutations in a high-throughput manner in crop plants, especially polyploids. In addition, we will evaluate the relative effectiveness of dsRNA-mediated transcriptional silencing as compared to post-transcriptional silencing for use in focused reverse genetic experiments, which are common components of plant functional genomics projects directed at specific families of related genes. Results and methods will be made available publicly via the RNAi tools section at The Plant Chromatin Database (ChromDB: http://chromdb.biosci.arizona.edu/), by presentation at appropriate scientific conferences, and by means of publication in scientific journals. Novel vectors will be made freely available via a public repository such as the Arabidopsis Biological Resource Center (ABRC: http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/abrchome.htm).Broader impacts: This project will fund an underrepresented minority high school student who will participate in the proposed research and receive independent study credit hours through the University of Arizona Honors College and Summer of Excellence program for superior high school students.