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

本研究的主要目标是最大限度地提高RNA干扰（RNAi）作为植物，特别是多倍体功能基因组学工具的效率和有效性。目前，RNAi技术受到一些限制，严重限制了其在全基因组范围内基因功能测定的有效性，以及针对基因组编码的特定基因亚群的更集中的反向遗传研究。通过RNAi实现高通量诱变是不可能的，因为必须在沉默构建体中插入相同序列的两个拷贝，这要求每个构建体都是单独制造的，这既费力又昂贵。本文提出的方法利用了感觉共抑制，这是一种通过靶序列的单个拷贝诱导RNA沉默的方法。这允许在池中同时为大量基因构建沉默结构（即，以大规模并行的方式生产沉默结构）。在SGER下进行这个小型的探索性项目的理由是，它为农作物，特别是多倍体，以高通量的方式产生突变的问题提供了一种新的方法。此外，我们将评估dsrna介导的转录沉默与转录后沉默的相对有效性，用于重点反向遗传实验，这是针对特定相关基因家族的植物功能基因组学项目的常见组成部分。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，在适当的科学会议上展示，并通过在科学期刊上发表的方式公开。新的载体将通过公共存储库免费提供，如拟南芥生物资源中心（ABRC: http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/abrchome.htm）.Broader)影响：该项目将资助一名未被充分代表的少数民族高中生，他们将参与拟议的研究，并通过亚利桑那大学荣誉学院和优秀高中生暑期计划获得独立学习学分。

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.