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

University Of Arizona, Tucson AZ

Investigators

Abstract

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.