Developing microRNA Vectors for Gene Suppression in Agricultural Plants

MicroRNAs (miRNAs) are newly discovered endogenous small non-coding RNAs (2]-25 nt) that target their complementary gene transcripts for degradation or translational repression in diverse eukaryotic organisms. Gene suppression is a powerful tool for functional genomics and elimination of specific gene products in plants and animals. However, the current gene suppression vectors have some drawbacks. For example, the progeny with long double-stranded RNA (dsRNA) mediated gene suppression often shows instability of the suppressed gene expression, which makes it difficult to generate a useful line for plant breeding. Nevertheless, the endogenous miRNAs show accurate and faithful regulation of their target gene expression temporally and spatially for finetuning development in plants. Our long-term objective is to understand miRNA-directed gene regulation in plants. The objective of this proposal is to develop new gene suppression technologies based on our deep understanding of the mechanisms of the miRNA-directed gene silencing, and to seek the potential applications in studying gene functions, functional genomics, and crop improvement. Specifically, this proposal will develop second-generation gene suppression vectors by dissecting the secondary structures of the plant miRNA precursor for gene silencing in crops. We will test the miRNA vectors for stable gene suppression first in model plants and then in one of the major crops, tomato. We expect to translate its application into other major crops in future. The new gene suppression vectors will be superior to and eventually replace the first-generation RNAi vectors (long dsRNA vector) in a wide range of applications in agricultural plants. The specific objectives of the proposed research are: (I) Analyzing the miRNA precursor structures of major agricultural plants for structural rules; (2) Synthesizing miRNA-like structures for silencing a few reporter genes and testing their gene silencing efficiency in vitro and in vivo; (3) Designing and testing miRNA-like vectors for stable silencing of a few reporter and endogenous genes in tomato plants. This project is proposed in relation to FY2006 Priorities for Research: I) Functional studies of agriculturally important genes and gene products, including the development of improved mutational and gene silencing approaches for these studies; and 2) Regulatory mechanisms of gene expression.