Arabidopsis 2010: A Transposon-Based System for Site-Specific Recombination in Arabidopsis

Iowa State University, Ames IA

Investigators

Abstract

The goal of this project is to develop a method for efficient homology-dependent site-specific recombination (SSR) in Arabidopsis. The approach used is based on the recombination-inducing properties of transposable elements, coupled with alternative methods of delivering recombination substrates to plant cells. In previous research, the PI and co-PI have shown that DNA and RNA transposable elements can stimulate recombination. First, excision of maize Ac/Ds DNA transposons greatly induces homologous recombination in plants, including Arabidopsis. Second, retrotransposons can generate high levels of cDNA that recombine readily with genomic sequences. This project will develop two-component (recipient and donor) transgene constructs that contain partially-overlapping visible and selectable marker gene fragments. The recipient component contains, in addition, a maize Ds element inserted between the marker genes. The recipient construct is integrated into the Arabidopsis genome; upon expression of Ac/Ds transposase, Ds excision generates a recombination hotspot in the recipient construct. The donor construct sequences are delivered into the plant cells via several alternative methods, including Agrobacterium T-DNA transformation, particle bombardment, ectopic chromosomal position, and retrotransposon-generated cDNA. Recombination of recipient and donor sequences can be detected in somatic cells by the visible marker, and events transmitted to progeny can be genetically selected. Heritable recombination events will be characterized by molecular and genetic analysis to gain a greater understanding of SSR mechanisms in plant cells. Project results will be available at the following web site: <http://www.public.iastate.edu/~voytas/rec_arab.html>. All reagents (seeds of transformed lines, plasmid vectors) will be made available upon request. Homology-dependent recombination is a powerful tool for genetic modification in many organisms; however, methods for routine SSR are not yet available for higher plants. An explicit goal of the Arabidopsis 2010 project is the development of methods for directed mutations and SSR. Successful completion of this project will provide an important new means for making precise changes in plant genomes for both fundamental research and practical applications.