Transposable Element-Based, Site-Specific Recombination System for Plants

Site-specific recombination (SSR) is a powerful tool for studying gene function, and it offers great promise for harnessing the biosynthetic capacity of plants to produce compounds of commercial value. Two limitations to the use of SSR in plants are 1) the low frequency by which it occurs and 2) the difficulty in delivering enough DNA to produce a desired recombination event. Phytodyne Inc. proposes to overcome these limitations through the use of transposable elements. Excision of DNA transposons increases SSR more than 1000-fold, thereby overcoming the inherently low frequencies of homologous recombination in plants. cDNA generated by retrotransposons - transposable elements that replicate by reverse transcription-is highly recombinogenic. Retrotransposons can be modified to carry any sequence of interest and to synthesize cDNA at high levels. Every plant cell, therefore, can be converted into a 'cDNA factory' to overcome limitations of DNA delivery. This proposal seeks to use retrotransposon cDNA to correct defective marker genes by SSR. Among the marker genes tested will be those from which DNA transposons are actively excising to increase recombination frequencies. Completion of these experiments will provide the foundation for a transposon-based, site-specific recombination system for altering gene sequences from a variety of important crop species.