TRANSPOSON TAGGING IN RICE USING A NOVEL Ds ELEMENT, Cre-lox SITE-SPECIFIC RECOMBINATION AND AN INDUCIBLE Ac TRANSPOSASE

¹ Department of Plant Pathology, Ohio State University, Columbus, OH 43210, USA

² Department of Plant Pathology, University of California, Davis, CA 95616, USA;

³ Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA

Transposon tagging is one of the widely used methods for generation of insertion mutants for gene functional analysis in plants. However, a major drawback of the current transposon tagging systems is the secondary transposition, which makes transposon insertions unstable and complicates gene tagging and cloning effort. We have developed eight Ac/Ds tagging vectors in which a novel Ds element, cre-lox site-specific recombination and an inducible Ac transposase are combined as a comprehensive approach to improve the current transposon tagging system in rice. The novel Ds element contains a hygromycin resistance gene (HPT) that has dual functions for transformation selection and preventing the Ds from excising from its original T-DNA site during tissue culture and plant regeneration period. The mutagenesis efficiency can be improved by creating many independent starter lines from the same transformed callus. In each starter line, the Ds element transposed from its donor site to different locations of the rice genome. We also use the cre-lox site-specific recombination system in which Ds transposition triggers cre recombinase expression and the deletion of Ac transposase. The permanent removal of Ac transposase helps stabilize the transposed Ds elements in rice genome. Additionally, a chemical inducible gene expression system has been used to control Ac transposase activity and Ds transposition. Over 200 transgenic rice lines transformed with these vectors have been generated. Ds transposition and cre-lox recombination in rice genome have been detected in the T1 and T2 populations. T-DNA flanking sequences in the transgenic lines are being isolated using TAIL-PCR method and in silico mapping results of the sequences on the rice genome will be presented. Since the novel Ds element also contains 4x CaMV 35S enhancers, our transposon tagging system provides a useful tool to create both knockout and gain-of-function mutants in rice.