inducible targeted tagging system for localized saturation mutagenesis in Arabidopsis

• Published in: Plant physiology (Bethesda) Vol. 137; no. 1; pp. 3 - 12
• Main Authors: Nishal, B, Tantikanjana, T, Sundaresan, V
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Biologists 2005; American Society of Plant Physiologists
• Subjects: Arabidopsis - genetics; Arabidopsis thaliana; beta-glucuronidase; Biological and medical sciences; Breakthrough Technologies; Chromosome Mapping; DNA Transposable Elements - genetics; DNA, Bacterial; Fundamental and applied biological sciences. Psychology; gene expression regulation; Genes; Genes. Genome; Genetic transposition; Genomes; Genomics; Hot Temperature; insertional mutagenesis; literature reviews; Molecular and cellular biology; Molecular genetics; molecular sequence data; Mutagenesis; Mutagenesis, Insertional - methods; nucleotide sequences; Phenotype; Plants; polymerase chain reaction; reporter genes; Seedlings; Seeds; Shock heating; Transformation, Genetic; Transposons; Arabidopsis thaliana; Gene; Cruciferae; Dicotyledones; Angiospermae; Spermatophyta; Gene expression
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.104.050633

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.