An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function

Abstract

SummaryTo devise a method for function-based gene isolation and characterization in barley, we created a plasmid containing the maize Activator (Ac) transposase (AcTPase) gene and a negative selection gene, codA, and a plasmid containing Dissociation (Ds) inverted-repeat ends surrounding the selectable herbicide resistance gene, bar. These plasmids were used to stably transform barley (Hordeum vulgare). In vitro assays, utilizing a Ds-interrupted uidA reporter gene, were used to demonstrate high-frequency excisions of Ds when the uidA construct was introduced transiently into stably transformed, AcTPaseexpressing plant tissue. Crosses were made between stably transformed plants expressing functional transposase under the transcriptional control of either the putative AcTPase promoter or the promoter and ®rst intron from the maize ubiquitin (Ubi1) gene, and plants containing Ds-Ubi-bar. In F 1 plants from these crosses, low somatic and germinal transposition frequencies were observed; however, in F 2 progeny derived from individual selfed F 1 plants, up to 47% of the plants showed evidence of Ds transposition. Further analyses of F 3 plants showed that approximately 75% of the transposed Ds elements reinserted into linked locations and 25% into unlinked locations. Transposed Ds elements in plants lacking the AcTPase transposase gene could be reactivated by reintroducing the transposase gene through classical genetic crossing, making this system functional for targeted gene tagging and studies of gene function. During the analysis of F 3 plants we observed two mutant phenotypes in which the transposed Ds elements co-segregate with the new phenotype, suggesting the additional utility of such a system for tagging genes.

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

“…The tendency of Ds to transpose to linked sites has also been exploited to disrupt genes present in tandem arrays (Takken et al 1999;Tantikanjana et al 2004) and to perform localized saturation mutagenesis (Nishal et al 2005). Ac/Ds mutagenesis platforms in Arabidopsis (Sundaresan et al 1995;Ito et al 1999;Muskett et al 2003;Kuromori et al 2004), tomato (Healy et al 1993;Knapp et al 1994;Meissner et al 2000), tobacco (Scofield et al 1992), rice (Chin et al 1999;Zheng-Ge et al 2003;Kim et al 2004;Kolesnik et al 2004), and barley (Koprek et al 2000;Cooper et al 2004) have been developed. Yet, despite the demonstrated utility of Ds in gene tagging in maize (Hake et al 1989;Colasanti et al 1998), a two-component Ac/Ds genetagging program for this important crop plant has not yet been developed.…”

Transposon-Mediated Single-Copy Gene Delivery Leads to Increased Transgene Expression Stability in Barley

“…We show that a single Ds-bar element can transpose from a multicopy locus to linked and unlinked sites. In the latter case the element can segregate away from the other copies of the Ds element and from the AcTPase gene (Koprek et al, 2000), physically stabilizing the element in its new location. The resulting single-copy element can be re-activated by providing AcTPase in trans.…”

“…The UbiIbar-nos cassette is flanked by a 254-bp 5Ј sequence and a 340-bp 3Ј sequence from Ds, derived from pDs7 (Wirtz et al, 1997) as a 0.59-kbp SalI-BamHI restriction fragment. The construction of the entire plasmid has been described in detail previously Koprek et al, 2000).…”

The Safety of Genetically Modified Foods Produced through Biotechnology

“…Even if the occasional transfer of resistance from plant to bacterium did occur, the practical impact would be negligible. However, since any increase in antibiotic resistance is recognized as undesirable and the technology is now available to omit the use of such marker genes, future genetically modified organisms are unlikely to contain them (e.g., see Goldsbrough et al, 1996;Koprek et al, 2000). Thus, concerns related to their use are likely to diminish.…”