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

Abstract

Instability of transgene expression in plants is often associated with complex multicopy patterns of transgene integration at the same locus, as well as position effects due to random integration. Based on maize transposable elements Activator(Ac) and Dissociation(Ds), we developed a method to generate large numbers of transgenic barley (Hordeum vulgare var Golden Promise) plants, each carrying a single transgene copy at different locations. Plants expressing Ac transposase (AcTPase) were crossed with plants containing one or more copies of bar, a selectable herbicide (Basta) resistance gene, located between inverted-repeatDs ends (Ds-bar). F1 plants were self-pollinated and the F2 generation was analyzed to identify plants segregating for transposed Ds-barelements. Of Ds-bar transpositions, 25% were in unlinked sites that segregated from vector sequences, otherDs-bar copies, and the AcTPase gene, resulting in numerous single-copy Ds-bar plants carrying the transgene at different locations. Transgene expression in F2 plants with transposed Ds-bar was 100% stable, whereas only 23% of F2 plants carryingDs-bar at the original site expressed the transgene product stably. In F3 and F4 populations, transgene expression in 81.5% of plants from progeny of F2plants with single-copy, transposed Ds-bar remained completely stable. Analysis of the integration site in single-copy plants showed that transposed Ds-bar inserted into single- or low-copy regions of the genome, whereas silencedDs-bar elements at their original location were inserted into redundant or highly repetitive genomic regions. Methylation of the non-transposed transgene and its promoter, as well as a higher condensation of the chromatin around the original integration site, was associated with plants exhibiting transgene silencing.

Molecular Dissection of the Gibberellin/Abscisic Acid Signaling Pathways by Transiently Expressed RNA Interference in Barley Aleurone Cells

“…Currently, barley is not an amenable genetic system for specific gene knockouts, and screens for mutants in specific genes can be very laborious and time consuming. Although transposon tagging and Agrobacterium tumefaciens-mediated transformation have been developed (Horvath et al, 2000;Koprek et al, 2001;Scholz et al, 2001), these methodologies still require a great deal of effort and time.…”

RT-PCR Analysis and Stress Response Capacity of Transgenic gshI-Poplar Clones (Populus × canescens) in Response to Paraquat Exposure

“…The instability of gene expression is also associates with transgene incorporation, as the results of multicopy integration of transgene at the same locus (Assaad et al, 1993;Atkinson et al, 1998), the position effects due to random integration, the AT/CG composition of the transgene (Matzke and Matzke, 1998), the presence of inverted repeats in the integration site (Stam et al, 1997), the overexpression of the transgene (Que et al, 1997), and the environmental conditions (Meyer and Heidman, 1994). All these factors can generate different expression patterns of the gene from an overexpression to the total gene silencing (Koprek et al, 2001).…”

Transposition-Based Plant Transformation

“…Such two-component systems have been introduced into a variety of plant species by transforming plants with T-DNAs carrying both Ds and the Ac-Tpa gene (Hehl and Baker, 1989). As opposed to the unpredictability of T-DNA integration, Ds elements frequently transpose from stably integrated T-DNAs into gene-associated sequences that are adjacent to predicted matrix-associated DNA regions (Meissner et al, 2000;Koprek et al, 2001;Ito et al, 2002;Kim et al, 2004;Pan et al, 2005;Zhao et al, 2006). The site preference for Ds integration has been linked to generally high expression levels of genes positioned within the element (Koprek et al, 2001).…”