Plant & Animal Genomes XVII Conference Plant & Animal Genomes XVII ConferenceJanuary 10-14, 2009
Town & Country Convention Center
San Diego, CA
Joanne G Bartlett , Silvia C Alves , Mark Smedley , John W Snape , Wendy A Harwood
Introns can have a significant effect on gene expression via intron-mediated enhancement. We have examined the effect of introns on transgene expression in barley by producing a large number of transgenic lines containing the luciferase transgene, either without an additional intron, or with a maize intron (RpoT-i4) or an Arabidopsis intron (UBQ10-i1) inserted within the coding sequence. The transgenic lines were produced using a highly efficient method of Agrobacterium-mediated transformation that yielded average transformation efficiencies of 25%. This resulted in the production of over 100 independent luciferase-expressing lines for each of the constructs. Both introns significantly increased the expression of the luciferase gene compared to the control. UBQ10-i1 enhanced luciferase expression to the greatest extent, increasing the accumulation of luc transcripts 9.6 fold in the single-copy primary transformants. Transgene copy number was determined for 265 lines, and an association between copy number and transgene expression was identified. A selection of single-copy lines were followed through to the T1 and T2 generations and patterns of expression through these generations were examined. In addition, transgene insertion was examined in detail in selected lines by the isolation of transgene flanking regions. Sixty-five percent of T-DNA flanking sequences obtained showed homology to known repeat elements. Integration of T-DNA into repetitive DNA did not appear to have an adverse effect on luciferase expression. This study has identified an effective means by which transgene expression can be upregulated in barley and contributed to our understanding of factors affecting transgene express