Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing

Abstract

Transgenic rice (Oryza sativa L.) lines were generated through particle-bombardment-mediated transformation. Hygromycin phosphotransferase (hpt) was used as a selectable marker gene on co-integrate plasmids containing either one or two unselected genes, the Bialaphos-resistance gene (bar) coding for phosphinothricin acetyltransferase and the β-glucuronidase gene (gusA), respectively. Transformants were analyzed to determine possible correlation between expression, integrated transgene copy number and/or complexity of integration patterns. We observed that an increase in transgene copy number did not always lead to a concomitant decrease in expression levels or to silencing through co-suppression. Transgenic lines with four to five copies of integrated transgenes expressed the protein product of both unselected genes stably and at levels comparable to transformants with one or two copies. In the majority of lines we analyzed, expression patterns of the two unselected genes were similar. In lines where transgene silencing was observed, this was independent of position effects. In specific cases, silenced transgenes could be reactivated by treatment with 5-azacytidine, suggesting methylation of cytosine residues. We report that methylation of cytosines may not spread to adjacent regions, hence other transgenes in the vicinity of the silenced transgene remain active. By comparing the structure of transgenic loci with expression patterns of introduced genes, we concluded that the integrity of integrated transgenes was a major factor in the onset of silencing. We observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA species, may be responsible for silencing.