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

Transgenic rice (Oryza sativa L.) lines were generated through particle-bom bardment-mediated transformation. Hygromycin phosphotransferase (hpt) was u sed 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 beta-glucuronidase gene (gusA), respectively. Transformants were analyzed to determine possible correlatio n between expression, integrated transgene copy number and/or complexity of integration patterns. We observed that an increase in transgene copy numbe r 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 copi es. In the majority of lines we analyzed, expression patterns of the two un selected genes were similar. In lines where transgene silencing was observe d, this was independent of position effects. In specific cases, silenced tr ansgenes could be reactivated by treatment with 5-azacytidine, suggesting m ethylation of cytosine residues. We report that methylation of cytosines ma y not spread to adjacent regions, hence other transgenes in the vicinity of the silenced transgene remain active. By comparing the structure of transg enic loci With expression patterns of introduced genes, we concluded that t he integrity of integrated transgenes was a major factor in the onset of si lencing. We observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA sp ecies, may be responsible for silencing.