Structural analysis of transgene rearrangements and effects on expression in transgenic maize plants generated by particle bombardment.

Abstract

We cotransformed maize embryogenic callus with two plasmids, one carrying the linked markers bar and gusA, and the other carrying the Bacillus thuringiensis cry1Ab gene. Molecular analysis of regenerated transgenic plants revealed a diverse range of transgene structures, including intact, truncated and internally-rearranged copies, and copies interspersed with genomic DNA. We found rearrangements associated with both functional and nonfunctional transgenes. We also found that rearrangements could affect the selected marker gene bar as well as the nonselected transgenes. Rearrangements often caused loss of transgene expression, but some rearranged transgenes were expressed normally. Conversely, while intact transgenes were often expressed, we also detected some nonexpressed copies, probably reflecting epigenetic silencing mechanisms. All the transgenic lines we studied contained multiple transgenes, generally comprising a mixed array of intact and rearranged copies. Rearrangement is rarely given serious consideration as a factor contributing to transgene silencing and variable transgene expression, but our data show that undetected rearrangements may have a major effect on transgene expression. This has significant implications, especially in studies attempting to correlate transgene copy number and expression levels.