Silencing of an aleurone-specific gene in transgenic rice is caused by a rearranged transgene

In rice, silencing of the aleurone-specific Ltp2-gus transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact Ltp2-gus gene and the other carried a rearranged transgene in which part of the gus gene was in the antisense orientation. Analysis of gus transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non-silenced lines. RNA analyses suggested that the Ltp2-gus silencing in the aleurone layer was post-transcriptional and that it may be caused by interaction of partial antisense gus transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post-transcriptional silencing is discussed.