Complex structures of transgene rearrangement implicate novel mechanisms of RNA-directed DNA methylation and convergent transcription

Abstract

An RNAi construct for silencing FtsZ gene functioning on plastid division in higher plants was transformed into tobacco genome. Sequencing of the flanking sequences of the T-DNA insertion site in a positive transgenic plant 2-3, which showed wildtype phenotype, revealed that the construct had rearranged, resulting in convergent transcription of nptII gene by 35S promoter and nos promoter with both transcripts expressing at low level. Although it is possible that the sense and antisense transcripts can form dsRNA, they did not trigger silencing of nptII gene derived from a FtsZ silenced plant 2-12 in the crossed plant 2-3×2-12. Furthermore, although small interfering RNA can trigger DNA methylation of the silencing locus, our investigation revealed that the crossed plant 2-3×2-12 showed partial methylation of the non-silencing transgene locus but full methylation of the non-rearranged silencing locus, suggesting that the efficiency of RNA-directed DNA methylation is associated with its original DNA locus.