Post‐transcriptional silencing of a neomycin phosphotransferase II transgene correlates with the accumulation of unproductive RNAs and with increased cytosine methylation of 3′ flanking regions

Abstract

The transgenic tobacco plant GVCHS(320)‐1 harbours several T‐DNAs with the neomycin phosphotransferase II (nptll)‐encoding chimeric gene under control of the cauliflower mosaic virus 35S promoter (CaMV 35S). These T‐DNAs are distributed over two loci, named A and B. The primary transformant GVCHS(320)‐1 had substantially reduced steady‐state nptll transcript levels due to the activity of a post‐transcriptional silencing mechanism. Silencing of the nptll‐encoding transgenes in the progeny of GVCHS(320)‐1 requires the presence of the A locus that consists of two physically separated T‐DNAs. Although the B locus contains three T‐DNAs in the same orientation, it gives rise to high steady‐state nptll mRNA levels and NPTII protein levels even in homozygous condition. The B locus only becomes silenced in trans in the presence of a silencing locus. The data suggest that silencing of the nptll transgenes is reinforced by ageing. It is also suggested that silencing is correlated with a reduced NPTII protein/nptll mRNA ratio, which may be interpreted as the accumulation of unproductive nptll RNA molecules in silenced plants. The data further demonstrate that the silenced state is correlated with extensive C‐methylation of diagnostic sites in the 3′ three‐quarters of the coding sequence and in the 3′ region up to 1400 bp downstream of the polyadenylation signal.