A transgene with repeated DNA causes high frequency, post-transcriptional suppression of ACC-oxidase gene expression in tomato

Abstract

Gene silencing with sense genes is an important method for down-regulating the expression of endogenous plant genes, but the frequency of silencing is unpredictable. Fifteen per cent of tomato plants transformed with a 35S-ACC-oxidase ( ACO 1) sense gene had reduced ACC-oxidase activity. However, 96% of plants transformed with an ACC-oxidase sense gene, containing two additional upstream inverted copies of its 5' untranslated region, exhibited reduced ACC-oxidase activity compared to wild-type plants. In the three plants chosen for analysis, there were substantially reduced amounts of both endogenous and transgenic ACO RNA, indicating that this was an example of co-suppression. Ribonuclease protection assays using probes spanning intron-exon borders showed that the reduced accumulation of endogenous ACO mRNA occurred post-transcriptionally since the abundance of unprocessed transcripts was not affected. The ACO1 transgene with the repeated 5'UTR also strongly inhibited the accumulation of RNA from the related ACO 2 gene in flowers, although there is little homology between the 5'UTRs of ACO 1 and ACO 2. These results indicate that although repeated DNA in a transgene greatly enhances the probability of gene silencing of an endogenous gene, it also involves generation of a trans -acting silencing signal produced, at least partly, from sequences external to the repeat.