Gene silencing and its reactivation in transgenic rice

摘要

Genome surveillance systems of higher organisms protect not only against intragenomic parasites such as retroposons and transposons but also against invasive DNA introduced via genome transformation, resulting in (trans)gene silencing at both the transcriptional and posttranscriptional levels. Transcriptional gene silencing of both bialaphos resistance and Btt CryIIIA crystal toxin genes was encountered in experiments targeted to provide resistance to the rice water weevil. Extensive methylation ofthe transgenes, especially of the promoter elements, was evident. Germination of seedlings from silenced lines in the presence of 5-azacytidine (which prevents cytosine methylation) led to the reactivation of bialaphos resistance in progeny of silencedplants. The CaMV 35S promoter has been implicated in many instances of silencing and the possibility that some promoters are especially prone to silencing is supported by the finding that RCg2, a rice root-specific promoter, is silenced in more than 80 percent of transformants. Genes flanking RCg2/uidA are rarely silenced, showing that silencing can be highly targeted. Multicopy insertions are very susceptible to silencing and epistatic interactions between multicopy and single-copy inserts are documented. Several strategies to alleviate silencing have been considered, such as sequence diversification and flanking transgene inserts with matrix attachment regions.