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BackgroundRNA interference (RNAi), a natural regulatory mechanism involved in gene regulation, transgene silencing and virus defense in plants, has been widely used for improving the quality of crops and for conferring resistance to plant pests and pathogens. For the sustained application of RNAi-based genetically modified (GM) crops in agriculture, stable expression of the silencing-inducing hairpin transgene is important. As RNA interference may be accompanied by RNA-directed methylation of homologous DNA, including the RNAi transgene, gene body methylation may spread into the promoter and eventually lead to progressive transcriptional silencing of the RNAi inducing transgene. This would be detrimental for the effective use of RNAi crops and may have safety implications if food allergens or antinutrients are the target. Methods and ResultsIn order to gain knowledge about the molecular structure of transgenes sensitive to gene silencing as well as about the early detection of unstable plant lines, an unstable GM tobacco line was analyzed with respect to the integration structure and methylation patterns. This plant line was selected because it lost reporter gene expression in progeny plants due to post-transcriptional gene silencing (PTGS) and was subject to transcriptional gene silencing (TGS) during prolonged tissue culture propagation of the primary transformant. It was found to contain an inverted repeat integration with transgene constructs separated by the bacterial transposon Tn5393 and to exist in two epigenetic states with respect to transgene methylation.ConclusionsInverted repeat integration containing bacterial transposon sequences may be a possible molecular trigger for the observed silencing of transgene expression in this unstable GM line. |
