Poster: Epigenetics & gene silencing
Abs #
826: High-frequency transgene silencing by hypermethylation of CaMV-35S promoter region in transgenic gentian plants
Transgenic gentian (Gentiana scabra x G. triflora) plants were produced via Agrobacterium-mediated transformation for modifying the floral phenotypes by introducing gentian-derived MADS-box genes. However, most of the resulting transformants exhibited no sign of transgene expression in their leaf tissues despite the use of constitutive CaMV-35S promoter. Interestingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed in these transformants. To investigate the possible factor that caused this transgene silencing, transgenic lines having single locus of transgene insertion were selected and subjected to methylation analysis. Methylation-specific sequence was analyzed after amplification of parts of the two transgene promoter regions from 35S-GtMADS and 35S-bar, by using genomic DNA treated with sodium-bisulfite. As a result, highly methylated cytosine residues of CpG and CpNpG sites were detected in both promoter regions in most lines. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. In the case of tobacco transformed with the same construct, cytosine methylation was rare and high levels of transgene expression were observed, implying that the transgene silencing was specific to gentian transformants. These results indicated that the cytosine methylation in the transgene promoter regions occurred exclusively after the selection of the gentian transformants and caused transcriptional gene silencing.
