Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

摘要

The complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa) that displayed aberrant segregation in R1 progeny was studied in herbicide (bialaphos [bilanafos])-sensitive and herbicide-resistant plants. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.