Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2003
Publication Date: 10/1/2003
Citation: Ling, M., Shibo, Z., Bregitzer, P.P., Peggy, L.G. 2003. Methylation of 5' untranslated exon and intron in ubil promoter complex is correlated with transcriptional transgene silencing in barley. Plant Molecular Biology. Interpretive Summary: Genetic biotechnology promises the development of barley plants with enhanced abilities to resist pests and environmental stress, and to produce food with better nutritional qualities. The procedures used to genetically engineer plants, however, require improvement. One problem is that transgenic plants often do not express the introduced transgenes, and thus cannot be used as sources of enhanced or unique characteristics. Gaining an understanding of the processes that are responsible for silencing transgenes will likely lead to better transformation methodologies that result in stable transgene expression, and also should contribute to a better understanding of how complex genetic systems function to control gene expression. In this study, two transgenic plants were identified that were selected from the same group of progeny plants; all plants within this group were derived ultimately from a single transgenic parent and contained the same transgene, which was expressed. However, from one generation to another, the transgene in one of these progeny plants was silenced, while in the other plant the transgene continued to be expressed. These two plants, and the progeny derived from them, are interesting subjects for study because, except for the silenced state of the transgene, they are are virtually identical. Thus, molecular differences between them are likely to be involved in the silencing phenomenon. Studies of the transgene in these two groups of plants showed that the silenced transgene was associated with several genetic changes that did not occur in the case of the expressed transgene. The control region of the silenced transgene was methylated, meaning that particular chemical groups were added to this particular region of the DNA. An association of transgene silencing with methylation has been noted in other studies, although the precise patterns of this methylation event differed slightly from that noted in some these other studies. The data from this study, in combination with data produced in other studies, are beginning to help us understand the precise molecular mechanisms that contribute to transgene silencing.
Technical Abstract: DNA methylation of the promoter is often associated with transcriptional silencing of transgenes. Here we report a case in which methylation of the first untranslated exon and intron in the maize ubiquitin promoter complex, but not of the promoter region itself, is correlated with differential transcriptional transgene silencing rates in T6 barley plants derived from a single transgenic barley line. Two sibling sublines were selected at random from the transgenic parental line GP724B-4-9 at the T3 generation and designated as T3-derived progeny sublines, T3#30 and T3#31. Subsequently it was discovered that expression of the ubiquitin-driven transgenes, bar and uidA, was stable in subline T3#30 but unstable in subline T3#31. No differences were detected between the two sibling sublines in terms of the nature of integration locus or its structure, based on fluorescence in situ hybridization and DNA hybridization analysis. However, differences were observed in the expression of the transgenes; all T6 progeny of subline T3#30 expressed bar and uidA, but both transgenes were silenced in T6 progeny of subline T3#31. Molecular analyses demonstrated that this silencing closely correlated with two events, methylation of the untranslated exon and intron of the promoter complex, but not the promoter per se, and the formation of compact heterochromatin in the region containing the transgenes.