T-DNA tagging of a seed coat-specific cryptic promoter in tobacco.

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.