Functional mapping of pea legumin upstream regulatory elements using TI plasmid vectors

Abstract

The leg A gene from Pisum sativum L. has been extensively characterised and a distinct pattern of developmental and organ-specific gene expression demonstrated. Homology between legumin genes from other species has given some indication of those sequences which may be responsible for the regulation at the level of transcription. This study was designed to provide a functional analysis of the upstream sequences. A number of plasmid vectors containing a maximum of 1.2 kb of upstream sequence from the leg A gene of Pisum sativum I., ligated to the coding region of the nopaline synthase (nos) gene, were constructed. The use of smaller promoter fragments and the insertion of spacer DNA within the promoter region was employed in an effort to localise the regions of 5' flanking sequence which may play a role in tissue specific expression. In a minority of tumours derived from tissue transformed with the vector containing the ' full-length ' leg A promoter, low levels of nopaline were detected, but not with those containing a shorter promoter fragment. Results from the analysis of Seed tissue indicates that 800 bp of the leg A promoter was insufficient to direct tissue-specific expression of the fused nopaline synthase gene in transgenic Nicotiana tabacum, although one individual plant showed a constitutive pattern of nopaline synthesis. However, published results obtained with legumin and other storage protein gene promoters would suggest that this promoter fragment should have been sufficient to confer seed-specific expression. This suggests that there may have been undesirable secondary structures, or small undetected rearrangements, introduced during the construction of the transcriptional fusions between leg A and nos. Alternatively the marker gene may be inadequately sensitive to permit detection of low levels of expression.