Examination of the Brassica napus β-Keto-acyl carrier protein reductase promoter for regulatory cis-acting elements
Major interest has focused on the identification of regulatory factors involved in lipid biosynthesis. This study examined the B.napus β-Keto-ACP reductase 5' sequence for potential regulatory cis-acting elements. The 5' sequence of the most highly expressed Brassica napus β-Keto-ACP reductase isoform was fused to the reporter gene β-glucuronidase (GUS) and its expression pattern examined within transgenic Arabidopsis. The construct was shown to act as a functional promoter and direct transcription within embryos, cotyledons and roots. There was no apparent staining within the true leaves, but staining was visible within the cotyledons. Overlapping fragments of the promoter were analysed in gel mobility shift assays and all six showed the formation of protein-DNA complexes. Competition analysis suggested that the same trans-acting factor binds to a number of regions along the promoter. The protein-DNA complex appeared to be competed away by the Arabidopsis enoyl-ACP reductase (EnR) promoter sequence, but not the lipid transfer protein (LTP) promoter. A common 9bp cis-element (CGCANTAAA) was identified in four of the six promoter fragments. Deletion analysis of the β-Keto-ACP reductase promoter intransient expression experiments into B.napus tissue, suggested the promoter could still direct transcription upon deletion to 132bp within embryos. The GUS expression appeared to show more than one decrease in expression upon subsequent deletions of the promoter within embryos, suggesting that more than one cis-element may be involved in the control of transcription. At least one of these suspected decreases con elated with the deletion of one of the 9bp boxes identified. Differences were observed for expression of the constructs within leaves and embryos suggesting that different elements may be involved in transcriptional control within these tissues. The identification of a potential cis-acting element within this study could be used to isolate a potential regulatory trans-acting factor that binds to the B.napus β-Keto-ACP reductase promoter.