A strong constitutive positive element is essential for the ammonium-regulated expression of a soybean gene encoding cytosolic glutamine synthetase.
In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.
Tercé-Laforgue
T
T
Laboratoire du Métabolisme et de la Nutrition des Plantes, INRA, Centre de Versailles, France.
Carrayol
E
E
Cren
M
M
Desbrosses
G
G
Hecht
V
V
Hirel
B
B
D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D012045:Regulatory Sequences, Nucleic Acid; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013025:Glycine max / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*
D018744:DNA, Plant; D000644:Quaternary Ammonium Compounds; D005974:Glutamate-Ammonia Ligase