Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity.
The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors. Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots. The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained. The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region. The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2 homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.
Macknight
R C
RC
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Reynolds
P H
PH
Farnden
K J
KJ
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics*; D018744:DNA, Plant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D007887:Fabaceae / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics*; D018517:Plant Roots / Q000502:physiology; D030821:Plants, Genetically Modified; D010946:Plants, Medicinal; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C045625:nodulin