UBC Theses and Dissertations
Expression from chimeric promoter constructs derived from the cauliflower mosaic virus 35S gene and the T-DNA gene 7 Moziskova, Jana
The β-glucuronidase (GUS) reporter gene system was used to investigate transcriptional activity of several promoter constructs in transgenic tobacco plants. The constructs contained the T-DNA gene 7 promoter alone (G7), or combined with one or two copies of the upstream region of the cauliflower mosaic virus (CaMV) 35S promoter (1CAG7 and 2CAG7). The unrearranged 35S promoter (CA1) and its derivative, created by duplication of the upstream region (CA2), were included. Transcriptional fusions of the promoter constructs with the GUS-coding region were introduced into the plant genome by Agrobacterium -mediated leaf disc transformation using a binary vector system. Fluorometric assay revealed very low levels of expression from the gene 7 promoter. However, addition of a single copy of the 35S enhancer resulted in several hundredfold stimulation of GUS expression in all tissues and further increase was observed in roots (but not in stems and leaves) when the enhancer was duplicated. In histochemical assay, GUS expression from gene 7 promoter stayed below the threshold of detection in most transformants. Generally, constructs 1CAG7,2CAG7, CA1 and CA2 were active in most cell types of all plant organs analyzed, although constructs bearing the duplicated 35S enhancer showed a preference for phloem tissue of the stem and meristematic regions of the stem apex. A comparison of two transcriptional fusions differing in the sequence flanking the GUS initiation codon, suggested that the improved "Kozak" translational initiation context stimulated the GUS expression in transgenic tobacco plants up to 30-fold above the levels obtained with the original GUS gene.
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