Structure, genetic mapping, and expression of the gene for pyruvate, orthophosphate dikinase from maize

Pyruvate, orthophosphate dikinase is a key enzyme in photosynthesis in some plants that exploit the C4 photosynthetic pathway for the fixation of CO2. The gene for this enzyme has been cloned and its primary structure has been analyzed. The sequence of the cloned genes spans about 12 kilobase pairs and consists of 19 exons. The site of initiation of transcription is located 211 nucleotides upstream from the first nucleotide of the initiation codon (position -211). Typical TATA and inverted CCAAT elements are present in the anticipated regions, as well as a sequence that is homologous to the binding site of Sp-1 protein (-51 to -42). Three long, direct-repeated sequences are present contiguously in the 5'-flanking region (-286 to -172), and two of the repeated sequences include sequences homologous to the core sequence of SV40 enhancer in the 3'-end portions. Southern blot analysis, coupled with mapping by analysis of restriction fragment length polymorphism, indicates that the gene for the enzyme involved in C4 photosynthesis is encoded by a single-copy gene and is located on chromosome 6. To test the promoter activity of the isolated gene, a chimeric gene composed of the 5'-flanking region of the gene and a structural gene for bacterial beta-glucuronidase was constructed and introduced into tobacco protoplasts by electroporation or used to transform tobacco plants by Agrobacterium-mediated transfer of the gene. In both cases, expression of the beta-glucuronidase gene was observed, indicating that the 5'-flanking region of the gene can act as a promoter in tobacco cells.