Molecular cloning and structural analysis of the cucumber (Cucumis sativus L) phosphoenolpyruvate carboxykinase (CsPCK) gene

Genomic sequence of the ATP-dependent phosphoenolpyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of the CsPCK gene. Analysis of a selected positive clone (lambdacscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5'-noncoding region of cucumber PCK cDNA, whereas Exon 2 comprises 12 nucleotides of the 5'-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of the CsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for both Arabidopsis Chromosome 4 (Atg4) PCK and the rice PCK genes, which contain 13 and 12 exons, respectively. Two additional Arabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. The CsPCK gene promoter has conserved plant-specific cis-acting elements within 2 kb of the 5' flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase (ms) gene promoters, identified in the CsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.