Molecular cloning and expression analysis of the gene encoding sedoheptulose-1, 7-bisphosphatase from Cucumis sativus

Sedoheptulose-1, 7-bisphosphatase (SBPase; EC 3.1.3.37) is a key enzyme in the photosynthetic carbon reduction cycle (Calvin cycle). In the present study, a full-length cDNA encoding SBPase (designated as CsSBP, GenBank accession no. FJ911553) was isolated from cucumber (Cucumis sativus L. cv. ‘Jinyou 3’) grown in solar-greenhouse by RT-PCR and RACE. The cDNA contained 1452 nucleotides with a complete open reading frame (ORF) of 1167 nucleotides, which was deduced for encoding a peptide of 388 amino acids whose molecular mass was inferred to be 42 kDa with its isoelectric point at 6.17. Sequence comparison analysis showed that the deduced amino acid of SBPase from cucumber leaves had high homology to that from other species. Expression analysis by real-time PCR and Western blot revealed that CsSBP mRNA and protein was most abundant in leaves, detectable in stems and fruits, but invisible in roots. A similar difference was found in SBPase activity among leaves, stems, fruits and roots. CsSBP expression at the mRNA and protein level was the highest in mid-position leaves (4th–8th apical expanded leaves) of plants, medium in base position leaves (12th), and the lowest in upper position leaves (1st). However, SBPase activity decreased successively as leaf position declined. The net photosynthetic rate (Pn) of mid-position leaves was also found to be the highest, but that of base-position leaves was lower than upper-position leaves. The diurnal variations of gene expression and SBPase activity in optimal functional leaf (6th leaf) was a single-peak pattern curve, which was similar to that of Pn, and the peak appeared at 12:00. These data indicate that CsSBP expresses only in green tissues of cucumber plants and there are close positive correlations between SBPase activity and gene expression at the mRNA and protein levels, and between SBPase activity and Pn in leaves of cucumber plants.