Cloning and expression analysis of transketolase gene in Cucumis sativus L.

Abstract

Transketolase (TK, EC 2.2.1.1) is a key enzyme in the photosynthetic carbon reduction cycle (Calvin cycle). A full-length cDNA encoding transketolase (TK, designated as CsTK) was isolated from cucumber leaves (Cucumis sativa L. cv 'Jinyou 3') by RT-PCR and RACE. The cDNA contained 2368 nucleotides with a complete open reading frame (ORF) of 2229 nucleotides, which was predicted to encode a peptide of 742 amino acids. Expression analysis by real-time PCR and western blot revealed that TK mRNA was abundant in cucumber leaves and detectable in stems, fruits and roots. TK activity and the gene expression at the mRNA and protein levels was higher in mid-position leaves (4th apical leaves) than in upper position leaves (1st) and base position leaves (12th). The diurnal variation of CsTK expression and TK activity in the optimal functional leaf (4th leaf) was a single-peak curve, and the peak appeared at 14:00 on a sunny day. Low temperature (5°C) and low light (100μmolm(-2)s(-1)) induced CsTK expression, but the expression level decreased after 24h of chilling stress. Over-expression of CsTK increased the TK activity, mRNA abundance and activities of other main enzymes in Calvin cycle, and net photosynthetic rate (Pn) in transgenic cucumber leaves. Transgenic plants showed a higher ratio of female flower and yield relative to the wild type (WT) plants. The decreases in Pn and carboxylation efficiency (CE) were less in transgenic plants than that in WT during low temperature and low light intensity.