Abstract:

Fructokinase (FRK) is of primary importance in phosphorylation of fructose in plants. Two genomic DNA fragments encoding putative fructokinase gene were isolated from Citrus unshiu Marc. using PCR, which were named Cufrk1 and Cufrk2. A cDNA sequence same to the exons’ sequences of Cufrk1 was amplified by RT-PCR and the full-length cDNA sequences of this gene was isolated using rapid amplifi-cation of cDNA ends (RACE) from mature fruit, named CuFRK1 (GenBank number: AY561840). Sequenc-ing analysis showed that the amino acid sequences were 68% identical between Cufrk1 and Cufrk2. The full-length cDNA of CuFRK1 was 1 459 bp in length and contained a complete ORF from 168 to 1 220 bp, encoding 350 amino acids with a calculated molecular weight of 37.5 kD and a predicted pI of 5.03. The deduced amino acids of CuFRK1 possessed two sugar-binding domains, three ATP-binding domains and were 62% to 78% identical to the previously characterized fructokinase genes of other plants. Northern analysis showed that transcripts of CuFRK1 (Cufrk1) and Cufrk2 were detected at a high level in leaves, fruit at early developmental stages, but undetectable in peels and stems. Their expression patterns were distinct in petals and mature fruit. Enzymatic analysis showed that the activity of citrus fructokinase was decreasing with the fruit development, coincided with the accumulation of fructose in fruit. There was a significantly negative correlation between the decreasing fructokinase activity and the increasing fructose content during fruit development.

Key words: citrus, fructokinase, gene cloning, expression analysis, fructose accumulation