Content area
Full Text
Mol Biol Rep (2011) 38:39353943 DOI 10.1007/s11033-010-0510-7
Cloning of phytoene desaturase and expression analysisof carotenogenic genes in persimmon (Diospyros kaki L.) fruits
Daqiu Zhao Chunhua Zhou Fen Kong
Jun Tao
Received: 26 August 2010 / Accepted: 13 November 2010 / Published online: 30 November 2010 Springer Science+Business Media B.V. 2010
Abstract Persimmon is a commercially important fruit crop, and the fruit is rich in different kinds of bioactive compounds, among which carotenoids contribute signicantly to its color and nutritional value. In this study, the cDNA of phytoene desaturase gene (PDS) was isolated by rapid amplication of cDNA ends (RACE) technique. Sequence analysis indicated that the full-length cDNA of PDS was 2064 bp, encoding 586 amino acids and containing one open reading frame (ORF) of 1761 bp. Homology analysis showed that DkPDS, which had been submitted in GenBank with accession number GU112527, shared high similarities of 8086% with PDS cloned from other plants. Prediction of deduced proteins showed that there was no signal peptide and transmembrane topological structure in DkPDS. It was a hydrophilic and stable protein, and located in chloroplast. To examine the specic expression patterns of carotenogenic genes we had cloned from persimmon, including phytoene synthase (DkPSY), DkPDS, f-carotene desaturase (DkZDS), lycopene b-cyclase (DkLCYB) and bcarotene hydroxylase (DkBCH), real-time quantitative PCR (Q-PCR) was performed in esh at ve different developmental stages. The results revealed that the expression levels of DkPSY, DkPDS and DkZDS gradually increased. Nevertheless, the expression level of DkLCYB was very low and maintained relatively stable. The expression level of DkBCH was also at a low level from stage 1 to 4, and then reached the maximum at stage 5. In addition, the expression level of DkZDS was higher than that of other genes. Carotenoid detection demonstrated that both b-cryptoxan-thin and total carotenoids increased with fruit development,
and zeaxanthin had little change, but with a sudden increase in nal stage.
Keywords Persimmon Cloning Expression
Phytoene desaturase Carotenoids
AbbreviationsABA Abscisic acidBCH b-Carotene hydroxylase
Ct Threshold cycleDa DaltonFAO Food and Agriculture Organization HPLC High performance liquid chromatography LCYB Lycopene b-cyclase
LCYE Lycopene e-cyclaseORF Open reading framePCR Polymerase chain reactionPDS Phytoene desaturasepI Isoelectric pointPSY Phytoene synthaseQ-PCR Real-time quantitative PCRRACE Rapid amplication of cDNA ends RT-PCR Semi-quantitative reverse transcription polymerase chain reactionUTR Untranslated regionZDS f-Carotene desaturase