Molecular cloning and expression analysis of a cytokinin oxidase (DhCKX) gene in Dendrobium huoshanense

Cytokinin oxidase plays an important role in the cytokinins regulatory processes. A putative cytokinin oxidase gene, Dendrobium huoshanense cytokinin oxidase (DhCKX), was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) of total RNA isolated from shoot apices of D. huoshanense plantlets in the presence of 20 mu mol l(-1) 6-benzylamino-purine (BAP). The full-length cDNA contains an open reading frame (ORF) encoding a putative protein of 537 amino acids with high homology to known CKX genes and with a predicted molecular mass of 60.383 kDa. Compared with the CKX sequences from other species, the DhCKX shows high identity with Dendrobium hybrid Sonia, 92.2% in nucleotide sequence and 89.9% in amino acid sequence, respectively. Based on the alignment of amino acid sequences, the phylogenetic tree of 15 CKX genes is constructed. Semi-quantitative RT-PCR analysis showed that the expression of the DhCKX gene was induced in roots, shoot apices and leaves, but not in stems, after treatment with 20 mu mol l(-1) BAP. The DhCKX gene showed maximum expression in shoot apices at day 12 during continuous treatment with 20 mu mol l(-1) BAP. Furthermore, CKX activity appears to play an important role in controlling the cytokinin levels and growth during shoot morphogenesis in D. huoshanense.