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, D endrobium h uoshanense 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 micromol 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 micromol l(-1) BAP. The DhCKX gene showed maximum expression in shoot apices at day 12 during continuous treatment with 20 micromol 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.
Wang
Ying
Y
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei, 230009, People's Republic of China.
Luo
Jian-Ping
JP
Wei
Zhao-Jun
ZJ
Zhang
Jing-Cheng
JC
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D031667:Dendrobium / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000032:analysis / Q000235:genetics*; D010802:Phylogeny; D018514:Plant Structures / Q000201:enzymology / Q000254:growth & development; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment
D018076:DNA, Complementary; D010088:Oxidoreductases; C019984:cytokinin oxidase