Using an Inverse PCR Method to Clone the Wheat Cytokinin Oxidase/Dehydrogenase Gene TaCKX1

The cloning and characterization of a novel gene TaCKX1 that encodes cytokinin oxidase/dehydrogenase (CKX) in wheat is described in this work. Using polymerase chain reaction (PCR) technology and degenerate primers derived from conserved regions of published CKX amino acid sequences, a TaCKX1 gene fragment was generated. The 5' and 3' flanking sequences of the gene was amplified using inverse PCR. Then, the full-length DNA and complementary DNA sequence of TaCKX1 were cloned. Comparison of the novel genes and wheat CKX gene expressed sequence tag (EST) nucleotide sequences shows that the cloned sequences was highly identical with previously named TaCKX1 EST. Comparison of amino acid sequences from maize, rice, barley, Arabidopsis thaliana, and Dendrobium shows that the TaCKX1 active sites are strongly conserved. The signal peptide, the theoretical pI/MW, the subcellular localization, and the function sites were analyzed. The TaCKX1 gene had been located on chromosome 3A using nulli-tetrasomic lines of Chinese Spring. Semi-quantitative reverse transcription polymerase chain reaction analysis showed that the TaCKX1 gene was expressed in root, sheath, and leaf. It could play important roles in wheat growth and development.