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Cytokinin is a sort of adenine derivatives that can promote cell division and differentiation, which is widely involved in plant organ morphogenesis and stress response. Cytokinin hydroxylase CYP735A belongs to the cytochrome P450 monooxygenase superfamily. It regulates the content of isoprenyl adenine and trans-zeatin in plants through hydroxylation reaction and plays an important role in plant growth and development. Up to now, there were few researches about gene cloning and analysis of cytokinin hydroxylase in woody plants. In this study, one CYP735A homologous gene was firstly cloned and identified in Pinus massoniana through the rapid amplification of cDNA ends(RACE). The results were as follows: The full-length of cDNA of PmCYP735A was 1 744 nucleotides, including 1 647 bp open reading frame(ORF), 44 bp 5'-untranslated region(UTR)and 53 bp 3'-UTR. PmCYP735A encoded a protein of 548 amino acids, and its secondary structure was rich in alpha helix and random coil. PmCYP735A protein did not contain either transmembrane region or signal peptidase cleavage site. But two conserved motifs of P450 superfamily such as ETLRLYP(ExxRxxP)and heme-binding region FSFGPRKCVG(FxxGxRxCxG)were existed in amino acid sequence of 399-406 aa and 475-484 aa, respectively. The phylogenetic tree showed that PmCYP735A was clustered into the same evolutionary branch with homologous of Arabidopsis thaliana, Oryza sativa and Zea mays, while CYP735A homologous proteins from Theobroma cacao, Populus trichocarpa and Jatropha curcas were classified in another evolutionary branch. The results of qRT-PCR showed that PmCYP735A expressed significantly higher in roots and stems than in leaves. Furthermore, the gene responded to exogenous auxin NAA with a increased firstly and then decreased gene expression trend over treatment time in all three tissues. These results will be valuable for further study of the biological function of CYP735A homologous genes in woody plants and enrich the excellent genetic resources of Pinus massoniana. [ABSTRACT FROM AUTHOR]