Scientia Agricultura Sinica ›› 2011, Vol. 44 ›› Issue (19): 4029-4036.doi: 10.3864/j.issn.0578-1752.2011.19.013

• HORTICULTURE • Previous Articles     Next Articles

Molecular Cloning of Isopentenyl Transferases Genes Family in Malus domestica Borkh. and a Preliminary Functional Analysis of MdIPT5a

 LI  Hao, ZHANG  Wen, ZHAO  Xu-Mian, REN  Xue-Qin, ZHU  Yuan-Di   

  1. 1.中国农业大学果树系
  • Received:2011-04-11 Online:2011-10-01 Published:2011-05-17

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Abstract: 【Objective】The aim of this study was to clone a multigene family encoding isopentenyl transferases in Malus domestica, and analyze the biological functions of MdIPT5a, which would provide a basis for in-depth studies of the functions of MdIPTs in cytokinin biosynthesis and genetic transformation. 【Method】The MdIPTs were isolated from apple cultivar ‘Fuji’ by RACE based on the information of apple genome. The subcellular localization of MdIPT5a was transient expressed in onion epidermal cells by the particle bombardment method and in Arabidopsis mesophyll protoplast by the PEG-calcium transfection. MdIPT5a was transferred into tobaccos (Nicotiana tabacum cv. Wisconsin 38) by Agrobacterium-mediated transformation. Transgenic tobaccos were examined by RT-PCR. 【Result】Ten cDNA sequences of MdIPTs were isolated from apple. Among them seven sequences encoding adenylate isopentenyl transferases that functioned in the major pathway of cytokinin biosynthesis shared a conserved domain of GxxGxGK[S, T] motif at N-teminal, and were located in chromosome No. 13, 16, 3, 11, 13, 16 and 6 respectively. They were designated as MdIPT1a, MdIPT1b, MdIPT3a, MdIPT3b, MdIPT5a, MdIPT5b and MdIPT7a. These genes encoded 284 to 370 amino acids without introns. The MdIPT5a-GFP fusion proteins were located in cytoplasm, but not in plastids. Overexpression of MdIPT5a in tobaccos in vitro induced the phenotypes of hardly rooting with more leaves and adventitious shoots. 【Conclusion】 The phenomenon that genes in pairs performed highly homologous occurred in MdIPTs, which was accordant with the theory that 17 chromosomes in the Pyreae were derived from nine ancestral chromosomes. MdIPT5a exhibited the catalytic function in cytokinin biosynthesis.

Key words: apple, cytokinin, isopentenyltransferase, subcellular localization, genetic transformation

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