苹果异戊烯基转移酶基因家族（MdIPTs）的克隆与MdIPT5a功能分析

 【目的】克隆苹果（Malus domestica Borkh.）异戊烯基转移酶（isopentenyltransferase，IPT）基因家族MdIPTs，分析MdIPT5a的生理功能，为深入研究MdIPTs在苹果细胞分裂素生物合成途径中的作用和基因的遗传转化提供理论依据。【方法】以‘富士’苹果为试材，利用RACE和苹果基因组信息克隆MdIPTs；利用洋葱表皮和拟南芥原生质体的瞬时表达研究MdIPT5a的亚细胞定位；通过农杆菌介导法遗传转化‘W38’烟草（Nicotiana tabacum cv. Wisconsin 38）过量表达MdIPT5a，RT-PCR鉴定转基因烟草。【结果】克隆了10个MdIPTs的cDNA序列，其中7个编码细胞分裂素生物合成主要途径中的腺苷-IPTs，具有N端的保守结构域GxxGxGKS,T]序列，分别位于苹果第13、16、3、11、13、16、6号染色体上，命名为MdIPT1a、MdIPT1b、MdIPT3a、MdIPT3b、MdIPT5a、MdIPT5b和MdIPT7a，均无内含子，编码284—370个氨基酸。MdIPT5a-GFP融合蛋白定位于细胞质中，但不定位于质体内。过量表达MdIPT5a的转基因烟草组培苗生根困难、叶片和不定芽增多。【结论】苹果中腺苷-异戊烯基转移酶基因具有成对高度同源现象，与苹果17条染色体起源于9条始祖染色体的同源起源学说相一致，MdIPT5a具有催化合成细胞分裂素的功能。

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

【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 GxxGxGKS, 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.