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以苹果砧木SH40[(Malus×domestica)×M.honanensis]为试材,采用RT-PCR结合RACE技术从SH40茎尖中克隆得到一个赤霉素合成关键酶基因(GA20–氧化酶基因),命名为MdGA20ox1,其cDNA全长1 579 bp,编码392个氨基酸。该基因在GenBank登录号为KC493633。氨基酸序列同源性分析表明:MdGA20ox1与玉米、拟南芥、梨等植物GA20ox具有55.9%~96.7%的相似性。洋葱表皮细胞瞬时表达显示,MdGA20ox1蛋白定位于细胞核与细胞质膜。植株生长量测定和相对定量表达结果表明,MdGA20ox1基因在砧木SH28、SH40和M26自根苗的表达呈先下降再上升然后下降的趋势,这与其生长动态基本一致。嘎啦/SH28的植株生长量和MdGA20ox1表达量最高,嘎啦/M26次之,嘎啦/SH40最低,初步表明该基因的表达有与苹果植株矮化程度呈负相关的趋势。MdGA20ox1在嘎啦/SH40和嘎啦/SH28不同组织中的表达模式一致,且半矮化类型砧木SH28嫁接‘嘎啦’,其茎尖、幼叶、成熟叶和枝皮中的表达量均高于矮化类型SH40嫁接‘嘎啦’。
A key gene for gibberellin synthesis (GA20-oxidase gene) was cloned from SH40 shoot tips by RT-PCR and RACE techniques using SH40 [(Malus × domestica) × M.honanensis] MdGA20ox1, its full-length cDNA is 1 579 bp, encoding 392 amino acids. The gene in GenBank accession number KC493633. Amino acid sequence homology analysis showed that MdGA20ox1 has 55.9% -96.7% similarity to GA20ox in plants such as corn, Arabidopsis thaliana and pear. Transient expression of onion epidermal cells showed that MdGA20ox1 protein localized in the nucleus and the plasma membrane. The results of plant growth and relative quantitative expression showed that the expression of MdGA20ox1 gene in rootstock SH28, SH40 and M26 rootstock firstly descended and then increased and then decreased, which is consistent with its growth dynamics. Plant growth and MdGA20ox1 expression were highest in Gala / SH28 plants, followed by Gala / M26 and lowest in Gala / SH40, suggesting that the expression of this gene was negatively correlated with the dwarfing degree of apple plants. The expression patterns of MdGA20ox1 in different tissues of Gaala / SH40 and Gaala / SH28 were the same, and the expression level of SH28 in the semi-dwarfing rootstock SH28 was higher than that in the shoot tip, young leaf, mature leaf and shoots Dwarf type SH40 grafted ’Gala’.