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NAD-malic enzyme(NAD-ME)是植物调控苹果酸代谢中的关键酶。以富士苹果(Malus×domestica Borkh.)叶片为试材,通过同源比对和RT-PCR技术,克隆获得2个NAD–苹果酸酶基因(MdNAD-ME1和MdNAD-ME2)。其ORF分别为1 785 bp和1 818 bp,推测其分别编码595和605个氨基酸的多肽。氨基酸序列和结构分析显示,含有5个保守的氨基酸区域(Ⅰ~Ⅴ),包含2个功能结构域:malic和NAD_bind_1_malic_enz。进化树分析结果显示,MdNAD-ME1属于α组双子叶亚组,MdNAD-ME2属于β组双子叶亚组。荧光实时定量结果显示,MdNAD-ME在被检测的组织中呈组成型表达,且在多种组织中MdNAD-ME1表达量高于MdNAD-ME2。在富士苹果果实不同发育阶段,MdNAD-ME1与MdNAD-ME2具有不同的表达模式。以上结果表明,克隆得到的MdNAD-ME1和MdNAD-ME2属于植物NAD-ME,在苹果的生长和发育过程中MdNAD-ME1和MdNAD-ME2可能起到不同作用。
NAD-malic enzyme (NAD-ME) is a key enzyme in plant regulation of malate metabolism. Two NAD-malic enzyme genes (MdNAD-ME1 and MdNAD-ME2) were cloned by homologous comparison and RT-PCR using leaves of Malus × domestica Borkh. The ORFs were 1 785 bp and 1 818 bp, respectively, suggesting that they encoded 595 and 605 amino acids of the polypeptide respectively. Amino acid sequence and structural analysis showed that there are five conserved amino acid regions (Ⅰ ~ Ⅴ), including two functional domains: malic and NAD_bind_1_malic_enz. Phylogenetic tree analysis showed that MdNAD-ME1 belonged to the α cotyledon subgroup and MdNAD-ME2 belonged to the β cotyledon cotyledon subgroup. Real-time fluorescence quantitative analysis showed that MdNAD-ME was constitutively expressed in the tested tissues, and MdNAD-ME1 was higher than MdNAD-ME2 in various tissues. At different developmental stages of Fuji apple fruit, MdNAD-ME1 and MdNAD-ME2 have different expression patterns. The above results showed that the cloned MdNAD-ME1 and MdNAD-ME2 belong to the plant NAD-ME, and MdNAD-ME1 and MdNAD-ME2 may play different roles in the growth and development of apple.