苹果果实中细胞质型苹果酸脱氢酶(cyMDH)的克隆与表达分析

【作者】 姚玉新； 李明； 刘志； 翟衡；

【机构】 山东农业大学园艺科学与工程学院； 辽宁省果树科学研究所；

【摘要】 苹果酸脱氢酶(MDH)广泛存在于动、植物和微生物中,主要负责催化草酰乙酸和苹果酸的相互转化。根据亚细胞定位苹果酸脱氢酶可分为五种类型,其中依赖于NAD的细胞质型苹果酸脱氢酶目前在植物上的研究比较少,cyMDH作为苹果酸代谢的关键酶之一, 其转录水平与苹果果实酸度的关系在苹果上目前未有报道。本研究从苹果果实中分离了cyM— DH基因的全长cDNA序列,命名为Mal—cyMDH(基因银行注册号为DQ221207),该序列含有一个996 bp的ORF,并且基因组序列分析表明该基因含有6个内含子。序列同源性分析表明Mal—cyMDH与其他物种cyMDH有较高的同源性。DNA杂交表明cyMDH在苹果基因组中可能存在三个拷贝。RNA杂交表明Mal-cyMDH在苹果的不同组织中呈现不同的转录水平, 其中在根中的转录水平最低;高酸和低酸果实中Mal-cyMDH表现出相同的表达模式,都随着果实的发育转录量逐渐增加直到趋于平稳;高酸和低酸个体之间的转录水平没有明显的差异。更多还原

【Abstract】 Malate dehydrogenase (MDH) ubiquitously exists in plants and catalyzes the in-terconversion from oxaloacetate to malate. Cytosolic NAD-dependent MDH gene (cyMDH) encodes a key enzyme crucial for malic acid synthesis in the cytosol, which has not been extensively characterized in plants. In this study, a full-length cDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3’and 5’ rapid amplification of cDNA ends, and designated as Mal-cyMDH (GeneBank accession No. DQ221207) . It contained a 996 - bp ORF with six introns in its genomic sequence. Sequence analysis showed a high similarity to other plant cyM-DHs. DNA gel-blot analyses suggested three copies of Mal-cyMDH in apple genome. Meanwhile, RNA gel-blot hybridization indicated it differentially expressed in tissues tested, with rather lower expression level in roots than in leaves, stems and fruits. An extremely low-acid hybrid and another high-acid one were chosen from offspring of cross ’Toko’ × ’ Fuji’ to investigate its expression during the period of fruit development. Its expression patterns were highly similar to each other in 2 hybrids. The transcript level was much higher in ripening fruits than in young fruits. However, the expression difference between 2 hybrids was inconsistent with the fact that malic acid content was much higher in high-acid genotype than in low-acid genotype at the same stage.更多还原