籽粒苋 AhNAD-ME 的序列特征与表达

摘要

NAD(P)-苹果酸酶催化苹果酸氧化脱羧,产生丙酮酸和 CO2,伴随 NAD(P)的还原。在 C4植物中,苹果酸酶参与了 C4光合作用。本研究对克隆的双子叶 C4植物籽粒苋 NAD-苹果酸酶基因(AhNAD-ME)编码的氨基酸序列进行了生物信息学分析,结果表明, AhNAD-ME 具有苹果酸酶的完整功能域,包括苹果酸 N 端结构域和苹果酸酶的 NAD结合结构域；进化树表明,该序列属于 NAD-ME 的α亚基,该亚基定位于线粒体基质中。半定量 RT-PCR 分析表明,该基因主要在叶片和茎中表达,表达量随光照时间延长而增加。将 AhNAD-ME 基因重组到原核表达载体 pEASY-E1中,电击法转化到大肠杆菌 Transette (DE3)菌株中, IPTG 诱导其高效表达,表达的融合蛋白的分子量与预期相符,主要以包涵体形式存在。%The NAD(P)-malic enzyme (NAD(P)-ME) found in many metabolic pathways catalyzes the oxidative decarboxylation of L-malate, which results in producing pyruvate, CO2 and NAD(P)H. In C4 plants, NAD(P)-ME plays a key role in photosyn-thetic carbon fixation. This study was aimed to characterize the AhNAD-ME in dicotyledonous C4 Amaranthus hypochondriacus by sequence analysis, examine the expression patterns of AhNAD-ME gene in different tissues and different durations of illumina-tion time, and construct a recombinant plasmid pEASY-E1 harboring the AhNAD-ME cDNA and then transform the plasmid into E. coli Transette (DE3) for prokaryotic expression after IPTG induction. The result showed that AhNAD-ME contains all of the motifs required for a complete and functional malic enzyme and is localized specifically to the mitochondrial matrix. Semi-quantitative RT-PCR results showed that AhNAD-ME was constitutively expressed in all examined tissues, with different expression levels, and strongly up-regulated under light in the leaf and stem. Results of SDS-PAGE demonstrated that the specific fusion protein with an expected molecular weight was successfully expressed in E. coli transette (DE3) induced by IPTG.