籽粒苋AhNAD-ME的序列特征与表达

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。

Sequence Characteristics and Expression of NAD-malic enzyme in Amaranthus hypochondriacus L.

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 photosynthetic 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 illumination 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