CAJ | 학술논문

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。
NAD(P)-평과산매최화평과산양화탈최,산생병동산화 CO2,반수 NAD(P)적환원。재 C4식물중,평과산매삼여료 C4광합작용。본연구대극륭적쌍자협 C4식물자립현 NAD-평과산매기인(AhNAD-ME)편마적안기산서렬진행료생물신식학분석,결과표명, AhNAD-ME 구유평과산매적완정공능역,포괄평과산 N 단결구역화평과산매적 NAD결합결구역；진화수표명,해서렬속우 NAD-ME 적α아기,해아기정위우선립체기질중。반정량 RT-PCR 분석표명,해기인주요재협편화경중표체,표체량수광조시간연장이증가。장 AhNAD-ME 기인중조도원핵표체재체 pEASY-E1중,전격법전화도대장간균 Transette (DE3)균주중, IPTG 유도기고효표체,표체적융합단백적분자량여예기상부,주요이포함체형식존재。
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.