Gallardo F, Miginiac-Maslow M, Sangwan R S, Decottignies P, Keryer E, Dubois F, Bismuth E, Galvez S, Sangwan-Norreel B, Gadal P
Institut de Biotechnologie des Plantes, Unité de Recherche Associée CNRS D 1128, Université Paris-Sud, Orsay, France.
Planta. 1995;197(2):324-32. doi: 10.1007/BF00202654.
Chloroplastic NADP(+)-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbon-fixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic protein-import machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of L-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.
叶绿体NADP(+)-苹果酸脱氢酶（cpMDH，EC 1.1.1.82）是一些C4植物如单子叶植物玉米或高粱碳固定途径中的关键酶。我们通过使用由花椰菜花叶病毒35S启动子控制的高粱cpMDH cDNA组成的基因，在转基因烟草（烟草）（双子叶C3植物）中表达了来自栽培高粱的cpMDH。在同基因双单倍体转基因烟草品系中观察到cpMDH mRNA的高稳态水平。在转基因叶片提取物中检测到高粱cpMDH蛋白，与对照烟草叶片相比，其cpMDH活性可测量到高三倍。重组蛋白的分子量和N端序列与高粱cpMDH相同。在转基因植物中未检测到具有不同N端序列的烟草cpMDH蛋白。免疫细胞化学分析表明，高粱cpMDH特异性定位于转基因烟草叶绿体中。这些数据表明，高粱cpMDH前体蛋白在烟草叶绿体中被有效合成、转运和加工。因此，C3-C4光合作用特化或单子叶-双子叶进化并未影响叶绿体蛋白导入机制。转基因叶片中较高水平的cpMDH导致L-苹果酸含量增加，表明高粱酶的表达改变了碳代谢。
