Regulatory Mechanism by Nitrogen of Gene Expression of Enzymes Involved in Photosynthesis and Nitrogen-Assimilation Pathways

本课题的研究结果总结如下：通过对近最佳光照和温度条件下生长的缺氮植株补氮过程中，氮在过渡状态下分配到rubisCO、PEP羧化酶和丙酮酸、Pi二激酶，研究了氮对玉米叶片碳同化酶基因表达的调控作用。在发育完全的最年轻叶片中，这三种酶在蛋白质和mRNA的积累上存在差异。在此基础上，认为PEP羧化酶是玉米叶片光合作用最受限制的酶。有证据表明，氮是光刺激这三种酶的mRNA和蛋白质积累所必需的调节因子。从C_4植物PEP羧激酶型Panicum maximum trichoglume的叶肉细胞中纯化出了天冬氨酸转氨酶。对该酶进行了部分表征，并制备了其特异性多克隆抗体。利用该抗体，从C_4植物NAD-苹果酸酶型Eleusine coracana mrna的DNA文库中制备了该酶的互补cDNA。同时制备了Gln合成酶和GOGAT抗体。通过对玉米叶片PEP羧化酶光/暗失活和光活化形式的分析，研究了其光/暗调控，并提出了其可能的机制。

The results obtained in this project were summarized as follows.1. The regulation of gene expression by nitrogen of carbon assimilating enzymes in maize leaf was studied by examining partitioning of nitrogen into rubisCO, PEP carboxylase, and pyruvate, Pi dikinase in transitional state during supplementing nitrogen to nitrogen deficient plant seedlings grown under near optimal light and temperature conditions. In a fully developed youngest leaf a differential accumulation of protein and mRNA was observed among these three enzymes. Based upon the data PEP carboxylase was judged as a most limiting enzyme to photosynthesis in maize leaf. An evidence was also provided to suggest that nitrogen is a regulatory factor essential for the light-stimulation of accumulation of mRNA and protein for these three enzymes.2. Aspartate aminotransferase was purified from the mesophyll cells of Panicum maximum trichoglume , a PEP carboxykinase type of C_4 plant. Partial characterization of the enzyme which appears to involve photosynthetic carbon assimilation was achieved and its specific polyclonal antibody was prepared. Using this antibody the complementary cDNA of this enzyme was prepared from DNA library of mRNAs derived from Eleusine coracana, a NAD- malic enzyme type of C_4 plant. Also, antibodies against Gln synthetase and GOGAT were prepared.3. Light/dark regulation of maize leaf PEP carboxylase was studies and its possible mechanism was proposed based upon analysis of dark-inactivated and light-activated forms.