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PEPC-specific protein kinase : Evaluation of physiological role in C4 photosynthesis and molecular mechanism of regulation

PEPC特异性蛋白激酶：评估C4光合作用的生理作用和调节的分子机制

基本信息

批准号：
14390030
负责人：
IZUI Katsura
金额：
$ 8.38万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Phosphoenolpyruvate carboxylase (PEPC) which catalyses an initial C0_2 fixation reaction in the C4 photosynthetic pathway, is regulated not only by allosteric effect but also by reversible phosphorylation. For this phosphorylation, a protein kinase whose substrate is confined to PEPC is involved. Thus this protein kinase is called PEPC kinase (PEPCk). We firstly had succeeded in the cloning of PEPCk involved in the C4 photosynthesis from a model C4 plant, Flaveria trinervia, and in its prokaryotic expression. By the use of this PEPCk (FtPEPCk) cDNA, we investigated the regulatory mechanisms of this kinase and evaluated its physiological significance. The results obtained are as described below.1)At least three copies of the PEPCk genes were found on the genome of Flaveria trinervia, and one of the genes was assigned to be involved in the C4 photosynthesis. The gene was expressed not only in leaves but also in other tissues at low levels.2)The recombinant FtPEPCk could successfully be obtained as a fusion protein with Nus tag protein. Similar to the case with PEPCk from maize (ZmPEPCk), FtPEPCk was also found to be subject to redox regulation. The candidate Cys residues participating in the disulfide bond formation upon oxidation were assigned.3)Several amino acid residues of PEPC required for the activation by phosphorylation were identified.4)By the use of protoplasts of mesophyll cells of maize, FtPEPCk with FLAG tag was transiently expressed and its degradation process was monitored. The Involvement of the ubiquitin/proteasome system in the degradation of FtPEPCk was demonstrated.5)To evaluate the physiological importance of PEPCk in vivo, PEPCk expression was suppressed by the use of an antisense method with Flaveria bidentis which is a transformable C4 plant. The transformants whose PEPC is not significantly phosphorylated were obtained, and its photosynthetic activities are under investigation.

磷酸烯醇式丙酮酸羧化酶(PEPC)在C4光合作用途径中催化最初的C0_2固定反应，不仅受变构作用的调节，而且受可逆的磷酸化作用的调节。对于这种磷酸化，涉及一种底物局限于PEPC的蛋白激酶。因此，这种蛋白激酶称为PEPC激酶(PEPCK)。我们首次成功地从模式C4植物黄花中克隆了参与C4光合作用的PEPCK基因，并在原核中进行了表达。利用该PEPCK(FtPEPCk)基因，我们研究了该激酶的调控机制，并评价了其生理意义。所获得的结果如下：1)在黄瓜基因组上至少发现了三个拷贝的PEPCK基因，其中一个基因被认为参与了C4光合作用。该基因不仅在叶片中表达，还在其他组织中低水平表达。2)成功地获得了与NUS标签蛋白融合的重组FtPEPCk。与玉米中的PEPCK(ZmPEPCk)类似，FtPEPCk也被发现受到氧化还原调节。3)鉴定了磷酸化激活PEPC所需的几个氨基酸残基。4)利用玉米叶肉细胞原生质体，瞬时表达了带有FLAG标签的FtPEPCk，并对其降解过程进行了监测。泛素/蛋白酶体系统参与了FtPEPCK的降解。5)为了评价PEPCK在体内的生理意义，利用可转化的C4植物黄花，通过反义方法抑制了PEPCK的表达。获得了PEPC没有明显磷酸化的转化子，其光合作用活性正在研究中。