Immunohistological studies on 14-3-3 proteins that regulate nitrate reductase.

硝酸还原酶（NR）活性在大麦植株光暗过渡过程中显著降低。这种失活是由于NR蛋白的磷酸化和14-3-3蛋白随后与磷酸化的NR结合。在本研究中，我们利用免疫技术研究了14-3-3蛋白在大麦植物中的组织分布和细胞定位。结果如下：以大麦叶片制备的总RNA为模板，采用RT-PCR方法克隆了大麦14-3-3蛋白cDNA。构建了重组14-3-3蛋白的质粒，并将其转化到大肠杆菌中。对大肠杆菌中表达的14-3-3重组蛋白进行了提取和纯化。通过免疫重组14-3-3蛋白制备了14-3-3蛋白的特异性抗体。研究14-3-3蛋白在组织中的分布。从大麦各种组织中提取蛋白质提取物，包括发芽种子的胚胎和胚乳，完全张开和未张开的叶片以及成熟的根，并进行免疫印迹分析。14-3-3蛋白在各组织中均存在，且含量相近。制备叶片和根组织切片，对14-3-3蛋白进行免疫组织学分析。推测14-3-3蛋白位于叶片维管束鞘细胞、根皮质和柱状叶外层细胞中。

Nitrate reductase (NR) activity was markedly decreased by light to dark transition of barley plants. This inactivation was due to phosphorylation of NR protein and subsequent binding of 14-3-3 proteins to phosphorylated NR.In this study, we investigated tissue distribution and cellular localization of 14-3-3 proteins using immunological techniques in barley plants. The results are the following.1.A cDNA for barley 14-3-3 protein were cloned by RT-PCR method using total RNA prepared from barley leaf as template. The plasmid that allows production of recombinant 14-3-3 protein was constructed and transformed into E.coli. The recombinant 14-3-3 protein expressed in E.coli was extract and purified. Specific antibody for 14-3-3 proteins was prepared by immunizing the recombinant 14-3-3 proteins.2. Tissue distribution of 14-3-3 proteins was investigated. The protein extracts were prepared from various barley tissues including embryo and endosperm of germinating seeds, fully expanded and unexpanded leaf brades and mature roots and subjected to immunoblot analysis. The 14-3-3 proteins were present in all tissues with similar amount.3. The sections of leaf and root tissues were prepared for immunohistological analysis of 14-3-3 proteins. It was suggested that 14-3-3 proteins located in vascular bundle sheath cells of leaf and in outer layer's cells of root cortex and stele.