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

硝酸还原酶（NR）活性在大麦植株的光暗转换过程中显著降低。这种失活是由于NR蛋白的磷酸化和随后的结合14-3-3蛋白磷酸化NR。在这项研究中，我们研究了组织分布和细胞定位的14-3-3蛋白在大麦植物中使用免疫技术。1.以大麦叶片总RNA为模板，通过RT-PCR方法克隆了大麦14-3-3蛋白的cDNA。构建允许产生重组14-3-3蛋白的质粒并转化到大肠杆菌中。在大肠杆菌中表达的重组14-3-3蛋白被提取和纯化。免疫重组蛋白14 - 3 -3制备14 - 3 - 3蛋白的特异性抗体.研究了14-3-3蛋白的组织分布。从各种大麦组织中制备蛋白质提取物，包括发芽种子的胚和胚乳、完全展开和未展开的叶布拉德斯和成熟根，并进行免疫印迹分析。14-3-3蛋白在所有组织中均有表达，且表达量相近.制备叶和根组织切片用于14-3-3蛋白的免疫组织学分析。推测14-3-3蛋白定位于叶片的维管束鞘细胞、根皮层和中柱的外层细胞。

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.