Functional analysis of rice proteins which bind to the α subunit of nuclear transport complex.

本研究的目的是通过分离与水稻Importinα1a(α1a)结合的蛋白来鉴定一个可能参与水稻光反应的新的核蛋白。以下是结果1.(1)全长序列的测定：IABP4的全长编码序列包含一个新蛋白的全长编码序列，该蛋白的预测分子量为12 2 kDa，并在拟南芥2号染色体上发现了该蛋白的同源基因。在含有TPR基序的N-末端区域，IABP4蛋白与小鼠核磷蛋白TSP具有较高的同源性，而在含有SH2结合区的小鼠TSP的C-末端区域的相似性较低，这表明该蛋白在功能上不一定与TSP同源。C-末端部分含有一个可能的核定位信号。(2)基因表达分析：RT-PCR结果表明，IABP4的转录水平随着光照水稻幼苗的增加而降低。(3)与IMPα1a的结合分析：Usins纯化的重组蛋白在大肠杆菌中表达，结果：1.天然聚丙烯酰胺凝胶电泳法证实，α蛋白的C-末端与GST-IMP蛋白1a形成了复合体，进一步的核定位和蛋白的内源功能有待进一步研究。用另一种亲和层析的方法从黄化水稻幼苗中分离到几个α1a结合蛋白，并测定了其中3个蛋白的N末端序列。对于一个与富含脯氨酸蛋白同源的蛋白质，分离到了相应的cDNA，并发现其在黑暗生长的幼苗中的表达受光照的下调。

The purpose of this research is to identify a novel nuclear protein which might be involved in the light-response in rice plants, by isolating proteins that bind to rice importin α 1a (IMP α 1a) whose expression is down-regulated by light. Below are the results.1. Analysis of an IMP α 1a-binding protein, IABP4, which has been isolated by cDNA screening by Far western method.(1) Determination of the full cDNA sequence : The IABP4 cDNA contained the entire coding sequence for a novel protein of predicted molecular mass of 122 kDa, and the homologous gene was found on the chromosome 2 of Arabidopsis thaliana.. In the N-terminal region containing TPR motifs, IABP4 protein shows high homology to a mouse nuclear phosphoprotein, TSP, whereas the similarity is low in the C-terminal region which contains the SH2-binding domain in mouse TSP, suggesting that the IABP4 protein is not necessarily homologous in function to TSP.The C-terminal part contains a putative nuclear localization signal.(2) Analysis of gene expression : The result of RT-PCR indicated that the IABP4 transcript level decreases by illuminating dark-grown seedlings of rice.(3) Analysis of binding to IMP α 1a : Usins purified recombinant proteins expressed in E.coil, the binding assay with native polyacrylamide gel electrophoresis demonstrated the complex formation between the C-terminal part of IABP4 and GST-IMP α 1a.Further experiments are required for determination of nuclear localization and the in planta function of the IABP4 protein.2. By another approach using affinity chromatography, several IMP α 1a-binding proteins were isolated from etiolated rice seedlings and the N-terminal sequences of three of them were determined. For one protein with homology to proline-rich protein, the corresponding cDNA was isolated and its expression was found to be down-regulated by light in dark-grown seedlings.