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水稻输入素α 1a（IMP α 1a）的结合蛋白来鉴定一种新的可能参与水稻光反应的核蛋白。下面是结果。1. IMP α 1a结合蛋白IABP 4的分析，IABP 4已通过远Western方法的cDNA筛选而分离。(1)全长cDNA序列的确定：IABP 4 cDNA包含一种新型蛋白质的完整编码序列，预测分子量为122 kDa，并且在拟南芥2号染色体上发现了该同源基因。IABP 4蛋白的N端含有TPR基序，与小鼠核磷蛋白TSP具有很高的同源性，而C端含有小鼠TSP的SH 2结合结构域，与TSP的同源性较低，表明IABP 4蛋白与TSP在功能上不一定同源，C端含有一个假定的核定位信号。(2)基因表达分析：RT-PCR结果表明，水稻幼苗在黑暗条件下，IABP 4基因的转录水平降低。(3)与IMP α 1a结合分析：纯化的重组蛋白在大肠杆菌中表达后，经聚丙烯酰胺凝胶电泳证实，IABP 4的C端与GST-IMP α 1a形成复合物，IABP 4蛋白的核定位及在植物体内的功能有待进一步研究.用亲和层析的方法从水稻黄化幼苗中分离得到了几种IMP α 1a结合蛋白，并测定了其中三种蛋白的N端序列。对于一个蛋白质的同源性脯氨酸丰富的蛋白质，相应的cDNA被分离出来，它的表达被发现在黑暗中生长的幼苗光下调。

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.