Komatsu Setsuko, Konishi Hirosato, Shen Shihua, Yang Guangxiao
Department of Molecular Genetics, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan.
Mol Cell Proteomics. 2003 Jan;2(1):2-10. doi: 10.1074/mcp.r200008-mcp200.
The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.
二维聚丙烯酰胺凝胶电泳蛋白质组分析技术能够监测组织和生物体蛋白质表达中发生的整体变化以及/或者在应激条件下发生的表达变化。在本研究中，构建了水稻蛋白质组目录，并对其中一些蛋白质进行了功能表征。从水稻组织中提取的蛋白质以及在各种应激条件下从水稻中提取的蛋白质通过二维聚丙烯酰胺凝胶电泳进行分离。使用图像分析仪在10种经考马斯亮蓝染色的二维聚丙烯酰胺凝胶上共显示出10589个蛋白质斑点。将分离的蛋白质电印迹到聚偏二氟乙烯膜上，并测定了905个蛋白质中272个蛋白质的N端氨基酸序列。在对蛋白质进行酶切后，使用蛋白质测序仪或质谱法测定了633个蛋白质的内部氨基酸序列。最后，构建了一个包含氨基酸序列和序列同源性信息的水稻蛋白质数据文件。利用蛋白质组学方法可以鉴定参与水稻生长和发育的主要蛋白质。其中一些蛋白质，包括一种被证明是钙网蛋白的钙结合蛋白和一种在水稻中为1,5-二磷酸核酮糖羧化酶/加氧酶激活酶的赤霉素结合蛋白，在信号转导途径中发挥作用。从水稻蛋白质组中获得的这些信息将有助于预测未知蛋白质的功能，并有助于它们的分子克隆。
