泛素/蛋白酶体途径在拟南芥根发育过程中的分布及其功能研究

The ubiquitin/proteasome pathway (UPP) is one of the most important proteolytic systems in eukaryotes， and is believed to involved in regulation of root development. But, to the best of our knowledge, the distribution and functions of the UPP during root development are still poorly understood. We have collected and identified 9 Arabidopsis mutants with the mutated subunits located in different parts of the 26 S proteasome. Preliminary studies with mutants and inhibitors revealed that different degree in proteasome malfunction has various effects on root growth, suggesting that the UPP does have a role in root development. To extend our knowledge of the involvement of the UPP in root development, the dynamic changes in activity and distribution of both ubiquitin/ubiquitinated proteins and proteasome in developing root are going to be studied by means of fluorogenic kinetic assays, immunoblotting,immunofluorescence and immunogold labeling. In addition, structural changes between wild type and mutant root in cell differentiation, and the morphology of organelle including the endomembrane, cytoskeleton, and cell wall will be observed by confocal laser scanning microscope and transmission electron microscope. Besides, effects of the UPP on the distribution and traffic of plant endogenous hormones and their receptors will also carried out using confocal and HPLC. Based on the results obtained by methods mentioned above, proteomic researches will be carried out with the purpose of providing further insights into the mechanism by which the UPP controls root development.

泛素/蛋白酶体途径（UPP）是真核细胞内蛋白质选择性降解主要方式之一。以往研究表明，UPP参与植物根发育调控，但UPP在根内的活性、分布模式及其在根发育过程中的功能至今仍不清楚。我们收集并鉴定了蛋白酶体不同位点发生突变的拟南芥突变体9个。初步结果表明蛋白酶体不同位点突变不同程度地影响拟南芥根的生长。不同浓度的蛋白酶体抑制剂处理也得到类似结果。为了深入探究UPP在根发育过程中的分布及其功能，本项目运用免疫印迹和免疫标记技术结合显微成像技术和荧光分光光度计等检测拟南芥根发育过程中蛋白酶体活性、含量及分布模式的变化趋势；运用各种荧光探针结合光学和电子显微技术以及高效液相色谱等分析蛋白酶体活性缺失对根细胞、细胞器以及内源激素及其受体的影响；并在上述工作的基础上再有针对性地开展蛋白质组学研究，以期揭示UPP调控根发育的机理，为农林育种提供了重要的理论依据和基因资源。

泛素/蛋白酶体途径（UPP）是真核细胞内蛋白质选择性降解主要方式之一。以往研究表明，UPP参与植物根发育调控,但UPP在根内的活性、分布模式及其在根发育过程中的功能至今仍有待进一步研究。在国家自然科学基金的资助下，经课题组成员的努力，本项目运用免疫印迹和免疫标记技术、光学和电子显微技术以及质谱分析等技术，比较系统地研究了泛素缀合蛋白和蛋白酶体的在拟南芥胚根生长、初生根发育以及侧根形成过程中的含量及分布模式；研究了蛋白酶体相关基因突变以及蛋白酶体抑制剂处理对根细胞、细胞器以及内源激素及其载体蛋白等影响。结果表明：1）泛素缀合蛋白和蛋白酶体在种子萌发和根发育过程中的含量及其分布模式具有发育相关的组织细胞特异性；2）UPP参与调节糖、蛋白质和油脂的代谢，进而影响种子萌发和根系发育；3）UPP参与QC的维持和分生区细胞分裂分化调控，进而影响主根的发育；4）UPP通过参与调控根重力信号的感受和激素信号的传递而调节根向地性；5）短时间适度的UPP活性缺失促进侧根的发生，但长时间剧烈的UPP活性缺失显著抑制侧根发育；6）蛋白酶体活性参与根毛极性生长过程中膜泡运输、细胞壁构建以及线粒体运动等。这些结果揭示了泛素蛋白酶体途径在跟发育过程中的作用，并为农林育种提供了重要的理论依据。