拟南芥ABCG14细胞分裂素转运蛋白复合体的鉴定和作用机理研究

项目名称： 拟南芥ABCG14细胞分裂素转运蛋白复合体的鉴定和作用机理研究

项目编号： No.31470370

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 张可伟

作者单位： 浙江师范大学

项目金额： 90万元

中文摘要： 细胞分裂素是调控植物生长发育、叶片衰老、抗病性以及非生物胁迫的重要激素，与作物产量、品质以及适应性等农艺性状密切相关。相对于对生长素转运分子机制的研究，人们对细胞分裂素转运的分子机理了解不多。在前期工作中，我们鉴定了植物第一个控制细胞分裂素向顶运输的转运蛋白AtABCG14，但其作为细胞分裂素转运蛋白的直接生化证据和其在地上组织的生物学功能尚不清楚。为解决以上重要问题，本项目拟利用免疫共沉淀结合质谱分析和膜蛋白酵母双杂交两种方法筛选AtABCG14的互作蛋白，研究互作蛋白的时空表达、亚细胞定位和生物学功能，进而利用异源表达系统检测转运蛋白复合体的转运活性及确切底物形式；同时，应用嫁接、细胞分裂素报告基因指示、同位素示踪等方法研究AtABCG14在地上组织的生物学功能。研究结果对于揭示细胞分裂素的运输机理和细胞分裂素在农业分子育种中的应用均具有重要的科学意义和实践价值。

中文关键词： 细胞分裂素；ABC；转运蛋白；互作蛋白；作用机理

英文摘要： Cytokinins are a major group of phytohormones regulating plant growth, development, leaf senescence, immunity and stress responses. In crops, cytokinins play crucial roles in different agriculturally important traits such as yield, quality and stress tolerance. However, in contrast to the well-defined polar transport of auxins, the molecular basis of cytokinin transporter is poorly understood. For the first time, we recently deciphered a half size ABC transporter AtABCG14 (Arabidopsis ATP-binding cassette transporter G family 14) as an effluxer controling the acropetal translocation of root-synthesized cytokinins in the root. But in contrast to its well-known physiological role in the root, the direct evidence of AtABCG14 as a cytokinin transporter and its function in the aerial part remain to be eluciated. As a half size ABC transporter, the AtABCG14 forms a full-size transporter complex with another partner. The preliminary data of a BiFC (Bimolecular fluorescence complementation) assay shows the AtABCG14 transporter complex is heterogeneous. To address above questions, we propose to identify and characterize the interaction proteins of AtABCG14 using the Co-IP/MS (Co-immunoprecipitation combined with Mass Spectrometry) method and the split-ubiquitin MYTH (membrane yeast two-hybrid) system. Then the gene expression patterns, protein subcellular localizations and biological functions of the candidate interaction proteins of AtABCG14 will be investigated to screen the genuine partner(s) to form an AtABCG14 cytokinin transporter complex. The candidate partner(s) will be co-expressed with AtABCG14 in a heterologous system such as saccharomyces cerevisiae cells, Sf9 cells, Hela cells or Xenopus laevis oocytes for the transporter activity assay in vitro. The substrates and substrate specificity of AtABCG14 transporter complex will be determined by the transportor assay system using different forms of cytokinins. Meanwhile, the grafting, isotope tracer and cytokinin profiling will be applied to discover the biological function of AtABCG14 and its complex in the aerial part of Arabidopsis. The comprehensive understanding of the mechanism of AtABCG14 complex for cytokinin transport will significantly contribute to the molecular breeding for improving the yield and quality of crops.

英文关键词： Cytokinin;ABC transporter;Interaction proteins;Mechanism