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Identification and functional characterization of protein components of the plant kinetochore complex

植物动粒复合体蛋白质成分的鉴定和功能表征

基本信息

批准号：
5397515
负责人：
Professor Dr. Ingo Schubert
金额：
--
依托单位：
Abteilung Züchtungsforschung
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2002
资助国家：
德国
起止时间：
2001-12-31 至 2006-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/5397515?language=en
关键词：
Identification functional characterization protein components

项目摘要

The goal is to identify and characterize several protein components of plant kinetochore complexes for better understanding of centromere functions and later approaches to construct de novo plant artificial chromosomes. We plan to elucidate the presence an functionality of candidates for chekpoint control proteins (Bub1, Bub3, Zw10, SKP1) and proteins interacting with spindle microtubuli (CBF5, Zw10) in Arabidopsis thaliana. We have already identified the Arabidopsis genes of the putative plant kinetochore proteins (KP) in silico. Arabidopsis transformants that express the KP-GFP fusion proteins will be generated to trace their cellular localization along the mitotic an meiotic cycle. Subsequently, by an inducible RNAi approach using the KP-GFP transformants as targets, the consequences of a loss or suppression of function will be studied. Recombinant KP candidates will be expressed and isolated to test their interaction with each other and with centromeric DNA by plasmon resonance. Antibodies against the KP will be produced to verify the GFP results and to study their phylogenetic conservation in plants. An existing collection of monoclonal antibodies against plant kinetochores may help to identify KP with less homology to non-plant KP either by screening an expression library or by immunoadsorption of chromosomal/ nuclear proteins.

目标是鉴定和表征植物着丝粒复合物的几种蛋白质成分，以便更好地了解着丝粒功能以及随后构建从头植物人工染色体的方法。我们计划阐明拟南芥中候选检查点控制蛋白（Bub1、Bub3、Zw10、SKP1）和与纺锤体微管相互作用的蛋白（CBF5、Zw10）的功能。我们已经在计算机上鉴定了假定植物动粒蛋白（KP）的拟南芥基因。将产生表达 KP-GFP 融合蛋白的拟南芥转化体，以追踪它们沿着有丝分裂和减数分裂周期的细胞定位。随后，通过使用 KP-GFP 转化体作为靶标的诱导性 RNAi 方法，将研究功能丧失或抑制的后果。重组 KP 候选物将被表达和分离，以通过等离子共振测试它们之间的相互作用以及与着丝粒 DNA 的相互作用。将生产针对 KP 的抗体来验证 GFP 结果并研究它们在植物中的系统发育保守性。现有的针对植物着丝粒的单克隆抗体集合可能有助于通过筛选表达文库或通过染色体/核蛋白的免疫吸附来识别与非植物 KP 具有较低同源性的 KP。