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

目的是鉴定和表征植物着丝点复合体的几种蛋白质成分，以便更好地了解着丝点的功能，并在以后构建植物人工染色体的方法。我们计划阐明拟南芥中检查点控制蛋白（Bub1, Bub3, Zw10, SKP1）和与纺锤体微管相互作用的蛋白（CBF5, Zw10）的存在和功能。我们已经在硅片上鉴定了拟南芥中假定的植物着丝点蛋白（KP）的基因。将产生表达KP-GFP融合蛋白的拟南芥转化子，以沿着有丝分裂和减数分裂周期追踪其细胞定位。随后，通过使用KP-GFP转化体作为靶标的诱导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，通过等离子体共振测试它们彼此之间以及与着丝粒DNA的相互作用。将产生针对KP的抗体来验证GFP结果并研究其在植物中的系统发育保守性。现有的植物着体点单克隆抗体可以通过筛选表达文库或通过染色体/核蛋白的免疫吸附，帮助鉴定与非植物KP同源性较低的KP。

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.