The impact of novel and confirmed cenH3 assembly factors on plant kinetochore formation

组蛋白H3被CENH3变体取代是迄今为止所研究的所有真核生物的着丝粒核小体的共同特征。尽管这一过程具有进化保守性，但尚不清楚是什么触发了CENH3沉积，以及它如何成为高度可变的着丝粒DNA序列的目标。这些知识是了解着丝粒形成和人工产生着丝粒的基础。在确定了有丝分裂周期中CENH3的装载时间后，本课题的目的是通过寻找相互作用因子及其引导CENH3进入着丝粒组装着丝粒的模式来深入了解CENH3在植物中的沉积。将通过EYFP融合构建体在体内和与特异性抗体的原位表达来分析假定的CENH3组装因子（Sim3， KNL2和Mis12）的动力学和亚细胞定位。将通过bic、酵母双杂交和IP方法检测CENH3组装因子与CENH3之间潜在的相互作用。将在T-DNA插入突变体和/或相应RNAi构建体转基因植物中研究敲除/敲低CENH3组装因子表达的后果。为了了解CENH3的N端和c端部件的功能需求，将分别测试它们与CENH3组装因子的相互作用。

Substitution of histone H3 by the variant CENH3 is a common feature of centromeric nucleosomes of all eukaryotes studied so far. Despite the evolutionary conservation of this process it is unknown what triggers CENH3 deposition and how it becomes targeted to the highly variable centromeric DNA sequences. Such knowledge forms the basis to understand centromere formation and to generate artificial centromeres. After having established the loading time during the mitotic cycle, the aim of this proposal is to get insight into CENH3 deposition in plants by finding out interacting factors and their mode to guide CENH3 to centromeres for kinetochore assembly. Kinetics and subcellular localization of putative CENH3 assembly factors (Sim3, KNL2 and Mis12) will be analysed via expression of EYFP fusion constructs in vivo and with specific antibodies in situ. Potential interactions between CENH3 assembly factors and CENH3 will be tested by BiFC, yeast two-hybrid and IP approaches. Consequences of knock out/knock down of CENH3 assembly factor expression will be studied in T-DNA insertion mutants and/or in plants transgenic for the corresponding RNAi constructs. In order to understand functional requirement of N and C-terminal parts of CENH3, they will be tested separately for interaction with CENH3 assembly factors.