有丝分裂检验点蛋白Bub1的功能研究存在较大争议。我们发现Bub1敲除细胞能够表达少量Bub1蛋白来支持有丝分裂。对敲除细胞进行RNAi能够完全清除Bub1蛋白。用该方法我们证实了Bub1对检验点激活的重要作用，也发现 Bub1对染色体移动，着丝粒与微管的结合起关键作用。这两个功能的机制尚不明确。我们提出假说：细胞进入有丝分裂时，Bub1-Mad1的结合帮助Mad1定位到KNL1表面，与BubR1和Cdc20接近并促进MCC形成。同时，Bub1通过自身或结合蛋白的活性促进染色体移动，着丝粒与微管的结合。这两种功能可能由结合不同蛋白的Bub1分别完成。为验证该假说我们将：(1)探明Bub1-Mad1激活检验点的机制；(2)明确Bub1促进染色体移动，着丝粒与微管结合的机制；(3)确认这两个功能协同作用的机制。该项目通过深入研究Bub1调控有丝分裂的机制，为开发抗肿瘤药物提供理论依据。

Activating mitotic checkpoint to arrest cells in mitosis is an efficient way to inhibit cancer cell amplification. Studying the molecular mechanism of the mitotic checkpoint will provide valuable information for pharmaceutical applications. Bub1 is a key component of the mitotic checkpoint. It also involves the chromosome congression and bi-orientation establishment with unknown mechanism. The difficulty of studying Bub1 is that very little Bub1 protein is enough to fully support its function. With CRIPR/Cas9 mediated gene knock out, we achieved several HeLa cell lines with very low Bub1 expression which is enough to supppor the viability of the cells. Further application of RNAi against Bub1 could fully abolish its expression. Using this method, we have successfully revealed Bub1's important roles in acitvating mitotic checkpoint and chromosome alignment. This project will further study the molecular mechanism of Bub1 in regulating mitosis based on this method.