ASK1-EBF1识别和结合EIN3的结构生物学研究

STRUCTURAL STUDY OF THERECOGNITION ANDBINDING MECHANISM OF ARABIDOPSISEIN3 BY THE ASK1-EBF1 COMPLEX

钟代靖

11649082

硕士

生物学

Dinshaw J.Patel

2018-06-01

2018-07-05

哈尔滨工业大学

深圳

乙烯是植物体内重要的内源性气态激素 ， 能够调节植物的生长发育，影响果实的成熟以及在胁迫响应中具有重要的作用。在拟南芥中，EIN3（ Ethylene Insensitive 3）是乙烯通路的主要转录因子，在没有乙烯时，EIN3 通 过 EBF1/2（Ethylene Insensitive 3 binding F-box protein 1/2）介导的泛素化降解途径进行降解；而在有乙烯的情况下，EBF1/2功能受到抑制，EIN3 蛋白在细胞核中积 累，启动相关基因的表达。乙烯信号通路的精确调节对植物的生长发育以及应激都非常关键，而EIN3 占据着乙烯信号通路调控的中心地位。所以理 解 EBF1/2 对 EIN3的 调 控 机制对理解乙烯信号通路的调控具有重大意义。本论文中，我们聚焦在 EBF1 是如何识别并结合转录因子 EIN3 的分子机制，并尝试用结构生物学的方法对该蛋白复合物进行三维结构的解析。我们通过凝胶过滤层析确定了 EIN3 上有两段可与 EBF1 发生相互作用，分别是1-166 号氨基酸和 313-501 号氨基酸段。同时合作实验室 使用酵母双杂交实验将相互作用区段精确到了 40-73 号氨基酸和 465-493 号氨基酸，我们进一步用凝胶过滤层析和共沉淀实验进行了验证并且进行了晶体筛选。目前，ASK1-EBF1 复合物的晶体 还需要进一步的优化。同时我们也对 ASK1-EBF1 进行了负染实验，结果显示该复合物可以形成均一密度的颗粒。

Ethylene is an important endogenous gaseous hormone in plants, which can regulate plant growth and development, affect fruit ripening and play an important role in stress response. In Arabidopsis thaliana, EIN3 (Ethylene Insensitive 3) is a major transcription factor controlled by ethylene. In the absence of ethylene, EIN3 is degraded through the EBF1/2(Ethylene Insensitive 3 binding F-box protein 1/2)-mediated ubiquitination degradation pathway, while in the presence of ethylene, the function of EBF1/2 is inhibited, and EIN3 protein accumulates in the nucleus and initiates the expression of related genes. The accurateregulation of the ethylene signaling pathway is critical to plant growth and stress, and EIN3 is the major component in the regulation of ethylene signaling pathways. Therefore, it is of great significance to study the recognition mechanism of EIN3 by the EBF1/2. In this paper, we focus on the molecular mechanism of how the EBF1 recognizes and binds the transcription factor EIN3, and try to solve the three-dimensional structure of this protein complex. We identified two segments of EIN3(1-166 and 313-501) that interact with EBF1 by gel filtration chromatography. Also our collaborator helps us map to more precise interaction region corresponding to EIN3 (40-73 and 465-493) by using yeast two-hybrid, which was further verified by gel filtration chromatography. Currently，the crystals of the ASK1-EBF1 complex needs to be further optimized and the negative staining of this complex show homogeneous particles.

乙烯 EIN3 EBF1

ethylene EIN3 EBF1

中文

联合培养

南方科技大学

钟代靖. ASK1-EBF1识别和结合EIN3的结构生物学研究[D]. 深圳. 哈尔滨工业大学,2018.