一种水稻体内泛素连接酶底物捕获技术

Ubiquitin-mediated proteolysis is involved in many biological processes in eukaryotes. F-box protein is a subunit of SCF complex, which are in charge of substrate specificity in the ubiquitin-proteasome pathway (UPP) and therefore play a pivotal role in many physiological activities such as cell-cycle progression, transcriptional regulation, programmed cell death and cell signal transduction. Many studied have shown that SCF-complex-mediated degradation of substrates is closely related to plant architecture and high-yield breeding of rice. So far, mapping and cloning genes by genetic approaches are main method to uncover substrates of F-box protein in rice. However, this method with time-consuming, manpower-costing and uncertainty has greatly restricted the excavation and functional study of substrates. Our project bases on the interaction between E3 ubiquitin ligase and its substrates, which can be able to be reinforced by fusing an ubiquitin associated domain (UBA) to E3 ligase. Simultaneously, we construct the 3×His-Ubq1 fusing protein and transform it into plant to combine with substrates of F-box protein. The polyubiquitinated forms of substrates are purified using affinity chromatography, and then identified by liquid chromatography-tandem mass spectrometry, LC-MS/MS. We develop a technique, called “Ubiquitin Ligase Substrate Trapping”, for quickly and efficiently isolating of substrates in complex with their F-box. Therefore, our works provide technical support for discovering key transcriptional regulatory factors and molecular breeding in rice.

F-box蛋白是SCF复合体的一个亚基，在泛素-蛋白酶体途径中因特异识别底物分子而参与细胞周期调控、转录调控、细胞凋亡、信号转导等生命活动。许多研究表明，该途径与水稻株型塑造和高产育种等方面密切相关。目前，水稻F-box蛋白底物主要基于遗传学方法定位和克隆获得，此方法耗时、费力，且具不确定性。本项目拟利用E3连接酶（F-box蛋白）与底物分子的互作关系，将E3与UBA（Ubiquitin associated domain）融合而加强与底物间的稳定性，并导入3×His-Ubq1融合蛋白用于结合底物分子，利用亲和层析技术将多泛素化底物纯化，并通过液相色谱串联质谱（LC-MS/MS）鉴定底物分子，从而建立一套快速、有效的F-box蛋白底物分子捕获技术，称为“Ubiquitin Ligase Substrate Trapping”。这将为水稻关键转录调节因子的发掘和分子育种提供技术支持。

泛素介导的蛋白降解途径参了许多生物学过程。F-box蛋白作为一类E3泛素连接酶，因其特异识别和降解底物分子，从而调控植物体生长发育的各个阶段。目前，水稻中F-box蛋白底物分子都是通过传统的遗传学方法克隆获得，缺少一中快速、有效地发掘底物分子的方法。本项目通过将F-box蛋白与polyubiquitin-binding domain（UBA）形成融合蛋白，从而在一定程度上能抑制其底物分子的降解，通过亲和层析纯化和质谱分析技术，在水稻中初步建立了一种能快速、有效地发掘底物分子的方法：在骨架载体pCAMBIA1300基础之上，通过改造构建了能过表达D3-3*Flag-UBA，D3-3*Flag，COI1b-3*Flag-UBA，GID2-3*Flag-UBA，LP-3*Flag-UBA（LP，Larger Panicle，底物分子未知）等融合蛋白的表达载体，通过农杆菌介导遗传转化水稻野生型Nipponbare，通过鉴定获得稳定表达的转基因材料。对转基因植株株高和分蘖数统计发现过表达D3-3*Flag-UBA融合蛋白能不同程度的降低株高及增加分蘖数，并且其程度跟融合蛋白的表达量呈正相关。通过水稻原生质体双荧光素酶降解实验，进一步证明当D3跟UBA形成融合蛋白后，能影响其底物分子D53的降解速度，因此，当F-box蛋白与UBA形成融合蛋白，一定程度上能抑制其底物分子的降解。在此基础之上，我们利用亲和层析，质谱分析技术和酵母双杂交实验，获得了一些能跟D3，COI1b以及 LP等F-box蛋白互作的底物分子，相关的功能研究正在进一步进行中。综上所述，本项目在水稻中初步建立一种快速，有效地发掘F-box蛋白底物分子的方法，为水稻关键转录调节因子的发掘和分子育种提供技术支持。项目资助一篇SCI文章正在投稿中。协助课题组培养博士生2名，其中1名已取得博士学位，另一名在读。