Arabidopsis 2010: Affinity Handle on Macromolecular Complexes

大分子相互作用网络是所有生物过程的基础。直到最近，拟南芥功能网络研究的进展主要是由基于遗传学的方法推动的。另一方面，定义大分子实体的高通量研究的巨大潜力尚未完全实现，主要是因为大多数拟南芥研究人员仍然无法获得可靠的亲和标签分离工具。为了促进这些研究，该项目将开发并提供一套高通量克隆适应载体，包括定量表征的串联亲和标签，用于快速蛋白质组学和RNomic分析拟南芥大分子复合物。此外，该项目将生成一套针对特定亲和标签的健壮的纯化协议。这些有形的成果将通过拟南芥生物资源中心（二元载体和植物品系）、专业出版物、TAIR以及项目网站（纯化协议）传播给拟南芥研究社区。这一贡献将使许多小组能够在植物生物学的各个领域进行系统级研究，目前由于进行内部优化研究的高昂成本而无法这样做。更广泛的影响。除了以载体、植物品系和实验方案的形式为拟南芥研究界提供切实的成果外，该项目还将为美国国家科学基金会整合研究、培训和教育的长期目标做出重大贡献。首先，它将为研究生和博士后提供优秀的跨学科培训，涉及广泛的方法，不同类型的专业知识，并在两个不同的地点吸引合作者。第二，每学期将有两名本科生被培训并纳入UMKC的项目团队，这是一所城市院校，其中20%的学生来自代表性不足的少数群体，即远高于全国平均水平。第三，该项目将与UMKC的城市教育研究所（IUE）合作，建立一个严格和创新的“湿凳/e凳”实践课程，根据IUE的需求和使命量身定制，专门培养科学教师，明确目标是缩小城市K-12学生的成就差距。

Networks of macromolecular interactions are fundamental to all biological processes. Until recently, the progress of functional networks studies in Arabidopsis has been propelled primarily by genetic-based approaches. On the other hand, the enormous potential of high-throughput studies of defined macromolecular entities has not been fully realized, largely because reliable affinity tag-enabled fractionation tools remain unavailable to most Arabidopsis researchers. To facilitate these studies, this project will develop and deliver a suite of high throughput cloning-adapted vectors incorporating quantitatively characterized tandem affinity tags, for rapid proteomic and RNomic analyses of Arabidopsis macromolecular complexes. In addition, the project will generate a set of robust purification protocols tailored to the specific affinity tags. These tangible deliverables will be disseminated to the Arabidopsis research community via the Arabidopsis Biological Resource Center (for binary vectors and plant lines), professional publications, TAIR as well as the project website (the purification protocols). This contribution will enable systems-level studies in a variety of areas of plant biology by many groups presently prevented from doing so by prohibitive cost of conducting the optimization studies in-house. Broader Impacts. In addition to the tangible deliverables for the Arabidopsis research community in the form of vectors, plant lines and experimental protocols, this project will also make significant contributions to long-standing goals of NSF of integrating research, training and education. First, it will provide an outstanding interdisciplinary training for graduate students and a postdoctoral associate that involves a wide range of approaches, different kinds of expertise, and engages collaborators at two different sites. Second, every semester two undergraduates will be trained and incorporated into the project team at UMKC - an urban institution with 20% students coming from underrepresented minority groups, i.e. considerably higher than national average. Third, the project will establish, in collaboration with the Institute for Urban Education (IUE) at UMKC, a rigorous and innovative "wet bench/e-bench" practical course that is custom-tailored to the needs and the mission of IUE to train science teachers specifically with the explicit goal of reducing the achievement gap for urban K-12 students.