A Toolkit for in Vivo Visualization/Modulation of Plant Cell Wall Polysaccharides

Pi：Michael G.Hahn(佐治亚大学)Copis：Geert-Jan Boons，Harry Gilbert，Christopher S.King，Malcolm A.O‘Neill和William S.York(佐治亚大学)目前还没有可用于对植物细胞壁多糖和糖蛋白进行活体成像的工具。该项目将提供一套单链抗体片段(ScFv)和碳水化合物结合模块(CBMs)形式的细胞壁多糖分子探针，作为使用显微镜对植物细胞壁进行活体成像的工具。具体目标是：1)从杂交瘤细胞系或从碳水化合物结合结构域产生一系列全面的标记分子探针，这些分子探针可以在植物细胞中表达并将在体内结合到细胞壁多糖上；以及2)证明所产生的探针将允许在活的植物细胞中定位不同的碳水化合物结构，或者通过选择性地破坏转基因植物中的多糖掺入细胞壁来改变细胞壁结构。预计在这个项目过程中产生的分子探针将有助于那些对植物细胞壁多糖动态感兴趣的人，其中一些探针将通过它们与多糖/糖蛋白的相互作用来破坏细胞壁的组装和结构，并作为发现细胞壁多糖在植物细胞生物学中的功能的有价值的工具。关于推广和培训，项目人员将教授为期一周的课程，该课程将在赠款期限内每年夏天在复合碳水化合物研究中心举行，向感兴趣的科学家传授多糖局部化方法。一项明确的努力将使本科生参与这项资助下进行的研究。通过佐治亚大学夏季本科生研究计划(SURP)(http://www.grad.uga.edu/outreach&amp；diversity/surp.html).招收本科生，将促进历史上代表性不足的群体参与研究所有学生和博士后助理都将受到密切指导，以鼓励和促进他们作为研究科学家的发展。通过这个项目产生的所有抗体、单链抗体和CBM的信息将通过张贴到基于网络的抗体数据库(http://www.wallmabdb.net).)立即提供给科学界所有分子探针将不受限制地通过碳源(http://www.carbosource.net).)提供给研究团体在分子探针功能验证研究中产生的质粒和转基因植物品系将通过拟南芥生物资源中心(http://abrc.osu.edu).)获得

PI: Michael G. Hahn (University of Georgia)CoPIs: Geert-Jan Boons, Harry Gilbert, Christopher S. King, Malcolm A. O'Neill, and William S. York (University of Georgia)There are currently no tools that can be used for in vivo imaging plant cell wall polysaccharides and glycoproteins. This project will provide a set of molecular probes for cell wall polysaccharides in the form of single-chain antibody fragments (ScFvs) and carbohydrate-binding modules (CBMs) as tools for in vivo imaging of plant cell walls using microscopy. The specific goals are: 1) to generate, either from hybridoma cell lines or from carbohydrate binding domains, a comprehensive series of tagged molecular probes that can be expressed in plant cells and will bind to cell wall glycans in vivo; and 2) to demonstrate that the probes generated will permit the localization of diverse carbohydrate structures within living plant cells or alternatively alter cell wall structure by selectively disrupting polysaccharide incorporation into walls in transgenic plants. It is anticipated that the molecular probes generated during the course of this project will be instrumental to those interested in the dynamics of plant cell wall polysaccharides and that some of these probes will disrupt cell wall assembly and structure through their interaction with the polysaccharides/glycoproteins and serve as valuable tools to discover the functions of cell wall polysaccharides in the biology of plant cells. With respect to outreach and training, project personnel will teach a one week course to be held during each summer at the Complex Carbohydrate Research Center during the tenure of the grant to teach polysaccharide localization methods to interested scientists. An explicit effort will be made to involve undergraduate students in the research carried out under this grant. The participation of historically underrepresented groups in the research will be fostered by recruiting undergraduate students through the University of Georgia Summer Undergraduate Research Program (SURP) (http://www.grad.uga.edu/outreach&diversity/surp.html). All students and post-doctoral associates will be closely mentored to encourage and foster their development as research scientists. Information about all antibodies, ScFvs, and CBMs generated through this project, will be made available immediately to the scientific community via posting to a web-based antibody database (http://www.wallmabdb.net). All molecular probes will be made available to the research community without restriction through CarboSource (http://www.carbosource.net). Plasmids and transgenic plant lines generated in the molecular probe function validation studies will be available through the Arabidopsis Biological Resource Center (http://abrc.osu.edu).