A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell Walls

植物细胞壁功能基因组学的单抗工具包资深人士项目摘要PI：Michael G.Hahn，CCRC和佐治亚大学(UGA)植物生物系Co-Pi：Geert-Jan Boons，CCRC和化学系，UGACo-Pi：Christopher S.King，兽医学院，CCRC，UGACo-Pi：Malcolm A.O‘Neill，CCRC，UGACo-Pi：William S.York，CCRC和UGA诱导生物化学和分子生物系植物细胞壁主要由多糖组成，由于多糖成分和糖之间的分支，许多结构是复杂的。现有证据表明，在一种特定的植物中，超过2000个基因可能编码参与细胞壁合成和修饰的酶。到目前为止，这些基因中只有一小部分从功能上被识别和表征。该项目将提供一个大型的、多样化的、具有良好特性的抗植物细胞壁多糖结构的单抗文库，作为对编码细胞壁生物合成和修饰酶的基因进行功能鉴定的工具；这些基因的替代分析方法有限。针对植物细胞壁多糖结构的多种抗体的可获得性将为植物细胞壁研究提供补充现有遗传和生化实验方法的额外工具。项目描述和预期结果具体目标是：1)从杂交瘤细胞系或噬菌体展示文库中分离识别植物细胞壁碳水化合物结构的单抗。用细胞壁提取物或从不同植物中分离的纯化多聚/低聚糖制备的各种新糖蛋白免疫小鼠，免疫动物的脾淋巴细胞将产生杂交瘤细胞系。由此产生的杂交瘤细胞系，或替代地，抗体噬菌体展示文库，将被筛选产生与植物碳水化合物结构反应的抗体。2)详细鉴定细胞壁反应性抗体识别的碳水化合物结构(表位)。获得的抗体将根据它们与一组来自不同植物的纯化壁多糖的结合能力进行分组。将详细描述每个反应组中选定的抗体识别的表位结构(S)。在这个项目过程中产生的100种独特的抗体将有助于那些对参与植物细胞壁生物合成和修饰的基因的功能表征感兴趣的人，也将成为植物细胞壁结构和功能的化学和生物学研究的有价值的工具。外展和培训将做出明确的努力，让本科生参与这项资助下进行的研究。通过UGA夏季本科生研究计划(www.gradsch.uga.edu/rr/)招收本科生，以及通过植物生物系、CCRC和亚特兰大莫尔豪斯学院之间新建立的互动，将莫尔豪斯本科生带到系/中心进行夏季研究项目，将促进历史上代表性不足的群体参与研究。获得预期结果通过这个项目产生的所有抗体的信息，包括每个抗体识别的多糖结构的细节，将通过张贴到美国国家科学基金会资助的植物细胞壁生物合成研究网络网站(http://xyloglucan.prl.msu.edu/).)上的抗体数据库立即提供给科学界复杂碳水化合物研究中心的网站(http://www.ccrc.uga.edu/).)上也将公布该数据库的链接这些抗体将通过设在乔治亚州雅典的CCRC和英国利兹大学的植物科学中心建立的抗体储存中心不受限制地提供给研究界。抗体可从碳源(http://www.ccrc.uga.edu/web/services/carbosource/CSS_home.html)和植物探针(http://www.plantprobes.co.uk/).)订购并将由其分发抗体的分发将通过向订购抗体的人收取适中的费用来自给。费用结构将张贴在碳源和植物探测器网站上。杂交瘤细胞株本身将被复制并存储在三个物理上分开的设施中：1)CCRC和2)兽医学院的单抗设施，两者都在UGA；3)与利兹大学的Paul Knox博士。

A Monoclonal Antibody Toolkit for Functional Genomics of Plant Cell WallsProject Summary List of Senior PersonnelPI: Michael G. Hahn, CCRC and Department of Plant Biology, University of Georgia (UGA)Co-PI: Geert-Jan Boons, CCRC and Department of Chemistry, UGACo-PI: Christopher S. King, College of Veterinary Medicine, UGACo-PI: Malcolm A. O'Neill, CCRC, UGACo-PI: William S. York, CCRC and Department of Biochemistry and Molecular Biology, UGAIntroductionPlant cell walls are composed primarily of polysaccharides, many of whose structures are complex due to multiple sugar components and branching between the sugars. Available evidence suggests that over 2000 genes in a given plant may encode enzymes involved in cell wall synthesis and modification. To date, only a handful of these genes have been functionally identified and characterized. This project will provide a large, diverse, well-characterized library of monoclonal antibodies against plant cell wall polysaccharide structures as tools for the functional identification of genes that encode cell wall biosynthetic and modifying enzymes; genes for which there are limited alternative assays. The availability of diverse antibodies against plant cell wall polysaccharide structures will provide additional tools that are complementary to existing genetic and biochemical experimental approaches for studies of plant cell walls. Project Description and Expected OutcomesThe specific goals are: 1) To isolate monoclonal antibodies that recognize plant cell wall carbohydrate structures either from hybridoma cell lines or from phage display libraries. Mice will be immunized with diverse neoglycoproteins prepared using cell wall extracts or purified poly/oligosaccharides isolated from diverse plants, and hybridoma cell lines will be generated from splenic lymphocytes of the immunized animals. Resulting hybridoma cell lines, or alternatively, antibody phage display libraries, will be screened for production of antibodies reactive with plant carbohydrate structures. 2) To characterize in detail the carbohydrate structure (epitope) recognized by the cell wall-reactive antibodies. The antibodies obtained will be grouped by their abilities to bind to a set of purified wall polysaccharides from diverse plants. The epitope structure(s) recognized by selected antibodies in each reactivity group will be characterized in detail. The ~100 unique antibodies generated during the course of this project will be instrumental to those interested in the functional characterization of genes involved in the biosynthesis and modification of plant cell walls, and will also serve as valuable tools for chemical and biological investigations of plant cell wall structure and function. Outreach and TrainingAn 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 UGA Summer Undergraduate Research Program (SURP) (www.gradsch.uga.edu/rr/) and through a newly established interaction between the Department of Plant Biology, the CCRC and Morehouse College in Atlanta that brings Morehouse undergraduates to the Department/Center for summer research projects. Access to Expected Outcomes Information about all antibodies generated through this project, including details about the polysaccharide structures recognized by each antibody, will be made available immediately to the scientific community via posting to an antibody database on the NSF-funded Plant Cell Wall Biosynthesis Research Network web site (http://xyloglucan.prl.msu.edu/). A link to this database will also be posted on the website of the Complex Carbohydrate Research Center (CCRC) (http://www.ccrc.uga.edu/). The antibodies will be made available to the research community without restriction through antibody stock centers established at the CCRC at UGA (Athens, GA) and the Centre for Plant Sciences at the University of Leeds (UK). Antibodies may be ordered from and will be distributed by CarboSource (http://www.ccrc.uga.edu/web/services/carbosource/CSS_home.html) and Plant Probes (http://www.plantprobes.co.uk/). The distribution of the antibodies will be self-supported by modest fees charged to those ordering the antibodies. The fee structure will be posted to the CarboSource and Plant Probes web sites. The hybridoma cell lines themselves will be replicated and stored in three physically separated facilities: 1) the CCRC and 2) the Monoclonal Antibody Facility of the College of Veterinary Medicine, both at UGA; and 3) with Dr. Paul Knox at the University of Leeds..