Collaborative Resarch: Development of Phytoplankton PCNA Antibodies for Use in Growth Studies: Emiliania huxleyi and Karlodinium micrum as Model Systems

合作研究：开发用于生长研究的浮游植物 PCNA 抗体：Emiliania huxleyi 和 Karlodinium micrum 作为模型系统

• 批准号: 0452780
• 负责人: Senjie Lin
• 金额: $ 27.94万
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0452780&HistoricalAwards=false
• 关键词: Collaborative; Resarch; Development; Phytoplankton; PCNA

Proliferating cell nuclear antigen (PCNA) is a cell cycle marker that is potentially useful for assessing phytoplankton growth status and estimating in situ growth rate. However, its application has been hindered by the lack of effective antibodies for phytoplankton PCNA. PCNA-encoding genes for major classes of phytoplankton have been cloned and sequenced recently, permitting production and characterization of effective antibodies. This project will produce a suite of ten phytoplankton PCNA antibodies. Three universal algal PCNA antisera and three dinoflagellate-specific PCNA antisera will be produced using synthetic peptides, in collaboration with an antibody producing company. This arrangement will reduce production costs and will provide a supply of antibody that can be made available to other researchers. Four antisera will be produced for the coccolithophorid species Eminiliania huxleyi (2) and the ichthyotoxic dinoflagellate Karlodinium micrum (2) based on purified PCNA overexpressed in bacteria from respective PCNA genes. This project will include the development of an optimized protocol for immunofluorescence using these antibodies, based on both epifluorescence microscopy and flow cytometry, with E. huxleyi and K. micrum as model systems. Expression of PCNA in E. huxleyi and K. micrum will be characterized using Western Blots. Association of PCNA with the cell division cycle will be characterized by means of immunofluorescence labeling of PCNA, coupled with flow cytometric, DNA-based analysis of the cell cycle. Comparisons of PCNA-based growth rates of these species to actual growth rates in culture (using cell counts) will allow assessment of the accuracy and precision of the new method. A total of 10 polyclonal antibodies will be available to interested researchers worldwide. These PCNA antisera will prove extremely useful for assessing the growth status of phytoplankton populations and for estimating in situ gross growth rates (GGR) using various detection formats atr the discretion of the researcher. Examples include Western Blotting, immunofluorescence, or potentially, enzyme linked immunoabsorbent assays (ELISA). The availability of PCNA antibody products will facilitate future applications including studies of the cell cycle and growth characteristics of the two model species and other phytoplankton, as well as field applications of the PCNA approach to in situ growth rate estimation. This project includes training for graduate and undergraduate students in molecular techniques and flow cytometric analyses. Travel between the two participating institutions will allow both PIs and their students to expand their expertise. Results of the research will be integrated in courses taught by the PIs. This project will also provide an excellent opportunity for the PIs to continue with their commitment to encourage minority and female students to participate in scientific research.

增殖细胞核抗原是一种细胞周期标记物，可用于评估浮游植物的生长状态和估计原位生长速度。然而，由于缺乏针对浮游植物增殖细胞核抗原的有效抗体，其应用一直受到阻碍。最近，主要种类的浮游植物的增殖细胞核抗原编码基因已经被克隆和测序，这使得有效的抗体的产生和鉴定成为可能。该项目将生产一套10种浮游植物的增殖细胞核抗原抗体。将与一家抗体生产公司合作，使用合成肽生产三种通用的藻类增殖细胞核抗原抗血清和三种甲藻特异的增殖细胞核抗原抗血清。这种安排将降低生产成本，并将提供可供其他研究人员使用的抗体。基于纯化的增殖细胞核抗原在细菌中的高表达，将产生四种抗血清，分别用于球虫类物种Eminiliania huxleyi(2)和鱼毒甲藻Karlowdium microrum(2)。该项目将包括开发使用这些抗体的免疫荧光的优化方案，基于荧光显微镜和流式细胞术，以E.huxleyi和K.microrum为模型系统。用免疫印迹法鉴定增殖细胞核抗原在湖北龙须草和微克雷伯菌中的表达。增殖细胞核抗原与细胞分裂周期的关系将通过免疫荧光标记，结合流式细胞术，基于DNA的细胞周期分析来表征。将这些物种基于增殖细胞核抗原的生长率与培养中的实际生长率(使用细胞计数)进行比较，可以评估新方法的准确性和精密度。共有10种多克隆抗体将提供给世界各地感兴趣的研究人员。这些增殖细胞核抗原抗血清将被证明是非常有用的，以评估浮游植物种群的生长状态，并在研究人员的自由裁量下，使用各种检测格式来估计原位总生长率(GGR)。例如，Western blotting，免疫荧光，或可能的，酶联免疫吸附分析(ELISA)。增殖细胞核抗原抗体产品的问世将促进未来的应用，包括研究这两个模式物种和其他浮游植物的细胞周期和生长特征，以及将增殖细胞核抗原方法用于现场生长速度估计。该项目包括对研究生和本科生进行分子技术和流式细胞仪分析方面的培训。两个参与院校之间的旅行将使PI和他们的学生能够扩大他们的专业知识。研究的结果将纳入私人投资督导员教授的课程。该项目还将为私人投资机构提供一个极好的机会，让他们继续致力于鼓励少数群体和女学生参与科学研究。