SGER: Use of the Tetracysteine Tag to Study Localization Dynamics of Hydroxyproline-Rich Glycoproteins (HRGP)

SGER：使用四半胱氨酸标签研究富含羟脯氨酸的糖蛋白 (HRGP) 的定位动力学

• 批准号: 0311972
• 负责人: Maura Cannon
• 金额: $ 7.63万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0311972&HistoricalAwards=false
• 关键词: SGER; Use; Tetracysteine; Tag; Study

A grant has been awarded to Dr. Maura Cannon of the University of Massachusetts to test the application of the recently developed 'tet-tag' technology for use in plant cells. This technology will be tested with a HRGP (hydroxyproline-rich glycoprotein) in the model plant Arabidopsis. HRGPs are a large family of cell wall proteins that occur in all known plants, and are involved in cell wall structure, cell shape, cell division, and plant growth and development. Although vitally important to plants, very little is known about HRGPs. To identify their localization dynamics at the cellular level, would be a valuable contribution to understanding their functions. In animal cells it has been shown that the biarsenical fluorophores, FLAsH or ReAsH, bind to recombinant proteins containing a tetracysteine sequence, CCXXCC, where X is a non-cysteine amino acid. When bound to the tet-tag, FLAsh and ReAsH fluoresce at different wavelengths to each other, therefore, a tet-tagged protein can be visualized in space and time, and older proteins can be distinguished from younger proteins. Antibodies are not required with these probes, thus making them very useful for localizing products of large gene families, where a specific antibody to an individual gene family member, can be impossible to obtain. Also, due to its small size, the tet-tag is less likely to hinder protein function, which is important in the case of structural proteins such as HRGPs. Results from this project will not only provide data concerning the HRGP in question, but it will enable other cell wall proteins to be probed by the methods developed. This is important because plant cell walls are a major component of food and animal feed, and are the starting material for most fuels. Data on the dynamics of essential components of cell walls will facilitate the improved production of plant biomass. Furthermore, the research in this proposal will provide training for students at the interface of molecular, cellular and developmental biology.

马萨诸塞州大学的莫拉·坎农博士获得了一笔赠款，用于测试最近开发的“tet-tag”技术在植物细胞中的应用。这项技术将在模式植物拟南芥中用HRGP（富含羟脯氨酸的糖蛋白）进行测试。HRGP是存在于所有已知植物中的细胞壁蛋白的大家族，并且参与细胞壁结构、细胞形状、细胞分裂和植物生长和发育。虽然对植物至关重要，但对HRGP知之甚少。在细胞水平上确定它们的定位动力学，将是理解它们功能的有价值的贡献。在动物细胞中，已经表明双砷荧光团FLAsH或ReAsH与含有四半胱氨酸序列CCXXCC的重组蛋白质结合，其中X是非半胱氨酸氨基酸。当与tet标签结合时，FLAsh和ReAsH在不同的波长下彼此发荧光，因此，tet标签的蛋白质可以在空间和时间上可视化，并且可以将较老的蛋白质与较年轻的蛋白质区分开来。这些探针不需要抗体，因此它们对于定位大基因家族的产物非常有用，而在大基因家族中，可能不可能获得针对单个基因家族成员的特异性抗体。此外，由于其小尺寸，tet标签不太可能阻碍蛋白质功能，这在结构蛋白如HRGP的情况下很重要。该项目的结果不仅将提供有关HRGP的数据，而且将使其他细胞壁蛋白能够通过开发的方法进行探测。这一点很重要，因为植物细胞壁是食物和动物饲料的主要成分，也是大多数燃料的起始材料。细胞壁基本成分的动态数据将有助于提高植物生物量的生产。此外，本提案中的研究将为学生提供分子，细胞和发育生物学方面的培训。