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

马萨诸塞大学的莫拉·坎农博士获得了一项资助，用于测试最近开发的用于植物细胞的“tet-tag”技术的应用。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。HRGPs是一个存在于所有已知植物中的细胞壁蛋白大家族，参与细胞壁结构、细胞形状、细胞分裂以及植物的生长发育。虽然对植物至关重要，但人们对HRGP知之甚少。在细胞水平上识别它们的定位动力学，对于理解它们的功能将是有价值的贡献。在动物细胞中，已经证明双砷荧光团，Flash或ReAsH，与含有四半胱氨酸序列CCXXCC的重组蛋白结合，其中X是非半胱氨酸氨基酸。因此，当与tet-tag、Flash和ReAsH结合时，彼此在不同波长的荧光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，在这种情况下，可能无法获得针对单个基因家族成员的特异性抗体。此外，由于它的小尺寸，tet-tag不太可能阻碍蛋白质的功能，这在结构蛋白质如HRGP的情况下是很重要的。该项目的结果不仅将提供有关所讨论的HRGP的数据，还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，也是大多数燃料的原料。有关细胞壁基本成分动态的数据将有助于提高植物生物量的生产。此外，这项计划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。

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.