Methods for Generation and Analysis of Fluorescent Protein-Tagged Maize Lines
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The use of fluorescent proteins to localize gene products in living cells has revolutionized cell biology. Although maize has excellent genetics resources, the use of fluorescent proteins in maize cell biology has not been well developed. To date, protein localization in this species has mostly been performed using immunolocalization with specific antibodies, when available, or by overexpression of fluorescent protein fusions. Localization of tagged proteins using native regulatory elements has the advantage that it is less likely to generate artifactual results, and also reports tissue-specific expression patterns for the gene of interest. Fluorescent protein tags can also be used for other applications, such as protein–protein interaction studies and purification of protein complexes. This chapter describes methods to generate and characterize fluorescent protein-tagged maize lines driven by their native regulatory elements.
Keywords:Confocal microscopy Gateway cloning™ Green fluorescent protein (GFP) Yellow FP (YFP) Red FP (RFP) Maize Native regulatory elements Protein localization Tissue-specific expression Triple-template PCR (TTPCR)
We thank members of our maize GFP tagging project for useful discussions. We acknowledge the Iowa State University Plant Transformation Facility for providing excellent transformation services to the public sector. Our research is funded by National Science Foundation Grant DBI # 0501862 to Dave Jackson, Anne Sylvester, and Agnes Chan.
- 9.Tian, G.-W., Mohanty, A., Chary, S. N., Li, S., Paap, B., Drakakaki, G., Kopec, C. D., Li, J., Ehrhardt, D., Jackson, D., Rhee, S. Y., Raikhel, N. V. and Citovsky, V. (2004) High-throughput fluorescent tagging of full-length arabidopsis gene products in planta. Plant Physiol. 135 25–38.PubMedCrossRefGoogle Scholar
- 12.Herrmann, M. M., Pinto, S., Kluth, J., Wienand, U. and Lorbiecke, R. (2006) The PTI1-like kinase ZmPti1a from maize (Zea mays l.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte. BMC Plant Biol. 6 22.PubMedCrossRefGoogle Scholar
- 14.Dresselhaus, T., Amien, S., Marton, M., Strecke, A., Brettschneider, R. and Cordts, S. (2005) Transparent leaf area1 encodes a secreted proteolipid required for anther maturation, morphogenesis, and differentiation during leaf development in maize. Plant Cell. 17 730–745.PubMedCrossRefGoogle Scholar
- 25.Rozen, S. and H. Skaletsky (1999) Primer3 on the WWW for general users and for biologist programmers. Meth. Mol. Biol. 132 365–386.Google Scholar