Abstract
The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.
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Kamiya, N., Physics and chemistry of protoplasmic streaming, Ann. Rev. Plant Physiol., 1960, 11: 323.
Adams, R. J., Pollard, T. D., Membrane-bound myosin-1 provides new mechanisms in cell motility, Cell Motility and the Cytoskeleton, 1989, 14: 178.
Kackar, B., Direct visualization of organelle movement along actin filaments dissociated from characean algae, Science, 1985, 227: 1355.
Steer, M. W., Role of actin in top growth, in Tip Growth in Plant and Fungal Cells, London: Academic Press, 1990.
Schmit, A. C., Lamber, A. M., Microinjected florescent phalloidinin vivo reveals the F-actin dynamics and assembly in higher plant mitotic cells, Plant Cell, 1990, 2: 129.
Yen, L. F., Shih, T. C., The presence of a contractile protein in higherplants, Scientia Sinica (in Chinese), 1963, 3: 490.
Kost, B., Spielhofer, P., Chua, N. H., A GFP-mouse talin fusion protein labels plant actin filamentsin vivo and visualizes the actin cytoskeleton in growing pollen tubes, The Plant Journal, 1998, 16(3): 393.
Kindle, K. L., Schnell, R. A., Fernandez, E. et al., Stable nuclear transformation ofChlamydomonas using the Chlamydomonas gene for nitrate reductase, The Journal of Cell Biology, 1989, 109: 2589.
Fang, Guochang, Su Ning, Zhang Zhonglin et al., Construction of chloroplast expression system inChlamydomonas, Chinese Science Bulletin (in Chinese), 1999, 44(12): 1301.
Detmers, P. A., Carboni, J. M., Condeelis, J., Localization of actin inChlamydomonas using antiactin and NBD-Phallacidin, Ce1l Motility, 1985, 5: 415.
Harper, J. D. I., McCurdy, D. W., Sanders, M. A. et al., Actin dynamics during the cell cycle inChlamydomonas reinhardtii, Cell Motility and the Cytoskeleton, 1992, 22: 117.
Sugase, Y., Hirono, M., Kindle, K. L., Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii, Gene, 1996, 168: 117.
Lee, V. D., Finstad, S. L., Huang, B., Cloning and characterization of a gene encoding an actin-related protein inChlamydomonas, Gene, 1997, 197: 153.
Minoura, T. K., Uryu, S., Hirono, M. et al., High divergent actin expressed in aChlamydomonas mutant lacking the conventional actin gene, Biochemical and Biophysical Research Communication, 1998, 251: 71.
Lilie, H., Schwarz, E., Rudolph, R., Advances in refolding of proteins produced inE. coli, Current Opinion in Biotechnology, 1998, 9: 497.
Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriaphage T4, Nature, 1970, 227: 680.
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Yang, Y., Liu, G. & Yan, L. Expression ofChlamydomonas actin-gfp fusion gene in tobacco suspension cell and polymerization of the actin-gfp proteinin vitro . Chin.Sci.Bull. 46, 806–810 (2001). https://doi.org/10.1007/BF02900428
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DOI: https://doi.org/10.1007/BF02900428