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Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plants cells

Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., Reiss, B., Maas, C.
Proceedings of the National Academy of Sciences of the United States of America 1996 v.93 no.12 pp. 5888-5893
Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, Zea mays, transgenic plants, gene expression, complementary DNA, Cnidaria, animal proteins, fluorescence, mutants, gene transfer, protoplasts, electroporation, genetic transformation, recombinant DNA, site-directed mutagenesis
The expression of the jellyfish green fluorescent protein in (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetrically determined fluorescence was raised by at least 2 orders of magnitude.