An improved green fluorescent protein gene as a vital marker in plants.
A synthetic green fluorescent protein (GFP) gene (pgfp) was constructed to improve GFP expression in plants. Corn and tobacco protoplast transient assays showed that pgfp gave about 20-fold brighter fluorescence than the wild-type gene (gfp). Replacement of the serine at position 65 with a threonine (S65Tpgfp) or a cysteine (S65Cpgfp) yielded 100- to 120-fold brighter fluorescence than wild-type gfp upon excitation with 490-nm light. Incorporation of a plant intron into the coding region yielded an additional 1.4-fold improvement, for a cumulative improvement of about 150-fold in fluorescence at 490-nm excitation. Various versions of pgfp were also stably introduced into corn, wheat, tobacco, and Arabidopsis plants. Bright-green fluorescence was observed with a fluorescence microscope in virtually all examined tissues of transgenic monocots and dicots. In the case of Arabidopsis, expression of the pgfp gene under the enhanced 355 promoter of the cauliflower mosaic virus produced green fluorescence that was readily detectable by eye using a hand-held, long-wave ultraviolet lamp and/or a black-light source.
Pang
S Z
SZ
Monsanto Company, St. Louis, Missouri 63198, USA.
DeBoer
D L
DL
Wan
Y
Y
Ye
G
G
Layton
J G
JG
Neher
M K
MK
Armstrong
C L
CL
Fry
J E
JE
Hinchee
M A
MA
Fromm
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017796:Caulimovirus / Q000235:genetics; D015870:Gene Expression; D005813:Genes, Synthetic; D005814:Genes, Viral; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D007438:Introns; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D037401:Scyphozoa / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism; D003313:Zea mays / Q000378:metabolism
D005819:Genetic Markers; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins