39.768
Plant Molecular Genetics
Lecture 4, part 2 of
4
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39.768
Plant Molecular Genetics |
NONDESTRUCTIVE MUG ASSAY [GUS Protocols p36, Fig. 1 ]
v. Photograph |
Arabidopsis strains used:
C24 - wild type
B33 - has chimeric patitin/GUS gene which gives expression primarily in roots
D4E - a deletion of patatin B33 promoter fused to CaMV 35S promoter, gives strong constitutive expression
Strong expression of GUS, as visualized in flourescence, is seen for D4E, very little for B33 and none for C24.
Legocki, R.P. et al.
(1986) Bioluminescence in soybean root nodules: Demonstration of a
general approach to assay gene expression in vivo by using
bacterial luciferase. Proc. Natl. Acad. Sci. USA 83:9080-9084.
FMNH2 + O2 + RCHO ---> FMN + RCOOH + H2O + hv490nm
Bacterial luciferase[alkanal, reduced
FMN:oxygen oxidoreductase (1-hydroxylating, lumenescing)] catalyzes
the flavin mediated hydroxylation of a long chain aldehyde to yeild
carboxylic acid and an excited flavin.; the flavin decays to ground
state with the emmision of light at 490nm.
Vibrio harveyi - source of
luciferase. Only luxA and luxB genes needed for
reaction.
Schneider, M., Ow, D.W., and
Howell, S.H., (1990) The in-vivo patern of firefly luciferase
expression in transgenic plants. Plant Mol. Biol. 14:935-947.
CONSTITUTIVE EXPRESSION OF
LUCIFERASE IN LUCIFERIN-IMBIBED LEAVES [Fig. 6]
Leaves from transgenic plants containing
35S-luciferin constructs were detached and 0.4mM luciferin was
imbibed through the petioles. Time lapse photography monitors the
uptake of luciferin into the leaves.
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Plant Molecular Genetics |
