nontarget or pleiotropic effects (El Ouakfaoui et al. 2005). It is the most frequently used
reporter gene in field trials so far. The greatest disadvantage is that both detection assays
are destructive to the cells. The substrates are also quite expensive.
The GUS reporter gene system has been most important in the study of gene regulatory
elements and mechanisms in plants. Generally, there is very little background activity in
plants compared with other organisms; specifically, plants do not normally turn blue
when exposed to X-gluc. It has been used in transcription fusions to study a wide range
of regulatory elements cloned from the plant genome (Fig. 9.1) and also for promoter-
trapping experiments (Fig. 9.5). It also forms stable translational fusions with proteins;
for example, fusions with thenptIIgene to generate bifunctional proteins that can be
used as a selectable marker and as a reporter (Fig. 9.8).
9.5.2 Luciferase
The firefly (Photinus pyralis) enzyme luciferase (EC 1.13.12.7) was one of the first useful
reporters for plants (Ow et al. 1986). Whereas GUS gives transgenic cells a blue color,
luciferase produces light (Fig. 9.9), an example of bioluminescence; however, it has not
been used as extensively as GUS. The enzyme catalyzes the ATP-dependent oxidative
decarboxylation of luciferin as substrate; therefore, it is another conditional nonselectable
marker system. A significant advantage is the sensitive, nondestructive monitoring
system that allows real-time analysis. Furthermore, the half-life of the luciferase protein
in plant cells is lower than that for GUS and may reflect transcriptional activity more accu-
rately. It is often used as an internal control in experiments that require the use of more than
one reporter system.
9.5.3 Green Fluorescent Protein
The Pacific jellyfish (Aequorea victoria) green fluorescent protein (GFP) is now becoming
the most important reporter gene system for plants [reviewed by Stewart (2001)].
Figure 9.8.Fusion of a reporter and selectable marker gene to create a bifunctional gene: (a)
GUS:NPTII fusion reporter system for plants that incorporates thenptIIgene for kanamycin selection
and the GUS reporter gene in a single module; (b) transformed tobacco shoots selected on kanamycin;
(c) shoots with roots regenerated on kanamycin; (d) a transgenic seedling after two generations
showing retention of GUS gene activity indicated by the histochemical staining with the GUS sub-
strate X-Gluc (provided by Raju Datla, Plant Biotechnology Institute, National Research Council
of Canada, Saskatoon, Canada). See color insert.
232 MARKER GENES AND PROMOTERS