Plant Biotechnology and Genetics: Principles, Techniques and Applications

Figure 9.4.TheuidAgene, coding for GUS, as an example of a reporter gene that has been
extensively used in plants. (a) Histochemical staining for GUS activity using the substrate
4-methyl umbelliferyl glucuronide (MUG) allows detection of gene activity in specific tissues of trans-
genic plants. Shown are the staining of cauliflower plantlets in which constitutive expression of GUS is
conferred by a strong constitutive promoter, tCUP (photo courtesy of Dan Brown, Agriculture and
Agri-Food Canada, London, Ontario, Canada); excised embryos from transgenic canola seeds in
which seed-specific expression is conferred by the napin promoter; and transgenic canola pollen in
which cell-specific expression is conferred by the pollen-specific promoter (Bnm1 promoter). Note
here that pollen cells are segregating as transformed and nontransformed cells indicated by the pre-
sence and absence of staining. (b) Measurement of GUS enzyme specific activity using the substrate
5-bromo-4-chloro-3-indolyl glucuronide (X-gluc). Each separate transgenic line of tobacco differs in
the level of gene expression due to variation in the influences on the inserted genes from the genetic
elements and chromatin environment at the different sites of insertion. These are often calledposition
effects(also see Fig. 9.5). To compare differences among genes and elements introduced into trans-
genic plants, analyses must account for a large number of transgenic lines to reduce the influence
of position effects. Reporter genes provide a valuable means for gathering large amounts of data.
Here, a comparison of the promoter strengths of the 35S(plant lines with theSdesignation) and
tCUP (plant lines with theTdesignation) constitutive promoters is inferred by comparing the activities
of the reporter gene. (c) To ensure that the reporter gene reflects transcriptional activity, RNase protec-
tion assays are used to measure the relative amounts of GUS mRNA accumulating in the transgenic
lines. This assay involves the formation of stable RNA duplexes with a radiolabeled antisense
RNA probe followed by RNase digestion of the single-stranded RNA molecules so that the protected
double-stranded RNA can be separated by gel electrophoresis and quantified.