- β-glucuronidase (GUS) is a commonly used reporter gene in plant molecular biology and genetic engineering to indicate successful introduction of foreign DNA into cells. - GUS expression can be detected through fluorometric or histochemical assays, allowing visualization of promoter activity, protein localization, and transgenic events. - The GUS gene is fused to genes of interest, and GUS activity is used to study processes like tissue-specific expression, response to stresses, and transformation efficiency. - While destructive, GUS is a stable and non-toxic reporter enabling versatile applications in fundamental and applied plant research.

1. Pradeep Kumar
MSc. (Ag.) Biotech.

2. • A selectable marker is a gene introduced into a cell, especially
a bacterium or to cells in culture, that confers a trait suitable
for artificial selection.
• They are a type of reporter gene used in
laboratory microbiology, molecular biology, and genetic engineering
to indicate the success of a transfection or other procedure meant to
introduce foreign DNA into a cell.
• Selectable markers are often antibiotic resistance genes; bacteria that
have been subjected to a procedure to introduce foreign DNA are
grown on a medium containing an antibiotic, and those
bacterial colonies that can grow have successfully taken up
and expressed the introduced genetic material.

3. • Normally the genes encoding resistance to antibiotics such
as ampicillin, chloroamphenicol, tetracycline or kanamycin, etc.,
are considered useful selectable markers for E.coli.
• Selectable marker genes can be divided into several categories
depending on whether they confer positive or negative selection
and whether selection is conditional or non-conditional on the
presence of external substrates.
• Positive selectable marker genes are defined as those that promote
the growth of transformed tissue whereas negative selectable
marker genes result in the death of the transformed tissue.

4.  Non-selectable marker genes or reporter genes have been very
important as partners to selectable marker gene systems.
 They have been used in co-transformation experiments to
confirm transgenic events where escapes may be common.
 They have been used to improve transformation systems and
the efficiency of recovering transgenic plants by allowing the
visual detection of transformed tissues.
 Non-selectable marker genes or reporter genes may aid in the
identification of the transformed cells.

5.  Green fluorescent protein (GFP) has been particularly
important in the development of these strategies, GFP has
become a valuable tool for monitoring gene expression in
field trials and for following pollen flow.
 Although destructive assays are needed to measure the
activity of reporter genes such as GUS,
 As a reporter, luciferase (LUC) can be monitored in living
tissue but this requires specialized detection equipment.

7.  In 1987, Richard Jefferson et.al, demonstrated the application of
a new reporter gene system in transgenic plants.
 The reporter gene was the uid A gene of Eascherichia coli that
encode the enzyme β-glucuronidase (gus).
 Uid A gene has become one of the most widely used reporter gene
in plant molecular biology and microbiology.
 The most frequent use of the gus gene is as a reporter gene for
promoter analysis, in both transient assays and in stable
transformed plants.

9.  It has been used to identified promoter elements involved
in many aspects of regulation of gene expression such as
tissue specific and developmental regulation hormonal
regulation, response to wounding and photoregulation.
 The gus gene has also been used as a reporter gene in
promoter trapping studies as a marker for the development
of plant transformation procedure and to study the
machanism of Agrobacterium tumefaciens mediated
transformation.

12.  There are different possible glucuronides that can be used as
substrates for the β-glucuronidase, depending on the type of
detection needed (histochemical, spectrophotometrical,
fluorimetrical).
 The most common substrate for GUS histochemical staining
is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc): the
product of the reaction is in this case a clear blue color.
 Other common substrates are p-nitrophenyl β-D-
glucuronide for the spectrophotometric assay and 4-
methylumbelliferyl β-D-glucouronide (MUG) for the
fluorimetrical assay.

14. GUS-system in Arabidopsis thaliana
Structure of GUS

16.  Harvest tissue and place in cold 90% Acetone on ice. This should
stay on ice until all samples are harvested. For sample containers,
Eppendorf tubes and glass scintillation vials work well.
 When all samples are harvested, place at room temperature (RT)
for 20 min.
 Remove acetone from the samples, and add staining buffer on ice.
 Add X- Gluc to the staining buffer to a final concentration of 2
mM from a 100 mM stock solution of X-Gluc in DMF- this must
be kept in the dark at -20 °C.
 Remove staining buffer from samples and add staining buffer
with X-Gluc on ice. Infiltrate the samples under vacuum, on ice,
for 15 to 20 min. Release the vacuum slowly and verify that all
the samples sink. If they don't, infiltrate again until they all sink
to the bottom when the vacuum is released.

17.  Incubate at 37 °C (I usually do it for 2 h for strong promotors
and up to overnight for weak promotors. It is not advisable from
my experience to go too long (over two days) as the tissue
seems to begin deteriorating during long incubations.
 Remove samples from incubator and remove staining buffer.
 Go through ethanol series from 10%, 30%, 50%, 70% (you may
heat the sample to 60 °C to get rid of chloroplasts), to 95%
(avoid light); 30 min each step and then finally 100%. You may
store at 4 °C for up to a month, seal well.
 Go to embedding procedure, or observe directly under
dissecting or light microscope. To mount, simply apply a few
drops of water to the samples.

18.  β-Glucuronidase (GUS) is a very versatile reporter of gene
expression that is frequently used in plant molecular biology.
 The diverse applications of the GUS gene fusion systems (Gallagher,
1992) are based on the detection of the enzymatic activity of GUS in
protein extracts or in tissues using fluorometric and histochemical
assays respectively.
 The histochemical assay has also been used for sub-cellular
localization of GUS fusion proteins, e.g. for the nuclear targeting of
important regulatory proteins (for review see Raikehl, 1994).
 A novel application of the GUS reporter was demonstrated for
protein fusions with the A. thaliana ATHSFI heat shock
transcription factor (Lee et al., 1995) using a fluorescence activity
staining protocol following gel electrophoresis.

19.  A useful feature of GUS is that it can be fused with other
proteins. Ex- GUS fusions with selectable marker genes
such as nptII allow the visualization of transformation in
addition to selection.
 GUS expression was used as a reporter to help detect
transformation events in tissue culture during the
production of a number of plant lines approved for
commercialization.

20.  Heat stress treatment- Approximately 2 g plant tissue (leaves) are
incubated at 37°C for 30 min. to two hours in an shaking water
bath in SIB-puffer. Control tissue is incubated in a buffer at 25 °C.
 Cell disruption- Tissue is washed in cold water, blotted dry with
paper towels and subsequently grind (0.3 g) in 100 µl extraction
buffer in a 1.5 ml cup.
 Centrifugation- Spin down in a microfuge for 10 minutes at
12000 rpm at 4 °C and collect the supernatant in a fresh cup.
 Native page - Pour 5% or 7% polyacrylamide gel in Tris pH 8.8
buffer, apply Tris/Glycine (6 g/ 15 g/l ) running buffer, load native
protein samples up to 50 µg/lane, and molecular weight standards
(Pharmacia) and run over night at 70 V.

21.  GUS activity staining (fluorescence method). Rinse gel with 50
mM Na-Phosphate buffer (PH 7) and incubate immediately in
a solution containing 0.5 mM MUG in phosphate buffer for 10
min, 37°C.
 Visualization, recording. Following electrophoresis the GUS
fusion proteins are visualized immediately by fluorescence of
the generated MU on a UV transilluminator at 360 nm. The
bands can be recordered photographically using Polaroid 667
film.

23.  The expression and regulation of a rice Glycine–rich cell wall
protein gene, osgrp1, transgenic rice plants were regulated that
contain the osgrp1 promoter or its 5’ deletions fused with the
bacterial β -glucuronidase (GUS) receptor gene.
 In root, of transgenic rice plants, GUS expression was specifically
located in cell elongation and differentiation regions and no gus
expression was detectable in apical meristem and the mature
region.
 In shoot, apices Gus activity was detected only in those leaf cells
which were starting to expend and differentiate and little Gus
activity was expressed in mature leaves or mature parts of
developing leaves.
 Gus expression was not detected in the apical meristem and the
young meristematic leaf primordia.
 Gus activity was highly expressed in the young stem tissue
particularly in the developing vascular bundles and epidermis.

24.  The expression of the osgrp1 gene is closely associated with cell
elongation expansion during the post-meiotic cell differentation process.
 The osgrp1 gus gene was also expressed in response to wounding and
down regulated by water stress condition in the elongation region of roots.
 Promoter deletion analysis
indicates that both positive and
negative mechanism are
involved in regulating the
specific expression patterns.

25.  GUS was expressed in transgenic yeast on a Multiplecopy vector
under the control of the alcohol dehydrogenase 1 (ADH1) promoter.
 GUS as a reporter for targeting proteins into different subcellular
compartments in vivo, we fused the presequence of the
mitochondrial tryptophanyl tRNAsynthetase gene (MSW) to the
amino terminus of GUS.
 Enzyme is stable in yeast and its activity may be monitored by very
sensitive colorimetric or fluorometric methods in extracts, or by the
histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X-
Gluc) on plates.

26.  GUS expression was used as a
reporter to help detect transformation
events in tissue culture during the
production of a number of plant lines
approved for commercialization.
 These lines include Bollgard II®
cotton, the glyphosate resistant sugar
beet line GTSB77 (variety
InVigorTM), papaya line 55-1, three
soybean lines with modified fatty
acid content (G94-1, G94-19, G168)
and two PPT tolerant soybean lines
(W62 and W68) .
 With 91 records, GUS is the most
frequently listed reporter gene in the
US field trials database in 2001 and
2002.

28.  GUS enzyme is very stable within plants and is non-toxic when
expressed at high levels.
 A useful feature of GUS is that it can be fused with other proteins i.e.
GUS fusions with selectable marker genes such as nptII allow the
visualization of transformation in addition to selection.
 Humans and animals are continuously exposed to GUS from bacteria
residing in their intestinal tracts and from non-transgenic food sources
without harmful effects; therefore, the low level of GUS protein from
genetically modified plants is not a concern with regard to toxicity or
allergenicity.

29.  The major drawback with the use of GUS as a reporter is that the
assays are destructive to the plant cells.
 Reporter assays in a given system can be readily adapted to almost
any gene of interest, without the need to develop separate assays
for individual gene products, which are sometimes very difficult or
laborious.

30. THANK YOU