COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
GUS
1. What are the benefits and limitations of the GUS Gene Reporter System in Plants?
Gene reporters enable valuable insight into gene expression. The GUS gene reporter
system is one of the popular and common plant reporter systems. GUS is short for
glucuronidase, an enzyme in the bacterium E. coli. GUS is a good reporter for plants, as it
does not occur naturally, and thus, has a low background. With some simple genetic
techniques, one can attach the promoter of the gene you want to investigate to the GUS
coding region. You can then transform your reporter construct into your plant species of
choice to monitor its expression. Transformation can be accomplished in plants via
methods like Agrobaterium-mediated gene transfer.
The GUS assay does not require the presence of any cofactors or ions for function. Beta-
glucuronidase can function through a wide range of pH values, and is fairly resistant to
thermal inactivation. However, GUS is susceptible to inhibition from certain heavy metal
ions, such as Cu2+ and Zn2+.
Additionally, the interpretation of the assay is limited by the movement of diX-indigo
throughout the cell. DiX-indigo, can associate with lipids to diffuse far from the site of
enzyme activity, which shows a lack of cytosolic localization and irregularity of substrate
penetration. This can potentially lead to an incorrect interpretation of GUS protein
localization. Despite a lack of cellular localization, nuclear localization of GUS has been
well observed. GUS assays can be carried out in the presence of potassium
ferricyanide to prevent the stain from diffusing.
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
2. What is GUS?
The GUS reporter system (GUS: β glucuronidase) is a reporter gene system, particularly
useful in plant molecular biology and microbiology. Several kinds of GUS reporter gene
assay are available, depending on the substrate used. The term GUS staining refers to the
most common of these, a histochemical technique.
3. What are substrates used for the β-glucuronidase?
There are different possible glucuronides that can be used as substrates for the β-
glucuronidase, depending on the type of detection needed (histochemical, fluorimetrical,
spectrophotometrical). The most common substrate for GUS histochemical staining is 5-
bromo-4-chloro-3-indolyl glucuronide (X-Gluc). X-Gluc is hydrolyzed by GUS into the
product 5,5'-dibromo-4,4'-dichloro-indigo (diX-indigo). DiX-indigo will appear blue, and
can be seen using light microscopy. This process is analogous to hydrolysis of X-
gal by Beta-galactosidase to produce blue cells as is commonly practiced in bacterial
reporter gene assays.
For other types of detection, common substrates are p-nitrophenyl β-D-glucuronide for
the spectrophotometric assay and 4-methylumbelliferyl-beta-D-glucuronide (MUG) for
the fluorimetric assay.
4. What is the GUS gene assay in transformed tissue?
In order to identify transformed cells or plants that have been growing on a selective
medium, it is necessary to have an easily assayable reporter gene. The most useful
reporter genes encode an enzyme activity not found in the organism being studied. A
number of genes currently are being used, however one of the most popular is the E. coli
glucuronidase. The protein has a molecular weight of 68,200 and appears to function as a
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
tetramer. It is very stable, and will tolerate many detergents, widely varying ionic
conditions, and general abuse. It is most active in the presence of thiol reducing agents
such as mercaptoethanol or DTT. It may be assayed at any physiological pH, with an
optimum between 5.2 and 8.0. The GUS gene is usually used in a gene fusion. This
means that the GUS coding sequence is under the direction of the controlling sequence of
another gene. For this exercise the GUS gene is under the control of the Cauliflower
Mosaic Virus 35S promotor.
The GUS gene was developed initially as a gene fusion marker in E. coli and in the
nematode C. elegans, but has more recently been used extensively to monitor chimeric
gene expression in plants. There is little or no detectable b-glucuronidase activity of
yeast, Drosophila, C. elegans, Dictyostelium, or in almost any higher plant.
Agrobacterium containing some of the GUS plasmids show significant GUS activity.
This seems to be due to in part read-through transcription from the gene into which the
GUS coding region might be located. Agrobacterium without these constructs shows little
if any detectable GUS activity. In order to solve this problem, one laboratory has
constructed GUS genes carrying an intron, which much be processed before expression
takes place. This totally eliminates expression in any untransformed system.
Histochemical assay
The best substrate currently available for histochemical localization of b-glucuronidase
activity in tissues and cells is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc). The
substrate works very well, giving a blue precipitate at the site of enzyme activity. There
are numerous variables that affect the quality of the histochemical localization, including
all aspects of tissue preparation and fixation as well as the reaction itself.
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
It is necessary to understand the nature of the reaction to better eliminate the variables.
The product of glucuronidase action on X-Gluc is not colored. Instead, the indoxyl
derivative produced must undergo an oxidative dimerization to form the insoluble and
highly colored indigo dye. This dimerization is stimulated by atmospheric oxygen, and
can be enhanced by using an oxidation catalyst such as a K+ ferricyanide/ferrocyanide
mixture. Without a catalyst, the results are often very good, but one must be concerned
about the possibility that localized peroxidases may enhance the apparent localization of
glucuronidase.
Fixation conditions will vary with the tissue, and its permeability to the fixative.
Glutaraldehyde which can be used, does not easily penetrate leaf cuticle, but is
immediately available to stem cross sections. Formaldehyde seems to be a more gentle
fixative than glutaraldehyde, and can be used for longer times.
Whole tissues, callus, suspension culture cells and protoplasts or whole plants or plant
organs, can be stained, but survival of the stained cells is not by means certain. After
staining, clearing the tissue with 70% ethanol seems to improve contrast in many cases.
Fluorimetric assay
Although spectrophotometric substrates for GUS are available, GUS activity in solution
is usually measured with the fluorometric substrate 4-methylumbelliferyl-b-D-
glucuronide (MUG). Fluorometry is preferred over spectrophotometry because of its
greatly increased sensitivity and wide dynamic range. The assay is highly reliable and
simple to use. Occasionally, endogenous compounds will interfere with the assay, either
by quenching or by producing high background fluorescence. In these situations,
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
fluorometric substrates with differing excitation and emission wavelengths are available
(the most popular is resorufin b-D-glucuronic acid). The substrate 4-
trifluoromethylumbelliferyl b-D-glucuronic acid (4-TFMUG) allows continuous
monitoring of GUS activity because, unlike MUG, it becomes fluorescent upon
hydrolysis at the assay pH. In contrast, after hydrolysis of MUG by GUS, the reaction
first must be terminated with basic solution. This not only stops the enzyme reaction, but
also causes fluoresce.
Spectrophotometric assay
A continuous spectrophotometric assay has been developed for detecting β-glucuronidase
activity. In the assay, Para-nitrophenyl β-D-glucuronide is cleaved to yield a
chromophoric product. With the commercial E. coli enzyme, it is demonstrated that the
reactions can be continuously monitored by the increase of absorbance at 405 nm. The
method is highly sensitive and able to detect less than 1.4 × 10-4 U/mL of the enzyme
activity in solution. Such a new assay offers significant advantages over the existing
discontinuous methods and should be useful for both routine enzyme assay and accurate
kinetic studies.
5. What are the target organisms suitable for GUS gene assay?
An organism is suitable for a GUS assay if it lacks naturally occurring β-glucuronidase
activity or if the activity is very low (background activity). For this reason, the assay is
not useful in most vertebrates and many molluscs. Since there is no detectable GUS
activity in higher plants, mosses, algae, ferns, fungi and most bacteria, the assay is ideally
suited for gene expression studies in these organisms, and considered the reporter gene of
choice for in plant science.
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR, DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE
Left: light grown and right: dark grown tobacco seedlings expressing the GUS gene
driven by the PAL1 promotor. Work of Dr. Tomoko Fugisaka Akada
References:
1. https://bitesizebio.com/33226/benefits-gus-gene-reporter-system-plants/
2. https://www.cas.miamioh.edu/~meicenrd/anatomy/Ch2_Ultrastructure/GUS_assay.html
3. https://www.future-science.com/doi/pdf/10.2144/01304rr02
4. https://en.wikipedia.org/wiki/GUS_reporter_system
(All the information is collected from above references and will be used only for teaching
and learning purposes)
BOATNY: SEM-VI, PAPER-CC14T: PLANT BIOTECHNOLOGY, UNIT 4: METHODS OF GENE TRANSFER