Tracking GUS Gene Activity in Tobacco Plants
Laura Cruz and Dr. Xing-Hai Zhang
Department of Biological Sciences, Florida Atlantic University
Question
Results
What is the effect of gene insertion location on GUS
activity?
Introduction
Figure 1. T-DNA with GUS gene driven by CaMV 35S promoter, with NOS terminator. NPTII gene confers
kanamycin resistance for plant cell selection. RB: right border; LB: left border. The size is not to scale.
• Out of the three GUS positive plant lines KEG-2
was confirmed to be leaf tissue specific for GUS
expression.
• Agrobacterium is a pathogenic bacterium that
has the ability to transfer a part of its DNA (TDNA) into a plant’s DNA [1]. In the lab it is used
for the genetic modification of plants.
• The other two GUS positive plants expressed
GUS activity in both leaves and roots.
• When using Agrobacterium to insert a gene of
interest into plant cells, the plant DNA will be
altered. The alteration is random and the location
of the newly inserted gene in the plant genome
is unpredictable.
• The ȕ-glucuronidase (GUS) gene was isolated
from Escherichia coli bacterium [2]. When the
gene product GUS reacts with a chemical
reagent it produces a blue pigment in the plant
visible to the naked eye. For this reason it is
widely used to track gene activity.
Method
1. The GUS gene and the kanamyacin resistance
gene were introduced into tobacco leaves by
cutting them into 1mm² disks and infecting
them with the Agrobacterium tumefaciens TDNA (pBI121) (fig.1).
2. The leaves were then regenerated in MS
media containing the antibiotic kanamyacin and
grown to plants (fig.2a,b).
3. Modified tobacco plants were confirmed by
using PCR analysis (fig. 3) and the GUS
reagent (fig. 2c).
4. The seeds from the modified plants were
cultured on Kanamyacin containing media.
5. The seedlings from each plant line were scored
either resistant or sensitive to kanamyacin and,
if resistant, tested for tissue specificity using
the GUS reagent (fig. 4).
6. Seedlings showing specificity were transferred
to pots and will be tested for GUS activity by
incubating samples of leaf, stem, root, flower,
and seed pod in the GUS reagent at 4, 8, and
12 weeks old.
7. DNA analysis of GUS expressing plants will be
done to identify the location of GUS gene
insertion.
• Three plant lines (LVC-1, KEG-2, LMC-5) were
revealed to be GUS positive when tested with
the GUS reagent (table 1).
a
• None of the GUS positive plants expressed the
gene in the stem.
b
• The HD plant line was able to thrive in the
kanamycin media but did not express any GUS
activity when tested with the GUS reagent.
Discussion
c
Figure 2. (a) Regenerated tobacco leaves on MS
regeneration media. (b) modified tobacco plant
growing on rooting media. (c) A piece of tobacco leaf
expressing the GUS gene product (left) and a piece of
leaf not expressing the GUS gene product (right).
Figure 3. GUS gene in transformed plants. A bright
band indicates the presence of 525 bp-long GUS
DNA. From left to right lanes: DNA ladders, Wt
(negative control), AP (positive control), and KEG-HD
(experimental transformations).
Table 1. Results for GUS gene expression in different
tissues for each plant line. S= strong expression, W =
weak expression, and N= no expression.
GUS Gene Expression
Plant Line
a
b
Figure 4. (a) Root sample from plant line LC-1
expressing the GUS gene product. (b) Root sample
from KEG-2 not expressing the GUS gene product.
• Results to date seem to indicate some variation
in tissue specificity of GUS expression between
plant lines. Specificity implies that the GUS gene
product is not present in all cells of the plant.
While the introduced gene may be present in the
cell, for unknown reasons it is not producing the
gene product.
• Plant line HD is a good example of this.
Although it contains the GUS gene in its leaves
(fig. 2) and thrives in the kanamyacin containing
media it does not express any GUS gene activity
when tested with the GUS reagent.
Roots
Stem
Leaves
LVC-1
S
N
W
HD
N
N
N
References
HD-1
N
N
N
KEG-2
N
N
S
LMC-5
S
N
S
[1] T. Tzfira, V. Citovsky, From host recognition to TDNA integration: the function of bacterial and plant
genes in the Agrobacterium-plant cell interaction,
Molecular Plant Pathology 2000, 1 (4), 201-212
JMR
N
N
N
Laura Cruz would like to thank the FAU Undergraduate Research Grant for funding the study, Dr. Xing-Hai Zhang for providing the
support needed throughout the research, Noah Kaplan for assisting with lab work, and Lorraine Chaljub for sharing her pictures.
• Identifying the location of insertion will help
define the mechanisms of GUS gene integration
in the plant genome, its activity in the plant cell,
and possible effects on the genomic structure.
[2] L.J.W. Gilissen, P.L.J. Metz, W.J. Stiekema, J-P.
Nap, Biosafety of E. coli ȕ-glucuronidase (GUS) in
plants, Transgenic Research 1998, 7, 157-163