The two most common reporters utilized in plant biology, (and the two we

will use in this lab), are the bacterial enzyme -glucuronidase (GUS) and the
jellyfish
protein Green Florescent Protein (GFP). A reporter gene can be fused to a
gene of
interest (or just to the gene of interests promoter) to aid investigation of
that gene
we will use a reporter gene to analyze the response of Arabidopsis seedlings
to the plant hormone Auxin
Auxins are a class of plant growth hormones that play roles in almost all
aspects of plant development and growth
and the most commonly used is the highly stabile 2,4-Dichlorophenoxyacetic acid (2,4-D).
The DR5 promoter is an artificially created promoter that was made by
modifying a few
bases in a naturally occurring AuxRE. AuxREs contain the Auxin-responsive
sequence
element TGTCTC, and DR5 contains two of these in a tandem repeat. The
DR5
promoter has greater activity and Auxin responsiveness than naturally
occurring
AuxREs, and has been heavily utilized as a marker for the presence of Auxin
in plant
tissues. We will use the DR5 promoter to drive the expression of two different
commonly used markers in plant research: GUS and GFP
-glucuronidase (GUS) is an enzyme from bacteria that catalyzes the
breakdown of
Carbohydrates. -glucuronidase
GFP can be used to label genes by attaching it to a gene or promoter of
interest to
monitor gene expression. If the gene is expressed, then the GFP will also be
expressed
and the organism (or tissue) will glow green in blue light. The expression of
GFP can
also be examined by western blotting
GFP is visualized by its
green florescence under UV or blue light. Some benefits of GFP are that it
can be
visualized in living tissue and can remain attached to your protein of interest,
revealing
the sub-cellular localization of your protein.

GUS staining in the transition zone (zone 2) in wildtype but not in aux1-T root tips.
The minor effects of ethylene treatment on the DR5:GUS maximum in aux1-T root
apexes were consistent with DR5:GUS expression in aux1-7 root apexes unaffected
by the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid
(Stepanova et al., 2007). Unexpectedly, GUS staining was not observed or was
extremely weak in aux1rcr1 root apexes, regardless of ethylene treatment
DR5:GUS expression is elevated in the root tip.
Of note, ethylene inhibited root elongation, and the region below the mature zone
was largely shortened compared with no-ethylene treatment
As a result, root cells grow faster with lower than with higher auxin concentrations,
and differential cell growth is facilitated. The differential root cell growth facilitates a
curvature formation that re-orients the root growth towards gravity (
GUS and GFP are competing DNA markers used in many studies, these reporter
genes can also be used to detect multiple integration and unstable transgenes
within a transformant (Filipecki and Malepszy, 2006). They work by attaching to a
fragment of DNA of interest,
The results of GUS-PCR was expected to be present in lanes 1 and 3, therefore a
band appeared at 401bp. These expected bands were shown in the PCR output. In
lane 1 there was also a band of smaller bp which indicates DNA fragments of a
smaller size were also present in this sample. Identically GFP-PCR in the second set
of wells, the GFP gene was expected to be seen in lanes 6 and 7, therefore a band
was produced at 714bp. The expected band was shown in lane 7 of the PCR output
but very faint in lane 6
. Light microscope examination of blue-coloured transgenic hairy roots suggested
that they expressing the blue stain constitutively confirming their transformation to
express 35S GUS gene
Microscopic examination of GFP stained tomato hairy roots were visualised under a
UV where by GFP expressing cells will fluoresce. These transgenic hairy roots
showed constitutive green fluorescence proved their successful transformation (Fig.
2D). These results demonstrated the incidence of transformation with 35S GFP
Intro: In response to the threats caused by phytopathogens and plant disease, this
investigation looks at the potential of genetic modification technology as a strategy
to protect plant crops globally. Specifically, it will explore the usefulness of GUS (Glucuronidase) and GFP (Green fluorescent protein) as reporter genes in
Agrobacterium-mediated transformation of tomato (Lycopersicum esculentum L.)
and potato (Solanum tuberosum L.) hairy roots. Transformation of Agrobacterium
with GUS and GFP will be analysed using PCR to evaluate the efficacy of this vector.
Compiling knowledge of genetic markers and transformation techniques will allow
for high yield and efficient crop plant transformations.
GFP transgenic potato roots did not show as much green fluorescence as tomato
roots, and some regions of roots showed up red colour indicating the absence of
transformation in those cells. However, microscopic observations indicated that
most of potato roots had successfully transformed
GFP shows low toxicity, no interference with normal cellular activities, and is easy to
assay
GFP has been used extensively in plant systems, in localization studies and as a
screenable marker for gene transfer