PROJECT
3
PLANT
TRANSFORMATION
This project is designed to introduce the
techniques of plant transformation and the use of reporter genes to externally
monitor the patterns or timing of expression of a
gene.
BACKGROUND:
Genetic
transformation
Transformation is the introduction of DNA
representing a cloned gene into a cell so that it expresses the protein encoded
by the gene. Although the physical insertion of DNA into a cell’s nucleus
is straightforward, the expression of proteins encoded by that DNA that is
not part of a chromosome is often only transient. Introduced DNA that is
inserted into one of the chromosomes will be passed during mitosis to all
subsequent daughter cells. It is this “stable” transformation that
will allow one to introduce one copy of DNA into one cell, and then allow
the one transformed cell to regenerate a complete organism, where each cell
contains a copy of that introduced DNA. The manipulation of an organisms
DNA by transformation allows unparalleled ability to determine the function
of a gene from levels of cell function, to organismal physiology to ecological
roles. It also provides a way to dissect the functional significance of parts
of the gene or specific amino acid residues of the resulting protein.
Transformation additionally allows the engineering of plants or animals to
produce novel proteins or specifically remove expression of certain
proteins.
There are a number of ways that cloned DNA
can be physically introduced into a cell. DNA can be micro-injected into
cells, or shot into the cell on the surface of microprojectiles, or enter
through holes in the cell membrane induced by a strong electric current.
Drosophila and C. elegans are usually transformed through
microinjection. Plant transformation can take advantage of a plant pathogenic
bacteria (Agrobacterium tumifaciens) that move DNA from a plasmid
it carries into plant cells as part of its life cycle (this is described
at http://www.ejbiotechnology.info/content/vol1/issue3/full/1/).
The mechanisms of this movement will be discussed
in class. However, it is possible to manipulate the plasmid such that a gene
of interest is placed into the plasmid in Agrobacterium so that the bacteria
will introduce this DNA into a plant cell.
To transform most plants using Agrobacterium,
a single plant cell that has received the new DNA from the bacteria has to
be regenerated into a whole plant. This process involves the culturing of
the transformed cell to provide replication of that cell. Levels of plant
hormones can be manipulated to cause this mass of cells to form roots and
shoots of a regenerated plant.This process can take weeks and the details
vary from plant to plant. For Arabidospsis, an alternate method has been
developed- called dip infiltration. In this method, Agrobacterium carrying
the modified plasmid is introduced into the whole plant by submerging the
plant in a bacterial solution. Applying a vacuum can help force the bacterial
solution into the inner air spaces between plant cells, but this was found
to be not necessary. Agrobacterium will move the DNA from its plasmid into
many of these cells in the plant. Some of these transformed cells will be
used to make the flowers of the plant, including the pollen and ovules. With
the self-fertilization possible in Arabidodpis, seeds produced from these
flowers will have the introduced gene at a low rate.
The major technical problem of transformation,
regardless of the method used, is the low frequency at which it occurs. Only
a small fraction of cells where the DNA has entered the nucleus does the
DNA get spliced into a chromosome. Thus, one needs a way of identifying those
cells or plants that contain the introduce DNA. Usually, one gene included
in the introduced DNA is a selectable marker gene - for example a gene that
confers resistance against a chemical that kills normal plant cells (antibiotic
inbroader sense). Kanamycin is one such antibiotic that kills plant cells.
Including a kanamycin resistance gene along with a gene of interest in the
Agrobacterium vector allows one to select transformed plants by growing them
on kanamycin. Only transformed plants will survive since they express the
introduced kanamycin resistance gene. For the Agrobacterium infiltration
method, the seed from the infiltrated plants are plated on agar containing
kanamycin – the low number of plants containing the introduced DNA will
germinate and grow on these plates.
We will use transformation to determine the function of genes through
reverse genetics. The cloned genes we will use are members of moderate to
large gene families in Arabidopsis. The encoded sequence of the genes clearly
suggest the molecular function by the presence of conserved protein motifs.
One gene is a cellulase that digests the cellulose in the cell walls of plants.
Arabidopsis has more than 12 cellulase genes. Another three genes we will
study are myb DNA binding proteins. Arabidopsis has 125 myb genes that are
expected to act as transcription factors. A fifth gene we will study is a
homeodomain protein also expected to be a DNA-binding transcription factor.
Although the biochemical activity can be predicted from the sequence, the
function of these genes would not be predictable from the sequence. To determine
the process in which these genes function, we will transform modified versions
of theses genes into Arabidopsis and determine how the phenotype of the plant
changes. This change in phenotype can then be linked to the function of the
genes.
Reporter genes
Arabidopsis contains nearly 26,000 genes.
Some of these genes are expressed at most times in every cell. However, the
majority of genes are only expressed in certain organs of the plant, either
causing that organ to be different than other organs, or adding function
to that organ. Further, many genes are only expressed under certain developmental
or environmental conditions, in response to internal or external cues. Since
the expression of genes is often regulated by transcription, the promoter
(section of DNA preceding the coding region), will contain the information
that allows the gene to be turned on or off in different organs or in response
to cues. The Cauliflower mosaic virus (CaMV)35S promoter is one of
the few plant promoters that is expressed in most every tissue at all times,
called constitutive.
An important clue to the function of a gene
is to determine where and when it is expressed. If it is expressed
only in flower stamens, then it is apparent that it has some role in male
gamete formation or stamen development. If it is only expressed under certain
conditions, such as after exposure to damaging UV light, it would be apparent
that the gene has a role in responding to such stress or repair. There are
several ways of determining where and when a particular gene is expressed
in a plant. One way is to use hybridization to detect the amount of mRNA
corresponding to a cloned gene in samples from different parts of a plant,
sampled after different treatments of the plant. This approach is quantitative
but is time-consuming and provides only as much time or organ resolution
as the researcher has patience for separating different parts of many plants
to gather sufficient quantities of mRNA samples. Another approach is the
use a reporter gene. A reporter gene produces a protein that is easily detectable
in transformed organisms. Often, the protein possesses an enzymatic activity
that can turn a colorless substrate into a colored product. Thus, one can
see the location and amount of gene expression in a transformed organism
by looking at the location and intensity of the colored product .
The b-galactosidase
(lacZ)
and b-glucuronidase (GUS) genes are two examples of these
reporter genes. When the reporter gene is fused to the promoter of the gene
of interest, the reporter gene will be expressed only at the times and locations
where that gene is expressed since the promoter often determines transcription.
This provides a method to detect a very limited expression of a gene, such
as in small patches of cells (like root tips or pollen) or at certain times
(such as after a certain stress or hormone treatment).
An important property of reporter genes is
that their activity is absent in the organism in which they will be used.
Both lacZ and GUS are genes from E. coli. Plants posses some LacZ
activity, and so it is difficult to use it as a reporter gene because one
doesn’t know if
the b-galactosidase staining if
from the introduced gene or the native plant gene. In contrast, GUS activity
is normally very low in plants, and so is a common reporter gene used in
plant studies.
cellulase -AT1G64390
Myb60
Homeodomain -AT1G79840.1
Gene Overexpression
Another method of determining a gene's function is to either mutate it so
that it is not expressed, or cause it to be overexpressed. Either should
perturb the process in which it participates and cause a change in phenotype.
Gene disruption can utilize transformation. However, redundancy in Arabidopsis
genes can compensate for the loss and phenoype changes can be too subtle
to detect. Overexpression of the gene offers fewer complications. The coding
region of the gene is fused to the constituitive CaMV 35S promoter
by manipulating the DNA sequences in E. coli. We will introduce these DNA
constructs into Arabidopsis through Agrobacterium and look for a change in
the phenotype of the plant. An alterered phenotype should result from
overexpression of the gene and so should be related to the function of the
gene.
MYB0
MYB75
OVERVIEW:
Transformation
1: Transformation of Arabidosis
plants
Students will use receive a culture of Agrobacterium that carries recombinant DNA.This culture will be used for infiltration to transform wild-type Arabidopsis. Two forms of recombinant DNA will be used, both containing a GUS reporter gene but it is fused to different promoters.
Read
instructions:Transformation of
Arabidopsis with infiltration
Description of agrobacterium
transformation:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc731/transgenic/transgenic2.htm
Use of reporter
genes:
http://www.ndsu.nodak.edu/instruct/mcclean/plsc731/transgenic/transgenic4.htm
Full Transformation
protocol:
http://plantpath.wisc.edu/~afb/protocol.html
When do you think the DNA is moved from
Agrobacterium to a plant cell in this process?
Although this may occur in multiple cells,
most will not matter. What is the one cell that will matter when it receives
the DNA so that the gene is passed onto the seeds?
Transformation
2: Collection and planting
of T1 seeds onto selective media
Students will collect the seed from the
transformed plants in lab 16 and let them dry one week.In this lab, sterilize
them and plant them onto kanamycin-containing agar medium.This will provide
a selection such that only transformed plants will
grow.
Read instructions:
Selection of transformed Arabidopsis
seedlings
A concern about transformed plants is that
kanamycin resistance is passed into the plant as well.What would you need
to do if you did not have a kanamycin-resistance gene in the transforming
DNA along with the reporter gene?
Web resources:
Picture of kanamycin selection:
http://www.bioinformatics.vg/Images/selectionpic.jpg
What frequency does a
transformed seed appear in all seed from infiltrated
plants?
The seedlings grown under inducing and
non-inducing conditions will be stained with X-Gluc, a histochemical stain
for GUS activity.Patterns of staining will be observed in lab
24.
Read
instructions: GUS
staining
X-gluc reaction:
http://www.biology.purdue.edu/people/faculty/karcher/blue2000/fig1.gif
Sample patterns of GUS expression in promoter
trap lines:
http://www.dartmouth.edu/~tjack/#Sample
Patterns
What tissues do you see staining in for each
plasmid- is there tissue specificity?
Does the pattern or intensity of blue color change with time?
Are there any tissues excluded from staining?
Could this be due to reasons other than the lack of promoter activity in
these?
What is the implication about function of these genes used with the reporter genes?
What has changed in the plants in which we overexpressed the two transcription
factors? What process might these transcription factors control?
1. Plant preparation. Arabidopsis seeds are
planted on top of cheese cloth held on top of soilless mix in a pot. Plants
have been grown already under a day/night period and at a low density so
as to create healthy plant. Plants need to have bolted.These primary bolts
are removed so that a secondary bolt has developed. Infiltration is done
3-5 days after clipping.
2. Bacterial preparation. Two Agrobacterium
strains have been grown, each carries a different plasmid.Liquid cultures
(300 mls) of each have been grown.
4. Invert the pot over a dish.Pour the bacteria
solution into the dish so that the bolts are immersed. Let sit for 5
minutes.
5. Remove the plant from the dish and let
it sit on its side in a tray over a paper towel so let excess solution fall
off. Place a plastic dome over the reclining plants. This will be removed
after 24 hours and the plants will be set upright.
6. The plants will be grown for 3-4 weeks
when the seeds will be collected.
1. Collect seed and weigh 0.1 gm of seed into
a 15 ml sterile test tube.
2. Add 10 mls of 70% ethanol and shake for
20 min
3. Decant off ethanol, add 10 mls 0.5% Tween,
remove, wash with 10 mls sterile distilled water.
4.Add to 8 mls of 1%
Agar.
5. Sow 5000 seeds under sterile conditions
on a 150x15 mm petri plate containing the kanamycin selective MS medium.Close
the dishes with only 2 pieces of adhesive tape to prevent high levels of
humidity.
6. Place the dishes at 4°C for 2
days.
7. Transfer the trays to the light rack-
preferably a higher shelf with warmer temperatures..
8. Transformants (green rooted plants- dark
green cotyledons and true leaves) are scored 8 days later for kanamycin
selection.
9. Carefully remove plants from the agar and
transfer onto agar containg different inducing
conditions.
Kirsten Bomblies, adapted from François
Parcy's protocol
1. 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.
2. When all samples are harvested, place at
room temperature for 20 minutes.
3. Remove acetone from the samples, and add
staining buffer on ice.
4. Add X- Gluc to the staining buffer to a
final concentration of 2mM - from a 100mM stock solution of X-Gluc in DMF-
this must be kept in the dark at -20°C .
5. Remove staining buffer from samples and
add staining buffer with X-Gluc on ice.
6. Infiltrate the samples under vacuum, on
ice, for 15 to 20 minutes. 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.
7. Incubate at 37°C (I usually do it
overnight, but it depends on transgene strength. It is not advisable from
my experience to go too long (over two days) as the tissue seems to begin
deteriorating during long incubations.
8. Remove samples from incubator and remove
staining buffer. Go through an Ethanol series in which samples are incubated
successively in 20%, 35% and 50% ethanol at room temperature for 30 minutes
each.
9. Incubate in FAA (recipe below) for 30 minutes
at room temperature to fix the tissue.
10. Remove FAA and add 70% ethanol. At this
point the tissue can be stored at 4°C for long periods, or examined
under the microscope
Staining Buffer (final
concentrations):
(make at time of use, do not prepare ahead
of time)
0.2% Triton X-100
50mM NaHPO4 Buffer
(pH7.2)
2mM Potassium
Ferrocyanide
2mM Potassium
Ferricyanide
Water to volume
10% Triton X-100
0.5M NaHPO4 Buffer
(pH7.2)
100 mM Potassium Ferrocyanide (Store in the
dark at 4°C)
100mM Potassium Ferricyanide (Store in the
dark at 4°C)
100mM X-Gluc (5-bromo-4-chloro-3-indolyl
ß-D-glucuronide cyclohexamine salt) in DMF
50% Ethanol
5% Formaldehyde
10% acetic acid
water to volume
