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11 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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BICD 123

Spring 2007






Yanofsky



Genetic Engineering of Arabidopsis


Overview:


Genetic modification of plants
, in the form of genetic engineering, offers tremendous
opportunities for the improvement of crop plants. These improvements include the
engi
neering of disease and insect resistance into plants as well as the possibility to greatly
increase crop yields. These improvements offer the possibly of reducing our reliance on
harmful pesticides and increasing food production to keep pace with a growin
g world
population. At the same time, there remains a great debate regarding the possible
negative impacts of genetically modified crop plants. Are they safe to eat? Will these
“transgenes” escape into the wild, making “superweeds”? These and other que
stions
offer a great opportunity to explore the positive and negative consequences of utilizing
this powerful technology.


While we are unlikely to solve this debate during this class period, we will demonstrate
the value of genetic engineering in terms of

its contributions to basic research. During
this class period, we will genetically engineer Arabidopsis plants using a “reporter”
construct that will allow us to monitor the activity of a specific promoter..


Agrobacterium

is a soil organism that has the

natural ability to genetically engineer host
plants. It does this by transferring a specific segment of DNA into the recipient plant cell.
Plant biologists take advantage of this natural process by tricking the Agrobacterium into
transferring a desired
segment of DNA. As you’ll see, the process of genetically
engineering Arabidopsis with Agrobacterium is extremely easy. Although
Agrobacterium can also be used to engineer a wide variety of host plants, the procedures
are much more time
-
consuming for pla
nts other than Arabidopsis, because other plants
typically require the use of tissue culture.


2

Analysis of expression patterns using the GUS reporter gene


During this ten
-
week lab course, we will use several different methods for analyzing gene
expression
, including the use of promoter
-
GUS fusions, analysis of RNA in situ
hybridizations and protein accumulation. Today we will begin the promoter
-
GUS fusion
experiment.


A commonly used technique for studying gene expression
involves

creating a
“reporter” co
nstruct where the promoter of the gene of interest is placed upstream of the
reporter gene. This construct is transformed into plants and the expression of the reporter
gene is monitored. If the promoter region contains all of the important regulatory
in
formation for the gene of interest, the expression of the reporter gene should mimic the
normal expression pattern of the gene of interest. In plants, GUS and GFP are commonly
used reporter genes. The GUS system is very similar to the LacZ (beta
-
galactos
idase)
system used in animals and bacteria. The GUS (beta
-
glucuronidase) enzyme cleaves the
colorless X
-
Gluc substrate to produce a blue precipitate, which is then visualized.


In today’s experiment, we will be studying the promoter region of an “unknow
n”
gene. In other words, it is your job to determine where this promoter is active by
generating transgenic plants and monitoring the activity of the GUS reporter gene. The
presumed promoter region of the
UNKNOWN

gene has been fused to the GUS reporter,
and the resulting construct is referred to as UNKNOWN::GUS. This construct has been
inserted into a typical plant transformation vector that is suitable for Agrobacterium
-
mediated plant transformation. This plant transformation vector also contains a sel
ectable
marker, kanamycin resistance, which will later allow us to identify transformed plants by
their ability to germinate in the presence of the kanamycin antibiotic. We will generate
kanamycin resistant plants that harbor the UNKNOWN::GUS reporter and

we will
monitor the activity of the
UNKNOWN

promoter by following the expression of the GUS
marker gene.



The plant transformation steps that you will be carrying out can be briefly summarized as
follows:


A.

Dip normal Arabidopsis plants into solution o
f Agrobacterium.

B.

Allow plants to grow to maturity and harvest the seeds.

C.

Germinate seeds in the presence of the antibiotic kanamycin to select for plants
that have been engineered.

D.

Analyze transgenic plants for GUS expression.


Note: Steps 1
-
3 wil
l be done in advance by the TAs


Plant growth

1. Grow about 5 plants per pot in small pots.


2. When plants begin to bolt, cut off the tip of the emerging inflorescence to induce the
growth of secondary inflorescences.




3

Agrobacterium infection

3. Ino
culate a culture of LB media (plus antibiotic) with the Agrobacteria carrying the
UNKNOWN::GUS construct you wish to introduce into the plants.


4. Harvest cells by centrifugation (5k, 10 min., preferably at room temp.) and resuspend
in 2 X volume infilt
ration medium.


5. Add 200 ml of Agrobacterium suspension to a 400 ml beaker or Magenta jar and invert
a pot with plants into the suspension. Be sure that entire plants are submerged and no
large air bubbles are trapped at the center of a rosette.


You ca
n insert a pipet along side of pot to hold pot in position.


Leave plants submerged for 30 seconds.


6. Remove pots from beakers, and lay them on their side in a plastic flat and cover with a
plastic dome to maintain humidity. After one day, uncover the
flat and set the pot
upright.


7. Grow 3 to 4 weeks.



8. Harvest seeds.


Selection of transformants

Seeds will be sterilized and plated onto petri dishes containing media and an antibiotic
which will only allow the growth of transformed plants.


1. Surfac
e
-
sterilize seeds.

Treat seeds:

2 min. in 70% ethanol 0.1% Triton
-
X



Remove 70% ethanol and wash with 95% Ethanol 2 times.

Suck up seeds and squirt on 3MM filter disc and let dry

Evenly sprinkle seeds on Petri dish


2. Seal the sides of the petri dish wit
h micropore tape.


Plant up to 100
µ
l dry seeds per plate. This will be the upper limit of density to have an
efficient screening.


3. Leave plates for 2 days in cold room. Move plates to growth room.


4. After 7
-
10 days, transformants can be easily ident
ified as
dark green

plants with
long

roots. Transfer these seedlings to soil. Keep covered for several days.



4

5. Use GUS
-
staining methods (provided separately) to monitor expression of the
UNKNOWN::GUS reporter.


Materials

Infiltration medium:

1/2 x Murash
ige
-
Skoog salts

5% sucrose


pH 5.7 with KOH

0.2% Silwet L
-
77 (Union Carbide Chemicals and Plastics)*




Selection plate:

1/2 x Murashige
-
Skoog salts


pH 5.7 with KOH

0.65% agar

50mg/L kanamycin



5

The Agrobacterium
-
plant interaction


Introduction:



Agroba
cterium tumefaciens is often called "nature's genetic engineer" because it
has the natural ability to genetically transform dicotyledonous plant hosts. It has only
been approximately twenty years that people have been capable of genetically engineering
pl
ants, whereas this remarkable bacterium has been successfully transforming plants for
millions of years. In fact, our ability to introduce foreign DNA into plant cells is largely
dependent on our knowledge of the Agrobacterium
-
plant interaction. What ben
efit does
Agrobacterium gain from genetically transforming plants? Remarkably, the bacterium
tricks the plant into providing a niche that only it can successfully colonize.



Agrobacterium is a pathogen of plants and induces tumors, or "galls", on infec
ted
plants. All virulent, or tumorigenic, strains of Agrobacterium harbor a large
tumor
-
inducing

(
Ti
) plasmid. During infection, a portion of this Ti plasmid, called the
T
-
DNA

(transferred
-
DNA), is physically transferred from the bacterial cell into the
plant cell,
where it becomes stably integrated into the plant chromosomal DNA. This T
-
DNA is
expressed in plant cells, resulting in the expression of several "bacterial" genes. Some of
these T
-
DNA genes code for enzymes that calalyze the synthesis of pla
nt hormones
(
phytohormones
)
auxin

and
cytokinin
. It is the overproduction of these hormones within
the transformed plant cells that results in the uncontrolled cell division characteristic of a
tumor. Overproduction of both of these phytohormones results

in an unorganized tumor.
However, when the auxin gene is mutated, the resulting Agrobacterium strain induces
"shooty" tumors due to the overproduction of cytokinin. Similarly, when the cytokinin
gene is mutated, the resulting tumors often display a "roo
ty" morphology due to the
overproduction of auxin. It is also important to recognize that many factors influence
tumor growth and morphology, such as the plant host and the site of infection.



The T
-
DNA also codes for enzymes that synthesize tumor
-
specif
ic compounds
called
opines
. Opines are usually simple derivatives of amino acids that can be utilized
by Agrobacterium as a sole source of carbon, nitrogen, and energy. Thus, Agrobacterium
has evolved a very sophisticated scheme to provide a niche for it
self. First it recognizes a
suitable plant host and transfers its T
-
DNA into the plant genome. This results in the
overproduction of phytohormones, and hence tumor formation. In addition, the plant
converts its photosynthate into the production of opine
s that the infecting bacterium has
the unique ability to catabolize. The bacteria then proliferate in this tumorous mass
utilizing the opines as a food source.



This remarkably simple system is actually very complex. How do the bacteria
specifically t
ransfer the T
-
DNA region into plant cells? A second region of the Ti
-
plasmid, called the VIR region is responsible for carrying out the T
-
DNA transfer
process. The VIR region contains many genes, called
VIR A
-
VIR G
, all of which are
required for efficien
t T
-
DNA transfer, and hence for tumor formation, to occur. Mutation
of any of these genes thus reduces or abolishes tumorigenesis.



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