Transcript Agrobacterium tumefaciens

_____________________22
Gene technology makes it possible to
alter plants to meet requirements of
agriculture, nutrition, and industry
1
• Recent years have witnessed spectacular
developments in plant gene technology.
• In 1984 the group of Marc van Montagu and
Jeff Schell in Gent and Cologne, and the group
of Robert Horsch and collaborators of the
Monsanto Company in St. Louis, Missouri
(United States) simultaneously published
procedures for the transfer of foreign DNA
into the genome of plants utilizing the Ti
plasmids of Agrobacterium tumefaciens (new
nomenclature:Rhizobium radiobacter).
2
• The Ti plasmid (tumor-inducing plasmid)
of Agrobacterium tumefaciens has been
developed as a vehicle for introducing foreign
genes into plants.
When Agrobacterium infects plants, a region
of the Ti plasmid called the T-DNA is taken up
by the plant cell and incorporated into one of
its chromosomes.
3
• This method has made it possible to alter the
protein complement of a plant specifically to
meet special requirements:
- for example, to render plants resistant to
pests or herbicides,
- to achieve a qualitative or quantitative
improvement in the productivity of crop plants,
- and to adapt plants so that they can produce
defined sustainable raw materials for chemical
industry.
4
22.1 A gene is isolated
5
• Let us consider the case where a transgenic
plant A is to be generated, which synthesizes a
foreign protein (e.g., a protein from another
plant B). For this,
• the gene encoding the corresponding protein
first has to be isolated from plant B.
• Since a plant probably contains between
25,000 and 50,000 structural genes, it will be
difficult to isolate a single gene from this very
large number.
6
A gene library is required for the isolation of a gene
• To isolate a particular gene from the great
number of genes existing in the plant genome,
it is advantageous to make these genes
available in the form of a gene DNA library.
• Two different kinds of gene libraries can be
prepared.
7
• To prepare a genomic DNA library, the total
genome of the organism is cleaved by
restriction endonucleases into fragments of
about 15 to several 100 kbp.
• Digestion of the genome in this way results in
a very large number of DNA sequences, which
frequently contain only parts of genes.
• These fragments are inserted into a vector
(e.g., a plasmid or a bacteriophage) and then
each fragment is amplified by cloning, usually
in bacteria.
8
• To prepare a cDNA library, the mRNA
molecules present in a specific tissue are first
isolated and then transcribed into corresponding cDNAs by reverse transcriptase.
• The cDNAs are inserted into a vector and
amplified by cloning.
• The mRNA is isolated from a tissue in which
the corresponding gene is expressed to a high
extent.
9
• In contrast to the fragments of the genomic
library, the resulting cDNAs contain entire genes
without introns and can therefore, after transformation, be expressed in prokaryotes to synthesize proteins.
• Since a cDNA contains no promoter regions, such
an expression requires a prokaryotic promoter to
be added to the cDNA.
• To prepare a cDNA library from leaf tissue, for
example, the total RNA is isolated from the
leaves, of which the mRNA may amount to only
2%.
10
• To separate the mRNA from the bulk of the
other RNA species, one makes use of the fact
that eukaryotic mRNA contains a poly(A) tail
at the 3’ terminus.
• This allows mRNA to be separated from the
other RNAs by affinity chromatography.
• The column material consists of solid particles
of cellulose or other material to which a polydeoxythymidine oligonucleotide [poly-(dT)] is
linked.
11
• When an RNA mixture extracted from leaves is
applied to the column, the mRNA molecules
bind to the column by hybridization of their
poly-(A) tail to the poly-(dT) of the column
material, whereas the other RNAs run through
(Fig. 22.1).
• With a suitable buffer, the bound mRNA is
eluted from the column.
12
Figure 22.1 Separation of mRNA from an RNA mixture by
binding to poly(dT) sequences that are linked to particles.
13
• To synthesize by reverse transcriptase a cDNA
strand complementary to the mRNA, a poly-(dT)
is used as a primer (Fig. 22.2).
• Subsequently, the mRNA is hydrolyzed by a
ribonuclease either completely or, as shown in
the figure, only partly.
• The latter way has the advantage that the mRNA
fragments can serve as primers for the synthesis
of the second cDNA strand by DNA polymerase.
• By using DNA polymerase I, these mRNA
fragments are successively replaced by DNA
fragments and these are linked to each other by
DNA ligase.
14
Figure 22.2 Transcription of mRNA to double stranded
cDNA.
15
• A short RNA section remains, which is not
replaced at the end of the second cDNA
strand, but this is of minor importance, since
in most cases the mRNA at the 5’ terminus
contains a non-encoding region.
• The double-stranded cDNA molecules thus
formed from the mRNA molecules are
amplified by cloning.
• Plasmids or bacteriophages can be used as
cloning vectors.
16
• Nowadays a large variety of made-to-measure
phages and plasmids are commercially
available for many special purposes.
• A distinction is made between vectors that
only amplify DNA and expression vectors by
which the proteins encoded by the amplified
genes can also be synthesized.
17
A gene library can be kept in phages
• Figure 22.3 shows the insertion of cDNA into
the DNA of a λ phage.
• In the example shown here, the phage DNA
possesses a cleavage site for the restriction
endonuclease EcoRI.
• To protect the restriction sites within the
cDNA, the cDNA double strand is first
methylated by an EcoRI methylase at the EcoRI
restriction sites.
18
• DNA ligase is then used to link chemically
synthesized double-stranded oligonucleotides
with an inbuilt restriction site, (in this case for
EcoRI) to both ends of the double-stranded
cDNA.
• These oligonucleotides are called linkers.
• The restriction endonuclease EcoRI cleaves
this linker as well as the λ phage DNA and thus
generates sticky ends at which the complementary bases of the cDNA and the phage
DNA can anneal by base pairing.
19
• The DNA strands are then linked by DNA
ligase, and in this way the cDNA is inserted
into the vector.
20
Figure 22.3 Insertion of cDNA in a λ-phage insertion vector.
21
• The phage DNA with the inserted cDNA is
packed in vitro into a phage protein coat (Fig.
22.4), using a packing extract from phageinfected bacteria.
• In this way one obtains a gene library, in which
the cDNA formed from the many different
mRNAs of the leaf tissue are packed in phages,
which, after infecting bacteria, can be
amplified ad libitum, whereby each packed
cDNA forms a clone.
22
Figure 22.4 A recombinant phage DNA is packed into a virus
particle.
E. Coli cells are infected with the formed phage and plated on agar
plates. The cells of the infected colonies are lysed by the
multiplying phages and show as transparent spots (plaques) in the
bacterial lawn.
23
• The bacteria are infected by mixing them with
the phages and they are then plated on agar
plates containing cultivation medium.
• At first the infected bacteria grow on the agar
plates to produce a bacterial lawn, but then
are lysed by the phages, which have been
multiplied within the bacteria.
• The lysed bacterial colonies appear on the
agar plate as clear spots called plaques.
24
• These plaques contain newly formed phages,
which can be multiplied further.
• It is customary to plate a typical cDNA gene
library on about 10 to 20 agar plates.
• Ideally, each of these plaques contains only
one clone.
• From these plaques, the clone containing the
cDNA of the desired gene is selected, using
specific probes as described later.
25
A gene library can also be kept in plasmids
• To clone a gene library in plasmids, cDNA is
inserted into plasmids via a restriction
cleavage site in more or less the same way as
in the insertion into phage DNA (Fig. 22.5).
• The plasmids are then transferred to E. coli
cells.
• The transfer is brought about by treating the
cells with CaCl2 to make their membrane more
permeable to the plasmid.
26
• The cells are then mixed with plasmid DNA
and exposed to a short heat shock.
• In order to select the transformed bacterial
cells from the large majority of untransformed
cells, the transformed cells are provided with
a marker.
• The plasmid vector contains an antibiotic
resistance gene, which makes bacteria
resistant to a certain antibiotic, such as
ampicillin or tetracycline.
27
• When the corresponding antibiotic is added to
the culture medium, cells containing the
plasmid survive and grow, whereas the other
non-transformed cells die.
• After plating on an agar culture medium,
bacterial colonies develop, which can be
recognized as spots.
28
Figure 22.5 cDNA can be propagated via a plasmid vector in E. coli. An
antibiotic resistance gene on the plasmid enables the selection of the
transformed cells.
29
• In order to check whether a plasmid actually
contains an inserted DNA sequence (insert),
plasmid vectors have been constructed in
which the restriction cleavage site for
insertion of the foreign DNA is located inside a
gene, which encodes the enzyme βgalactosidase (Fig. 22.6).
• This enzyme hydrolyzes the colorless
compound X-Gal into an insoluble blue
product.
30
• When X-Gal is added to the agar plate culture
medium,
• all the clones that do not contain a DNA insert,
and therefore contain an intact β-galactosidase
gene, form blue colonies.
• If a DNA segment is inserted into the cleavage
site of the β-galactosidase gene, this gene is
interrupted and is no longer able to encode a
functional β-galactosidase.
• Therefore the corresponding colonies are not
stained blue but remain white (blue/white
selection)
31
Figure 22.6 To check whether the plasmid of a bacterial colony carries a DNA
insert, the cleavage site of the plasmid vector is contained within a βgalactosidase gene.
32
A gene library is screened for a certain gene
• Specific probes are employed to screen the
bacterial colonies or phage plaques for the
desired gene.
• A blot is made of the various agar plates by
placing a nylon or nitrocellulose membrane on
top of them.
• Some of the phages contained in the plaques, or
the bacteria contained in the colonies, bind to the
blotting membrane, although most of the
contents of the plaques and the colonies remain
on the agar plate.
33
• Two kinds of probes can be used to screen the
phage or bacterial clones bound to the
blotting membrane:
1. Specific antibodies to identify the protein
formed as gene product of the desired clone
(Western blot); and
2. Specific DNA probes to label the cDNA of the
desired clone by hybridization (with
radioactivity).
34
A new plant is regenerated following
transformation of a leaf cell
35
1. Transformation by A.
Tumefaciens
36
• Agrobacterium tumefaciens attacks plants at
wounds.
• Leaf discs therefore, with their cut edges, are
a good target for performing a transformation
(Fig. 22.16).
• The leaf discs are immersed in a suspension of
A. tumefaciens cells transformed by the
vector.
37
• After a short time, the discs are transferred to
a culture medium containing agarose, which,
besides nutrients, contains the phytohormones cytokinin and auxin to induce the
cells of the leaf disc to grow a callus.
• The addition of the antibiotic kanamycin kills
all plant cells except the transformed cells,
which are protected from the antibiotic by the
resistance gene.
• The remaining agrobacteria are killed by
another antibiotic specific for bacteria.
38
• The cut edges of the leaf discs are the site where
the calli of the transformed cells develop.
• When the concentrations of cytokinin and auxin
are appropriate, these calli can be propagated
almost without limit in tissue culture on agarose
culture media.
• In this way transformed plant cells can be kept
and propagated in tissue culture for very long
periods of time.
• If required, new plants can be regenerated from
these tissue cultures.
39
40
• To regenerate new plants, cells of the callus
culture are transferred to a culture medium
containing more cytokinin than auxin, and this
induces the callus to form shoots.
• Root growth is then stimulated by transferring
the shoots to a culture medium containing more
auxin than cytokinin.
• After plantlets with roots have developed and
grown somewhat, they can be transplanted to
soil, where in most cases they develop to normal
plants, capable of being multiplied by flowering
and seed production.
41
• The pioneering work of Jeff Schell, Marc van
Montagu, Patricia Zambryski, Robert Horsch,
and several others has developed the A.
tumefaciens transformation system to a very
easy method for transferring foreign genes to
cells of higher plants.
• Nowadays it is often possible even for
students to produce several hundred different
transgenic tobacco plants with no great
difficulty.
42
• Using this method, more than 100 different
plant species have been transformed
successfully. Initially, it was very difficult or even
impossible to transform monocot plants with
the Agro-bacteriumsystem.
• Recently, this transformation method has been
improved to such an extent, that it also can now
be successfully applied to transform several
monocots, such as rice.
43
2. physical gene transfer
44
• An alternative way to transform plant cells is a
physical gene transfer, the most successful
being the bombardment of plant cells by
microprojectiles.
45
Plants can be transformed by a modified
shotgun
• Transformation by bombardment of plant cells
with microprojectiles was developed in 1985.
The microprojectiles are small spheres of
tungsten or gold with a diameter of 1 to 4mm,
which are coated with DNA.
• A gene gun (similar to a shotgun) is used to
shoot the pellets into plant cells (Fig. 22.17).
46
Figure 22.17
Transformation of a
plant by a gene gun.
Gold or tungsten
spheres are
coated with a thin
DNA layer by a
deposit of CaCl2. The
spheres are inserted
in front of a plastic
projectile into the
barrel of the gun.
47
• Initially, gunpowder was used as propellant, but
nowadays the microprojectiles are often
accelerated by compressed air, helium, or other
gases.
• The target materials include calli, embryonic
tissues, and leaves.
• In order to penetrate the cell wall of the
epidermis and mesophyll cells, the velocity of
the projectiles must be very high and can reach
about 1,500 km/h in bombardments with a
gene gun in a vacuum chamber.
48
• The cells in the center of the line of fire may
be destroyed during such a bombardment but,
because the projectiles are so small, the cells
nearer the periphery survive.
• The DNA transferred to the cells by these
projectiles can be integrated not only in the
nuclear genome, but also in the genome of
mitochondria and chloroplasts.
• This makes it possible to transform mitochondria and chloroplasts.
49
• In some plants, the gene gun works especially
well.
• Thus, by bombardment of embryonic callus
cells of sugarcane, routinely 10 to 20 different
transformed plant lines are obtained by one
shot.
50