Download Agrobacterium tumefaciens

TRANSGENIC TECHNOLOGY
Traits that plant breeders
would like in plants
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High primary
productivity
High crop yield
High nutritional
quality
Adaptation to intercropping
Nitrogen Fixation
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Drought resistance
Pest resistance
Adaptation to
mechanised farming
Insensitivity to
photo-period
Elimination of toxic
compounds
Plant transformation
getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Requirement
1. a suitable transformation method
2. a means of screening for transformants
3. an efficient regeneration system
4. genes/constructs
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Vectors
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Promoter/terminator
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reporter genes
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selectable marker genes
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‘genes of interest’
Transformation methods
DNA must be introduced into plant cells
Indirect
- Agrobacterium tumefaciens
Direct
- Chemical method
- Electrical method
- Physical methods
Chemical Method
1. Use of PEG (Polyethylene glycol (PEG)-mediated )
2. Protoplasts are incubated with a solution of DNA
and PEG
Electrical method
1. Electroporation (electropermeabilization)
2. Cells or protoplast are subjected to short electrical
pulse
Physical Methods
1. Particle bombardment
2. Microinjection
3. Silicon Carbide whiskers
Agrobacterium-mediated
transformation
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A natural genetic
engineer
2 species
• A.tumefaciens
(produces a gall)
• A. rhizogenes
(produces roots)
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Oncogenes (for
auxin and cytokinin
synthesis) + Opines
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In the presence of
exudates (e.g.
acetosyringone) from
wounded plants,
Virulence (Vir) genes
are activated and cause
the t-DNA to be
transferred to plants.
Everything between the
left and right border is
transferred.
BACTERIAL GALL DISEASES
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Galls:
overgrowth or proliferation of tissue, primarily due
to increased cell division (hyperplasia) and
increased cell size (hypertrophy).
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Bacterial Galls:
induced by bacteria in 3 different genera.
• Agrobacterium
• Pseudomonas
• Clavibacter
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Genes for plant hormone production found on
bacterial plasmids!
Crown Gall Disease:
Agrobacterium tumefaciens
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Gram Dicots
Worldwide
Disease Cycle
Agrobacterium tumefaciens
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Characteristics
• Plant parasite that causes Crown Gall Disease
• Encodes a large (~250kbp) plasmid called
Tumor-inducing (Ti) plasmid
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Portion of the Ti plasmid is transferred between
bacterial cells and plant cells  T-DNA (Tumor
DNA)
Agrobacterium tumefaciens
T-DNA integrates stably into plant genome
Single stranded T-DNA fragment is
converted to dsDNA fragment by plant cell
Then integrated into plant genome
 2 x 23bp direct repeats play an important role in
the excision and integration process
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Agrobacterium tumefaciens
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Tumor formation = hyperplasia
Hormone imbalance
Caused by A. tumefaciens
• Lives in intercellular spaces of the plant
• Plasmid contains genes responsible for the
disease
 Part of plasmid is inserted into plant DNA
 Wound = entry point  10-14 days later,
tumor forms
Agrobacterium tumefaciens
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What is naturally encoded in T-DNA?
• Enzymes for auxin and cytokinin synthesis
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Causing hormone imbalance  tumor
formation/undifferentiated callus
Mutants in enzymes have been characterized
• Opine synthesis genes (e.g. octopine or nopaline)
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Carbon and nitrogen source for A. tumefaciens growth
Insertion genes
• Virulence (vir) genes
• Allow excision and integration into plant genome
Ti plasmid of A. tumefaciens
1. Auxin, cytokinin, opine
synthetic genes
transferred to plant
2. Plant makes all 3
compounds
3. Auxins and cytokines
cause gall formation
4. Opines provide unique
carbon/nitrogen
source only A.
tumefaciens can use!
Agrobacterium tumefaciens
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How is T-DNA modified to allow genes of
interest to be inserted?
• In vitro modification of Ti plasmid
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T-DNA tumor causing genes are deleted and replaced with
desirable genes (under proper regulatory control)
Insertion genes are retained (vir genes)
Selectable marker gene added to track plant cells
successfully rendered transgenic [antibiotic resistance
gene  geneticin (G418) or hygromycin]
Ti plasmid is reintroduced into A. tumefaciens
A. tumefaciens is co-cultured with plant leaf disks under
hormone conditions favoring callus development
(undifferentiated)
Antibacterial agents (e.g. chloramphenicol) added to kill A.
tumefaciens
G418 or hygromycin added to kill non-transgenic plant cells
Surviving cells = transgenic plant cells
Agrobacterium and genetic engineering:
Engineering the Ti plasmid
Co-integrative and binary vectors
LB
RB
Co-integrative
Binary vector
Agrobacterium-mediated transformation
Agrobacterium tumefaciens
cause ‘Crown gall’ disease
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants
Expose wounded plant cells to transformed
agro strain
Electroporate TDNA vector into
Agrobacterium
and select for tetr
Induce plant regeneration and select
for Kanr cell growth
Electroporation
Explants: cells and protoplasts
Most direct way to introduce foreign DNA into the
nucleus
Achieved by electromechanically operated devices that
control the insertion of fine glass needles into the nuclei
of individuals cells, culture induced embryo, protoplast
Labour intensive and slow
Transformation frequency is very high, typically up to ca.
30%
Microprojectile bombardment
• uses a ‘gene gun’
• DNA is coated onto
gold (or tungsten)
particles (inert)
• gold is propelled by
helium
into plant
cells
• if DNA goes into the
nucleus
it can be
integrated into the
plant chromosomes
• cells can be
regenerated
to
whole plants
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In the "biolistic" (a cross between biology and ballistics )or
"gene gun" method, microscopic gold beads are coated with
the gene of interest and shot into the plant cell with a
pulse of helium.
Once inside the cell, the gene comes off the bead and
integrates into the cell's genome.
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Model from BioRad:
Biorad's Helios Gene
Gun
Microinjection
Most direct way to introduce foreign DNA into the
nucleus
Achieved by electromechanically operated devices that
control the insertion of fine glass needles into the nuclei
of individuals cells, culture induced embryo, protoplast
Labour intensive and slow
Transformation frequency is very high, typically up to ca.
30%
Silicon Carbide Whiskers
 Silicon carbide forms long, needle like crystals
Cells are vortex mixed in the present of whiskers and
DNA
DNA can be introduced in the cells following penetration
by the whiskers
Competent cells
Not all cells take up DNA & not all cells can regenerate
Need an efficient regeneration system and transformation
system i.e. lots of cells take up DNA and lots of cells
regenerate into a plant
to maximize chance of both happening
regenerable cells
Transformed cells
Cells containing new DNA that are able
to regenerate into a new plant
Screening technique
There are many thousands of cells in a leaf disc or callus
clump - only a proportion of these will have taken up the
DNA
therefore can get hundreds of plants back - maybe only 1%
will be transformed
How do we know which plants have taken up the
DNA?
Could test each plant - slow, costly
Or use reporter genes & selectable marker genes
Screening
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Transformation frequency is low (Max 3% of all cells)
and unless there is a selective advantage for
transformed cells, these will be overgrown by nontransformed.
Usual to use a positive selective agent like antibiotic
resistance. The NptII gene encoding Neomycin
phospho-transferase II phosphorylates kanamycin
group antibiotics and is commonly used.
Screening (selection)
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Select at the level of the intact plant
Select in culture
• single cell is selection unit
• possible to plate up to 1,000,000 cells on a
Petri-dish.
• Progressive selection over a number of
phases
Selection Strategies
Positive
 Negative
 Visual
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Positive selection
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Add into medium a toxic compound e.g.
antibiotic, herbicide
Only those cells able to grow in the presence
of the selective agent give colonies
Plate out and pick off growing colonies.
Possible to select one colony from millions of
plated cells in a days work.
Need a strong selection pressure - get
escapes
Negative selection
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Add in an agent that kills dividing cells
e.g. chlorate / BUdR.
Plate out leave for a suitable time, wash
out agent then put on growth medium.
All cells growing on selective agent will
die leaving only non-growing cells to now
grow.
Useful for selecting auxotrophs.
Visual selection
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Only useful for coloured or fluorescent
compounds
Plate out at about 50,000 cells per
plate.
Pick off coloured / fluorescent
compounds
Possible to screen about 1,000,000 cells
in a days work.
Positive and Visual Selection
Regeneration System
How do we get plants back from cells?
We use tissue culture techniques to regenerate
whole plants from single cells
getting a plant back from a single cell is important so
that every cell has the new DNA
Regeneration
Plant tissue culture uses growth regulators and
nutrients to regenerate plants in vitro
Regeneration of shoots from leaf protoplasts in
Arabidopsis thaliana
Somatic embryogenesis in peanut
Organogenesis
Gene construct
BamHI
P SAG12 ipt
nptII
LB T 35S
P 35S
gus-intron
T nos T 35S
P 35S
RB
Cloning
Clone: a collection of molecules or cells, all
identical to an original molecule or cell
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To "clone a gene" is to make many copies of it - for
example, in a population of bacteria
Gene can be an exact copy of a natural gene
Gene can be an altered version of a natural gene
Recombinant DNA technology makes it possible
Plasmids
Naturally occurring extrachromosomal
DNA
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Plasmids are circular dsDNA
Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends
of plasmid
Restriction Enzyme
 Molecular scissors; isolated from bacteria
where they are used as Bacterial defense
against viruses.
 Molecular scalpels to cut DNA in a precise and
predictable manner
 Members of the class of nucleases
Nuclease
Breaking the phosphodiester bonds that link
adjacent nucleotides in DNA and RNA
molecules
 Endonuclease
Cleave nucleic acids at internal position
 Exonuclease
Progressively digest from the ends of the
nucleic acid molecules
Endonuclease
Type
Characteristics
I
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II
III
Have both restriction and modification activity
 Cut at sites 1000 nucleotides or more away from
recognition site
 ATP is required
 It has only restriction site activity
 Its cut is predictable and consistent manner at a site
within or adjacent to restriction site
 It require only magnesium ion as cofactor
Have both restriction and modification activity
Cut at sites closed to recognition site
ATP is required
Restriction Enzyme
 There are already more than 1200 type II enzymes isolated
from prokaryotic organism
 They recognize more than 130 different nucleotide sequence
 They scan a DNA molecule, stopping only when it recognizes
a specific sequence of nucleotides that are composed of
symetrical, palindromic sequence
Palindromic sequence:
The sequence read forward on one DNA strand is identical to
the sequence read in the opposite direction on the
complementary strand
 To Avoid confusion, restriction endonucleases are named
according to the following nomenclature
Nomenclature
 The first letter is the initial letter of the genus name of the
organism from which the enzyme is isolated
 The second and third letters are usually the initial letters of
the organisms species name. It is written in italic
 A fourth letter, if any, indicates a particular strain organism
 Originally, roman numerals were meant to indicate the order
in which enzymes, isolated from the same organisms and
strain, are eluted from a chromatography column. More
often, the roman numerals indicate the order of discovery
Nomenclature
EcoRI
BamHI
HindIII
E : Genus Escherichia
co: Species coli
R : Strain RY13
I : First endonuclease isolated
B : Genus Bacillus
am: species amyloliquefaciens
H : Strain H
I : First endonuclease isolated
H : Genus Haemophilus
in : species influenzae
d : strain Rd
III : Third endonuclease isolated
Specificity
Enzyme
BamHI
BglII
EcoRI
EcoRII
HaeIII
HindII
HindIII
HpaII
NotI
PstI
Source
Bacillus amyloliquefaciens H
Bacillus globigii
Escherichia coli RY13
Escherichia coli R245
Haemophilus aegyptius
Haemophilus influenzae Rd
Haemophilus influenzae Rd
Haemophilus parainfluenzae
Nocardia otitidis-caviarum
Providencia stuartii 164
Sequence
GGATCC
AGATCT
GAATTC
CCTGG
GGCC
GTPyPuAC
AAGCTT
CCGG
GCGGCCGC
CTGCAG
End
Sticky
Sticky
Sticky
Sticky
Blunt
Blunt
Sticky
Sticky
Sticky
Sticky
Restriction enzymes
Restriction enzymes can be grouped by:
 number of nucleotides recognized (4, 6,8 base-cutters
most common)
 kind of ends produced (5’ or 3’ overhang (sticky), blunt)
 degenerate or specific sequences
 whether cleavage occurs within the recognition
sequence
Become familiar with the back of your molecular
biology catalog!
A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic
sequence (5’GAATTC)
3. Cuts within the
recognition
sequence (type II
enzyme)
4. produces a 5’
overhang (sticky
end)
Restriction enzymes
Cloning Vectors
Plasmids that can be modified to carry
new genes
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Plasmids useful as cloning vectors must have
• a replicator (origin of replication)
• a selectable marker (antibiotic resistance
gene)
• a cloning site (site where insertion of foreign
DNA will not disrupt replication or inactivate
essential markers
A typical plasmid vector with a
polylinker
Chimeric Plasmids
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Named for mythological beasts with body
parts from several creatures
After cleavage of a plasmid with a restriction
enzyme, a foreign DNA fragment can be
inserted
Ends of the plasmid/fragment are closed to
form a "recombinant plasmid"
Plasmid can replicate when placed in a
suitable bacterial host
Directional Cloning
Often one desires to insert foreign DNA in a
particular orientation
 This can be done by making two cleavages
with two different restriction enzymes
 Construct foreign DNA with same two
restriction enzymes
 Foreign DNA can only be inserted in one
direction
Promoter
1.
2.
3.
4.
5.
6.
A nucleotide sequence within an operon
Lying in front of the structural gene or genes
Serves as a recognition site and point of attachment
for the RNA polymerase
It is starting point for transcription of the structural
genes
It contains many elements which are involved in
producing specific pattern and level of expression
It can be derived from pathogen, virus, plants
themselves
Types of Promoter
 Promoter always expressed in most tissue (constitutive)
-. 35 s promoter from CaMV Virus
-. Nos, Ocs and Mas Promoter from bacteria
-. Actin promoter from monocot
-. Ubiquitin promoter from monocot
-. Adh1 promoter from monocot
-. pEMU promoter from monocot
 Tissue specific promoter
-. Haesa promoter
-. Agl12 promoter
 Inducible promoter
-. Aux promoter
 Artificial promoter
-. Mac promoter (Mas and 35 s promoter)
Reporter gene
easy to visualise or assay
- ß-glucuronidase (GUS)
(E.coli)
-green fluorescent protein (GFP)
(jellyfish)
- luciferase
(firefly)
GUS
Cells that are transformed with GUS will form a
blue precipitate when tissue is soaked in the
GUS substrate and incubated at 37oC
this is a destructive assay (cells die)
The UidA gene encoding activity is commonly used.
Gives a blue colour from a colourless substrate (X-glu)
for a qualitative assay. Also causes fluorescence from
Methyl Umbelliferyl Glucuronide (MUG) for a
quantitative assay.
GUS
Bombardment of GUS gene
- transient expression
Stable expression of
GUS in moss
Phloem-limited expression of GUS
HAESA gene encodes a receptor protein kinase that
controls floral organ abscission. (A) transgenic plant
expressing a HAESA::GUS fusion. It is expressed in
the floral abscission zone at the base of an Arabidopsis
flower.
Transgenic plants that harbor the
AGL12::GUS fusions show rootspecific expression.
Inducible expression
GFP (Green Fluorescent Protein)
 Fluoresces green under UV illumination
 Problems with a cryptic intron now resolved.
 Has been used for selection on its own.
GFP glows bright green when irradiated by
blue or UV light
This is a nondestructive assay so the same
cells can be monitored all the way through
GFP
protoplast
colony derived
from protoplast
mass of callus
regenerated plant
Selectable Marker Gene
let you kill cells that haven’t taken up DNA- usually genes
that confer resistance to a phytotoxic substance
Most common:
1. antibiotic resistance
kanamycin, hygromycin
2. herbicide resistance
phosphinothricin (bialapos); glyphosate
Only those cells that have
taken up the DNA can
grow on media containing
the selection agent
Gene of interest
Sequence of DNA which will be inserted to
the host cell and its product will be studied
or beneficial for mankind
Origin of gene interest:
1.
Non plant genes
2.
Plant genes
pathogen-derived genes
Exogenous genes
(non-plant genes)
bacterial genes
any other organism
Endogenous genes
(Plant genes)
Enzymes in biochemical pathway
Natural resistance genes
The desired DNA produced by the above methods
can then be introduced into a host cell by a
number of methods.
The desired DNA can be inserted into cloning vectors
such as plasmids and viral genomes. The desired DNA can
be spliced into isolated plasmids using restriction
endonuclease enzymes.
The desired DNA can be spliced into the genome of
viruses capable of inserting their genome into the
chromosomes of the host cell, such as modified
retroviruses or temperate bacteriophages. Infection of
the host cell then allows for the insertion of the viral
genome (now containing the "desired" DNA) into the host
cell's DNA.
 The desired DNA can be introduced into plant cells by protoplast
fusion. With protoplast fusion, the plant cell wall is enzymatically
removed to create protoplasts. Polyethylene glycol is then used to
enable the protoplasts to fuse together.
 The desired DNA can be introduced into cells by microinjection
and electroporation. In the case of microinjection, micropipettes
are used to inject the DNA into the cell's cytoplasm. With
electroporation, high voltage electrical impulses are used to
destabilize the cytoplasmic membrane and permit entry of DNA.
 The desired DNA can be introduced into plant cells by
Agrobacterium or using gene guns. With agrobacterium, the plant
cell is cocultivated with agrobacterium for a second. Agrobacterium
will integrate the gene of interest to plant chromosome. Gene guns
use helium bursts to propel microscopic gold or tungsten particles
coated with DNA through plant cell walls.
T-DNA
binary
vector
A. tumefaciens