Agrobacterium and genetic engineering - Faperta

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12 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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Plant Genetic Engineering

1.
a suitable transformation method

2.
a means of screening for transformants

3.
an efficient regeneration system

4.
genes/constructs


Vectors


Promoter/terminator


reporter genes


selectable marker genes


‘genes of interest’


Requirement

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

Electroporation (electropermeabilization)

Physical Methods

1.
Particle bombardment

2.
Microinjection

3.
Silicon Carbide whiskers

Agrobacterium tumefaciens


Plant parasite that causes Crown Gall Disease


Encodes a large (~250kbp) plasmid called
Tumor
-
inducing (Ti) plasmid


Portion of the Ti plasmid is transferred between
bacterial cells and plant cells


T
-
DNA (Tumor
DNA
)


T
-
DNA integrates stably into plant genome


Single stranded T
-
DNA fragment is converted
to dsDNA fragment by plant cell



1. Then integrated into plant genome



2. 2 x 23bp direct repeats play an important role in


the excision and integration process

Agrobacterium tumefaciens


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


What is naturally encoded in T
-
DNA?


Enzymes for auxin and cytokinin synthesis


Causing hormone imbalance


tumor
formation/undifferentiated callus


Mutants in enzymes have been characterized


Opine synthesis genes (e.g. octopine or
nopaline)


Carbon and nitrogen source for
A. tumefaciens

growth


Insertion genes


Virulence (vir) genes


Allow excision and integration into plant genome

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

and genetic engineering:

Engineering the Ti plasmid

Co
-
integrative and binary vectors

Binary vector

LB

RB

Co
-
integrative


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%

Electroporation

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

Pressure gauge

Disk with DNA
-
coated particles

Stop plate

Sample goes here

Vacuum line

Gas line

Rupture disk

Vacuum chamber


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.


Model from BioRad:

Biorad's Helios Gene
Gun


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%

Microinjection

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 technique

Screening


Transformation frequency is low (Max 3% of all cells)
and unless there is a selective advantage for
transformed cells, these will be overgrown by non
-
transformed.


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)


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

Positive selection


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

Positive and Visual Selection

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 System

Transformation series of events

Transform
individual cells

Callus formation

Auxins

Cytokinins

Remove from sterile conditions


easy to visualise or assay


-

ß
-
glucuronidase (GUS)



(E.coli)

-
green fluorescent protein (GFP)

(jellyfish)

-

luciferase





(firefly)

Reporter gene

GUS

Cells that are transformed with GUS will form a
blue precipitate when tissue is soaked in the

GUS substrate and incubated at 37
o
C

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
M
ethyl
U
mbelliferyl
G
lucuronide (
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

AGL
12
::
GUS

fusions

show

root
-
specific

expression
.

Inducible expression

GFP (Green Fluorescent Protein)

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


Fluoresces green under UV illumination


Problems with a cryptic intron now resolved.


Has been used for selection on its own.

GFP

protoplast

colony derived
from protoplast

mass of callus

regenerated plant

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

Selectable Marker Gene

Only those cells that have
taken up the DNA can
grow on media containing
the selection agent

A. tumefaciens

binary

vector

T
-
DNA