T
-
DNA Mutagenesis
and Plant Genetic
Engineering
Purpose:
Determine
gene function to
produce better plants
for society
Mutagenesis:
Chemical or physical treatment
that changes the nucleotide sequence of DNA.
The altered DNA sequence may be passed on
to the next generation.
Mutant:
An organism that differs from the
“normal” or wild type by one or more changes
in its DNA sequence.
Mutagenesis
-
Single nucleotide change G
--
> A
Mutagenesis
-
creating mutants
ATTAG
G
CTACCGT
TAATC
C
GATGGCA
ATTAG
A
CTACCGT
TAATC
T
GATGGCA
-
Or delete or add a nucleotide
Normal: Wild type
Mutant
Mutagenesis
-
Delete a segment of DNA
-
many nucleotides
Mutagenesis
-
larger mutations
Insert a segment of DNA = “Insertional”
X
X
Insertion tagging
•
Principle:
A DNA fragment (with a
known
sequence
) is allowed to insert into the genome
(it usually causes a recessive, loss of function
mutation).
•
Similar to ligating an insert into LacZ
-
alpha
Insertion tagging
•
Advantages:
–
tags or marks the gene.
–
Provides a powerful way to identify or fish the
gene out
.
•
Disadvantages:
–
Cannot knock out essential genes.
–
Other redundant genes mask loss of disrupted
gene.
–
May disrupt non
-
functional sub
-
region of gene.
Is it useful?
•
Highly and broadly useful
•
Applied to most organisms.
•
Mice, bacteria, yeast and plants have had their
genes inactivated by DNA insertions
-
> knockouts.
T
-
DNA Mutagenesis:
A method of disrupting genes
in plants with a “T
-
DNA” to “knock
-
out” gene
function and activity.
T
-
DNA
=
T
ransfer
DNA
a segment of DNA derived from the T
i
plasmid
contained inside the bacterium,
Agrobacterium
tumefaciens.
“Agro” = plant pathogen
Transferred
from the bacterium to the plant.
Randomly integrated into chromosomal sites in the
nuclei.
A type of insertion mutagenesis
Agrobacterium tumefaciens
-
and Ti Plasmid
Soil Bacterium infects
plants through wounds &
openings
Causes crown gall
tumors….
E
硰敳獥猠来湥猠潮oa
T
i
plasmid
-
Tumor
i
nducing Plasmid
Ti Plasmid
•
Contains genes for:
Plant growth hormones
-
cytokinins and auxins.
-
stimulate undifferentiated growth
Opine biosynthesis
-
food for Agro.
Opine catabolism
-
convert opines into E
Acetosyringone receptors
Plant wound produces
acetosyringone
Bacteria is attracted to wound
-
receptor tells bacteria to swim to wound
=
Bacterial T
-
plasmid encodes
receptors for acetosyringone
Bacterial cell
T
-
DNA is excised from Ti plasmid and integrates into
plant genome.
Genes on T
-
DNA are activated and stimulate cell
proliferation.
Opine genes produce bacterial nutrients “Opines”
Tumor
-
producing
genes
Virulence region
Opine
catabolism
ORI
T
-
DNA
region
IDEA: Ti
-
Plasmid,
Tumor producing genes can be
Replaced with other genes. New genes will be transferred!
Left & right borders
must be retained.
Tumor
-
producing
genes
Virulence region
Opine
catabolism
ORI
T
-
DNA
region
Ti
-
Plasmid
-
delete genes for tumor and Agro nutrients
X
X
X
X
Virulence region
Opine
catabolism
ORI
T
-
DNA
region
Ti
-
Plasmid
-
delete genes for tumor and Agro nutrients
New Gene
New foreign genes can be carried as passengers
when the T
-
DNA integrates into plant genome.
No tumors formed when auxin and cytokinin
genes are replaced
-
plant has taken up
T
-
DNA but no disease!
= Disarmed Ti Plasmid
What kind of genes can be added to T
-
DNA?
-
Any gene
-
Selectable marker
Kanamycin Resistance
Hygromycin R “
-
reporter gene, marks cells
to show they are transformed.
Not always used.
-
genes for crop improvement,
disease & insect resistance, new proteins,
Vitamins, many possibilities
Left
border
Right
border
HygR
GFP
Plants will be hygromycin resistant and
express green fluorescent protein.
Modified T
-
DNA for GFP Expression
•
Green fluorescent protein (GFP)
From luminescent jellyfish
Aequorea victoria.
Produces green fluorescence under
blue and UV light
Root Root Hair cotyledon
Light
Dark
Redistribution of GFP
-
2SC in the Light
GFP
-
2SC moves from vacuole to
ER and golgi, from Dark to Light
Protoplasts: plants with cell walls removed.
Left
border
Right
border
KanR
Plants will be Kanamycin resistant.
Might disrupt a gene or spacer DNA.
Modified T
-
DNA for Mutagenesis
Transformation with Disarmed Ti
-
plasmid
in Agrobacterium
-
Mix Agro containing Ti
-
plasmid with:
-
Wounded leaf
-
Plant cells in culture
-
Floral dip under vacuum
-
plant cells or seeds on growth media containing
selection antibiotic (i.e. Kan).
-
Only engineered plants grow
Genome
-
wide insertional mutagenesis of
Arabidopsis thaliana
(2003)
•
Objective: create loss of function mutations for all
genes.
•
Strategy: use T
-
DNA (with kanamycin
-
resistance
gene as selectable marker) to generate collection of
150,000 T1 transformants.
•
> 225,000 independent T
-
DNA integration events thus
far.
Arabidopsis
•
Genome size = 125,000 kb; Avg gene length = 2 kb
•
Random distribution of insertion events, predicts
96.6% probability of finding an insertion in a gene,
•
To determine the site of integration of each T
-
DNA,
junction sequences were analyzed and 88,122 sites
were proven to be at a single genomic location
•
Of the 29,454 annotated genes, 21,799 (74%) were hit,
•
Create catalog and allow researchers to order seeds
for their favorite gene disruption on
-
line.
2000 bp
CNGC10
Not all genes can be knocked out.
T
-
DNA
Distribution of T
-
DNAs showed hot spots (in gene
-
rich
regions) and cold spots (in centromere and
Peri
-
centromeric regions)
T1 generation
-
first generation after T
-
DNA insertion
Single T
-
DNA insertion
T
-
DNA
-
heterozygous
-
1 normal gene
-
1 disrupted gene
Obtaining Homozygous
-
2 T
-
DNAs in same gene
Heterozygous is self
-
pollinated
N T
N
T
N T
NN
NT
TN
TT
25% homozygous TT
Need homozygous
-
both copies knocked out
T
-
DNA
-
Homozygous
Screen for homozygotes by PCR using
combinations of primers to the T
-
DNA
and to the target gene to be knocked out
Want to know precise location of the T
-
DNA
T
-
DNA
-
Homozygous
Where is it exactly within a gene or
near a gene?
Normal gene
T
-
DNA
How can PCR be used to verify copy # and
location of the T
-
DNA?
Gene 3’
Gene 5’
PCR screen T
-
DNA mapping
No PCR product
with this primer
T
-
DNA
Normal gene
Non
-
perfect, but usable, results
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