The Biotechnology Age: Issues and Impacts

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