Transcript The Biotechnology Age: Issues and Impacts

T-DNA Mutagenesis
Purpose: Determine
gene function to
produce better plants
for society
Mutagenesis
Mutant: An organism that differs from the
“normal” or wild type by one or more changes
in its DNA sequence.
Mutagenesis: Chemical or physical treatment that
changes the nucleotide sequence of DNA. Can lead
to changes in DNA sequence passed on to the next
generation.
Mutagenesis- creating mutants
- Single nucleotide change G --> A
Mutant
Normal: Wild type
ATTAGGCTACCGT
TAATCCGATGGCA
Mutagenesis
-Or delete or add a nucleotide
ATTAGACTACCGT
TAATCTGATGGCA
Mutagenesis- larger mutations
- Delete a segment of DNA - delete many nucleotides
X X
Insert a segment of DNA = “Insertional”
Insertion tagging
• Principle: A DNA fragment (with a known
sequence) is allowed to insert into the genome
(when it lands in a gene, it usually causes a
recessive, loss of function mutation).
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 knock-out.
– 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 knockouts.
T-DNA Mutagenesis: A method of disrupting genes in
plants with a “T-DNA” to “knock-out” gene function and
activity.
T-DNA = Transfer DNA
a segment of DNA derived from the Ti 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.
Agrobacterium tumefaciens - and Ti Plasmid
Soil Bacterium infects plants
through wounds & openings
And causes crown gall
tumors….
by expressing genes on a
Ti plasmid - Tumor Inducing
Plasmid
Ti Plasmid
• Contains genes for:
Plant growth hormones
- cytokinins and auxins.
- stimulate undifferentiated growth
Opine biosynthesis - food for Agrobact.
Opine catabolism
Acetosyringone receptors
Plant wound produces
acetosyringone
Bacterial T-plasmid produces
receptors for acetosyringone
Bacteria is attracted to wound
- receptor tells bacteria to swim to wound
T-DNA is excised from Ti plasmid and integrates into
plant genome.
Genes on T-DNA are activated and stimulate cell
proliferation.
and Opine genes produce bacterial nutrients “Opines”
IDEA: Ti- Plasmid, Tumor producing genes can be
Replaced with other genes. New genes will be transferred!
T-DNA
region
Left & right borders
must be retained.
Tumorproducing
genes
Opine
catabolism
Virulence region
ORI
Ti- Plasmid - delete genes for tumor and Agro nutrients
T-DNA
region
X
X XX
Tumorproducing
genes
Opine
catabolism
Virulence region
ORI
Ti- Plasmid - delete genes for tumor and Agro nutrients
T-DNA
region
New Gene
Opine
catabolism
Virulence region
ORI
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
Modified T-DNA for GFP Expression
Left
border
HygR
GFP
Right
border
Plants will be hygromycin resistant and
express green fluorescent protein.
• Green fluorescent protein (GFP)
From luminescent jellyfish Aequorea victoria.
Produces green fluorescence under
blue and UV light
Redistribution of GFP-2SC in the Light
Root
Dark
Light
Root Hair
cotyledon
GFP-2SC moves from vacuole
to ER and golgi, from Dark to Light
Protoplasts: plants with cell walls removed.
Modified T-DNA for Mutagenesis
Left
border
KanR
Right
border
Plants will be Kanamycin resistant.
Might disrupt a gene or spacer DNA.
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; Average gene length = 2
kb
• Random distribution of insertion events, predicts
96.6% probability of finding an insertion in an average
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 a catalog and allow researchers to order
seeds for their favorite gene disruption on-line.
Not all genes can be knocked out.
CNGC10
2000 bp
T-DNA
T1 generation - first generation after T-DNA insertion
Single T-DNA insertion
Distribution of T-DNAs showed hot spots (in gene-rich
regions) and cold spots (in centromere and
Peri-centromeric regions)
T-DNA - heterozygous
- 1 normal gene
- 1 disrupted gene
Obtaining Homozygous - 2 T-DNAs in same gene
N
T
Heterozygous is self-pollinated
N
T
N NN
NT
TN
TT
T
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
PCR screen T-DNA mapping
Gene 5’
T-DNA
Gene 3’
No PCR product
with this primer
Non-perfect, but usable, results