Transcript a Decreasing -Gliadins in Wheat Grains using RNAi

Decreasing a-Gliadins in
Wheat Grains using RNAi
Shannon Bell
Dr Oscar Riera-Lizarazu, Dr Andrew Ross, and Christy Watson
Dept of Crop Science, Oregon State University
Background
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Wheat
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Major world staple crop
Used for many food products
Celiac Disease
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An immune response to proteins
found in wheat, barley, and rye
Approximately 1 in 200 people in
larger populations suffer
Effects mainly children
Symptoms include diarrhea,
vomiting, failure to flourish
Wheat Storage Proteins
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Key protein in flour is Gluten
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Composed of ~30% Glutenin and ~70% Gliadin
Gliadin
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Composed of 4 main classes: a,b,g and w
The a gliadins have been shown to cause
strongest allergy response in Celiac disease
patients
Region of Interest
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The noted region of interest is a gliadin
peptide 33 amino acids long proven to be
indigestible by stomach acids
It is this region which acts as an antigen
stimulating the T-cell response and causing
the allergic reaction in those with Celiac
disease
RNA Interference
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RNAi
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Method of Post Transcriptional Gene Regulation
Basic Mechanism
Inverted repeats
Double Stranded
RNA
mRNA sequence
Single Stranded
siRNA
Gene
expression
stopped
Objectives
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We will
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Use RNAi to decrease the levels of a gliadin in the
wheat cultivar Bobwhite
Analyze the transformed wheat for its
protein content and end-use properties
Plan of Action
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Transform explant using particle bombardment
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Gliadin inverted repeat with herbicide resistance
GFP construct to monitor shooting success
Select for herbicide resistance and/or GFP
expression
PCR, Southern blot, and Northern blot analyses
Transgenic Plants
Protein profile
The Gene Gun
Helium Delivered
at set Pressure
Hits DNA coated
Gold
Gold is Forced
into Explants
Dish with Explant Material
2 days after bombardment
Parameters for Optimization
Amount of Gold
Stage of Explant
Gold Particle Size
Pressure
Pressure and Amount Gold
Set B, 1550psi, 1µm gold, 750
µg
Set J, 1100psi, 1µm gold, 118
µg
Stage and Amount Gold
Set I using 1 µm
Gold and 1100psi
Calli, 360 µg
Calli, 118 µg
Immature embryo, 118 µg
60 days after bombardment
Leaves
GFP
Expressing
Root
Set H, Shot 6/8/04
picture taken 8/6/04
How the Performance is Gauged
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Relative amount of GFP
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Score of 0(none) - 10(all) area of explant expressing
GFP
Monitor decline over time (Initial % - Final %)
Response to herbicide selection
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In media
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Yellowing, failure to develop
In soil
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Stripe test (scored)
Current Progress
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Collecting immature seeds and harvesting
embryos
Embryo, Scale
is 1mm
Current Progress
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Collecting immature seeds and harvesting
embryos
Have shot 23 sets of material, Sets H-W in
tissue culture stage. A-G showed no
transformation.
Current Progress
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Collecting immature seeds and harvesting
embryos
Have shot 23 sets of material, Sets H-W in
tissue culture stage. A-G showed no
transformation.
Continued selection of herbicide resistance
and GFP activity
High GFP Activity, Good Growth
Hb, Shot 6/8/04, 1550 PSI, 1µm gold,
Photo taken 8/6/04
Root expressing
GFP
Current Progress
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Collecting immature seeds and harvesting
embryos
Have shot 23 sets of material, Sets H-W in
tissue culture stage. A-G showed no
transformation.
Continued selection of herbicide resistance
and GFP activity
Developed two PCR tests
Currently 5 possible transgenics
Next Steps
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Generate multiple plants
showing herbicide resistance
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Analyze the results of the
optimizations
Concluding Remarks
This project will provide:
 A better understanding of the role gliadins
play in seed formation and end use qualities
of wheat
 Optimization of transformation process in
wheat
 A possible application to treat Celiac disease
Acknowledgements
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Dr Oscar Riera-Lizarazu and Christy Watson
for their time, assistance, and expertise
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Members of Cereal Biotechnology for their time
and advice
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HHMI and NSF for funding