nasa's institute for advanced concepts -- home - a flexible … · goal: 30,000 est sequences...
TRANSCRIPT
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A Flexible Architecture forPlant Functional Genomics
in Space Environments
Dr. Terri LomaxDepartment of Botany and Plant Pathology
Oregon State University
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• Remotely measure the response of plants to any unique space condition
• Determine gene function
• Optimize plant performance under space conditions
Goals:
A Flexible Architecture for Plant Functional Genomics
in Space Environments
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SpaceEnvironmental
Condition
Test Plant RNA
DNAMicroarrays
GeneIsolation
GeneDisruption
GeneModification
OptimizedPerformance
FunctionalAnalysis
Basic Architecture for Plant Functional Genomics
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The “NIAC Dilemna”
“Planning for things that will be practical in 2010 - 2040but needing to demonstrate practibility now”
Dr. Steven Dubowsky, MIT(NIAC Fellow)
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Model System to Test Architecture Feasibility
Gravistimulationof
Arabidopsis
DNAMicroarrays
ArabidopsisGenomicDatabase
Moss GeneDatabase
MossGene Disruptionby HomologousRecombination
GravitropismAssays
OptimizedPerformance
ArabidopsisRNA
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• model plant system
• defined gravitropic response
• genome sequence complete
• targeted gene knock-outs inefficient
Arabidopsis thaliana:
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Physcomitrella patens :
• gene knockout efficiencies > 90% viahomologous recombination
• defined gravitropic response
• more closely related to dicots than monocots
• similar DNA usage
• used to determine cellularfunction of an Arabidopsis gene
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DNAMicroarrays
Basic Architecture for Plant Functional Genomics
SpaceEnvironmental
Condition
Test Plant RNA
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Microarray Technology
• temporal and spatial gene expression
• Affymetrix Arabidopsisgene chips with over 8200 genes
• provides information on gene involvement in a process or pathway
A Flexible Architecture for Plant Functional Genomics
in Space Environments
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Basic Architecture for Plant Functional Genomics
GeneIsolation
DNAMicroarrays
SpaceEnvironmental
Condition
Test Plant RNA
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Data Mining:Plant Genome Sequence Databases
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Moss EST Sequence Databases
The Physcomitrella EST Program
Goal: 30,000 EST sequences To Date: 14,410 moss EST sequences on GenBank
Physcomitrella Genomics Program
University of Leeds, UK/ Washington University,
St. Louis, MO
To Date: 120,000 EST sequences representing 22,000 different genes
University of Freiburg/BASF
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Basic Architecture for Plant Functional Genomics
SpaceEnvironmental
Condition
Test Plant RNA
DNAMicroarrays
GeneIsolation
GeneDisruption
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Functional Genomics
Gene function - analysis of gene malfunction
Conventional Approaches:• manipulate the level of gene expression• block the expression of a gene
Problems:• non-directed integration of transgenes• positional effects• gene silencing due to co-suppression• incorrect spatial and temporal expression
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plasmidmossgenome
mossgenome
a b c b c dp
mossgenome
plasmida
bc
dbcp
Targeted Gene Knock-out through Homologous Recombination
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Regeneration following transformation of protoplasts
No selection Antibiotic selection
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Basic Architecture for Plant Functional Genomics
FunctionalAnalysis
SpaceEnvironmental
Condition
ab
cdbc
p
GeneDisruption
GeneIsolation
DNAMicroarrays
Test Plant RNA
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Transfer strips of
mutagenized
tissue and allow
to regenerate
gravity vector
Grow in darkness
Transfer to
unilateral
lightlight source
wwr
agr
ptr
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agr wwr
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QuickTime™ and aCinepak decompressor
are needed to see this picture.
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Untransformed moss protoplastswith no fluorescein-tagged oligo
Untransformed moss protoplastswith fluorescein-tagged oligo
Moss protoplasts transformedwith fluorescien-tagged oligo
Feasibility of Antisense Knockouts:Can we get the oligos in?
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Architecture Issues & Accomplishments
√ Test plant Arabidopsis thaliana as model system to identify genes involved in the gravitropic response
√ Optimize sample preparation for differential
expression analysis
√ Affymetrix Microarray analysis
√ Identify genes whose expression increases
Arabidopsis
DNAMicroarrays
ArabidopsisDatabase
ArabidopsisRNA
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√ Establish Physcomitrella patens model system to determine function of genes
√ propagation and maintenance √ protoplast isolation, transformation, and
regeneration
√Collaborative agreements with two sources for moss gene databases
√Assemble molecular tools for gene knock-outs √ cDNA and genomic libraries √ two types of vectors
Architecture Issues & AccomplishmentsMoss
System
MossDatabase
GeneDisruption
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What’s Next?
Ø Increase repetitions for microarray analysis to confirm differential gene expression
Ø Construct replacement and knock-out vectors using available genomic and cDNA sequences
Ø Generate transformed moss and score for phenotype
Near Future:
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The Future: 2002-2020
Ø Introduce additional environmental conditions into architectureØ space-flown plant material
Ø Expand collaborations with Affymetrix and AVI Biopharma to develop remote technologies
Ø Adapt architecture to accommodate additionalplant systems as data and technologies evolve
Ø Work with engineers to optimize architecture for space flight/habitat