agricultural and pharmaceutical nanotechnology plate stackers allow printing up to 20,000 dna oligo...
TRANSCRIPT
Agricultural and Nanotec
Lila VDepartment of
University
NSF I/UCRC PlaAug. 31-Se
University of Il
Pharmaceutical chnology
odkinCrop Sciences
y of Illinois
anning Workshopept. 1, 2009llinois, Urbana
• Applications of NanotechnoloPlant Genomics
• Builds on current plant gene currently in use including the
•Microarrays (extensive data sets)•Small RNAs (extensive data sets)•Digital Gene Expression and RNA•Transformation •Characterization of phenotype and
• Current collaboration with Brinvestigate photonic crystal sfluorescence detection of gen
:
ogy to Gene Expression and
discovery tools for soybean e following:
A-Seq
d metabolic profiles
rian Cunningham of MNTL to surfaces for enhancing ne expression with microarrays
Transcript Profiling of Seed
Lila Vodkin, Orlando Gonzale
Matt Hunt, Anne Marie Boone, Crop Sciences Department, Unive
Acknowledgements
From ISU Extension Report #53
and Seedling Development
ez, Sarah Jones, Gracia Zabala,
Lindsay Freeberg, Jigyasa Tutejaersity of Illinois, Urbana-Champaign: NSF, USB, USDA, ISA
Genetix Arrayer for PrintingConventional Superamine Co
g Large Numbers of DNAs on oated or Photonic Crystal Slides
DNAs Are Printed on Either ConvenCrystal Slides to Compa
ntional SuperAmine Glass or Photonic are Sensitivity of Detection
Soybean cDNA and 70-meas a Commun
• Assembled a “unigene” set of 36,sequenced the 3’ ends and printe(Vodkin et al., BMC Genomics 5:7award.
• Designed and synthesized (via Illu(70-mers) and printed these on glaGonzalez and Vodkin, BMC Genom
For availability of the cDNA or long o
Contact: Lila Vodkin at l-vodkin@uiDepartment of Crop SciencUniversity of Illinois
NSF Soybean Functional Genomics and United Soybean Board SuVodkin Lab
er Oligo Microarray Slides nity Resource
,000 low redundancy cDNAs and ed these on slides for microarrays 73, 2004). Supported by prior NSF
umina, Inc.) 38,400 long oligos ass slides for microarray analysismics 2007) Supported by USB
oligo microarray slides
iuc.educes
upport
Uses of the Soybean cDArrays for Global Dev
• Used microarrays for comprehensiveof somatic embyos during tissue culPhysiology 132:118-136, 2003)
• Global transcript profiles of normal c(Jones et al, 2008 and in preparation
• Functional transition of cotyledon de(Gonzalez and Vodkin, BMC Genomioligo array)
• Examined challenge of soybean planglyphosate other (collaboration withInteraction, 18:1161-1174, 2005; Zabaet al. J. Agri Food Chem, 2008, Brech2008; Li et al. New Phytol, 2008).
• Effect of elevated CO2 on soybeans gAinsworth, USDA/ARS, Univ of Illino
Soybean Functional Genomics
DNA and 70-mer Oligo velopmental Profilese developmental study of the induction lture (Thibaud-Nissen et al., Plant
cotyledon and seed coat development n)
evelopment during germinationcs 8: 468 (2007) uses the new 70mer
nts by pathogens P. syringae, aphids, Clough lab, Zou et al Plant Microbe ala et al BMC Plant Biology, 2006); Zhu henmacher et al Plant Microb Inter,
grown in SoyFACE (with Lisa ois; Plant Physiol. 2006)
• Current collaboration with Brinvestigate photonic crystal sfluorescence detection of genmicroarrays
• Uses a custom oligo array thaglass superamine substrates surface
• 40 replicates of each DNA oligexpression and increased sencrystal is observed (see preseCunningham and his laborato
rian Cunningham of MNTL to surfaces for enhancing ne expression detection by
at is printed on conventional or photonic crystal containing
go are assayed for gene nsitivity of using the photonic entation from Brian ory at MNTL)
Ba
Example: SB0032
Blocks 11 and 35:Well 519
Blocks 12 and 36:Well 495
Blocks 15 and 39:Well 520
Blocks 16 and 40:Well 496
Custom Oligo Array
For a total of 40 spots from SB0032
arcode
5
55
5
5
5 5
5
Field 2
Field 1
Field 2
Block 28
The Custom Oligo 1 slides are “subarrays” containwe want to confirm expression data from specific e
The Custom Oligo 1 slides are divided into two ideexample, blocks 4 and 28 are replicates of each o
The Custom Oligo 1 slides containg many repeatsrepeated 40 times (20 repeats in each field) whichlevel of each oligo.
Bar
code
end
Slide bar code 13140155
Field 1
Block 4
ning genes from the 38,000 oligos chips for which experiments.
entical fields containing 24 blocks each. For ther.
s of 192 specific 70 mer long oligos that are each h will yield statistical validataion of the expression
Barcode
Gene 1 Gene 2
Gene 4 cont.
Custom Oligo Array
Five replicates in a row o
16 colum
2 Gene 3 Gene 4
of the same gene
mns
10 rows
Day 0Fertilization/Flowering
4 DAFGlobular Stage
8-10 DAFHeart Stage
12-14 DAFDay 0Fertilization/Flowering
4 DAFGlobular Stage
8-10 DAFHeart Stage
12-14 DAF
Global Expression AnalyseSmall RNAs for the Ve
Development P
• Total RNA from whole seeds was exthe Epicentre TargetAmp kit from nan
• Four replicates including two biologstage, including dye swap against ref
• Arrays of all stages and high throug12 million raw reads for 4 DAF, 12-14
•Small RNA data for 12-14 DAF
5-6mgReference
17-19 DAF 22-24 DAF 5-6mgReference
17-19 DAF 22-24 DAF
es using Arrays, RNA-seq and ery Early Stages of Seed Post-Fertilization
xtracted and amplified to mRNA using nogram amounts of total RNA
gical replicates hybridized at each ference stage (5-6 mg fresh wt per seed
ghput Illumina paired end reads of over DAF, and 5-6 mg
•The mid-maturation stages of cotyledon devanalyses using the 100-200 mg stage as refe•RNA seq is also in progress on several stag•Small RNA populations on seed coats versu
velopment were subjected to microarray erencegesus cotyledons
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set4 (305 genes) colored by calculated
75-100 vs. 100-200
1.02.03.04.05.06.07.08.0 Normalized
Intensity
set1 (323 gen
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set7 (252 genes) colored by calculated
75-100 vs. 100-200
1.02.03.04.05.06.07.08.0 Normalized
Intensity
set6 (410 gen
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set11 (325 genes) colored by calculated
75-100 vs. 100-200
1.02.03.04.05.06.07.08.0 Normalized
Intensity
set5 (247 gen
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set9 (455 genes) colored by calculated
75-100 vs. 100-200
1.02.03.04.05.06.07.08.0 Normalized
Intensity
set10 (220 gen
3757 genes in cotyledons that meet our
RNA Condition
nes) colored by calculated
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set8 (587 genes) colored by calculated
RNA Condition
nes) colored by calculated
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set2 (537 genes) colored by calculated
RNA Condition
nes) colored by calculated
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
set3 (96 genes) colored by calculated
RNA Condition
nes) colored by calculated
75-100 vs. 100-200
RNA Condition1.02.03.04.05.06.07.08.0 Normalized
Intensity
unclassified (23,852 genes)
r criteria (11 k-means clusters)
Transcript profiles of immaturSarah Jo
4 k-means cluster sets of 11 sets are shown belMany of the cDNAs have no known function or mMany transcription factors show developmental
re seed cotyledon developmentones, Ph.D. ow for 27,000 cDNAs on arraysmatch to other organisms l changes
Omega-3 v
0
0.5
1
1.5
2
25-50mg avg 75-100mg avg 40
Rat
io• Expression profiles of omega-3 (red) mRNAs in the cotyledons
• Profiles for many enzymes involved indiscerned from the microarray data of
vs. Omega-6
00-500mg avg yellow avg dry avg
and omega-6 (green) fatty acid desaturase
n compositional traits such as oil can be cotyledon development.
27k cDNA array ratios
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
25-50mg 75-100mg 4
Rat
io
Trancription factors that increa
: Transcription Factors
400-500mg yellow dry seed
Stages
ase during late seed maturation
Stage 1 Reference
Tissue Stage 2 Stage 3 Stage 4
Stage 2 vs 1 Stage 3 vs 1 Stage 4 vs 1
2 Slides 2 Slides 2 Slides Cy3/Cy5 Cy3/Cy5 Cy3/Cy5
2 Slides 2 Slides 2 Slides Dye Swap Dye Swap Dye Swap
2 Slides 2 Slides 2 Slides Cy3/Cy5 Cy3/Cy5 Cy3/Cy5
2 Slides 2 Slides 2 Slides Dye Swap Dye Swap Dye Swap
Biological Replicate #1
Biological Replicate #2
Yellow Cotyledons FunctionalYellow- Gree
Experimental Design U
Specific elements of the glyoxylate pfunctional transition of the soybean co
Gonzalez and Vodkin, BM
Stage 5 Stage 6 Stage 7
Stage 5 vs 1 Stage 6 vs 1 Stage 7 vs 1
2 Slides 2 Slides 2 Slides Cy3/Cy5 Cy3/Cy5 Cy3/Cy5
2 Slides 2 Slides 2 Slides Dye Swap Dye Swap Dye Swap
2 Slides 2 Slides 2 Slides Cy3/Cy5 Cy3/Cy5 Cy3/Cy5
2 Slides 2 Slides 2 Slides Dye Swap Dye Swap Dye Swap
l Transition en Cotyledons
Green Cotyledons
Using 56 Oligo Arrays
pathway play a significant role in the otyledon during seedling developmentMC Genomics 8: 468 2007
Oligo Microarray Va
RNA Blots Q Chlorophyll a/b Binding Protein
0
2
4
6
8
10
12
2-ΔΔC
T
S1/S1 S2/S1 S3/S1 S4/S1 S5/S1 S6/S1 S7/S1
Stage Comparison
Auxin Down Regulated ADR 12-2
0
50
100
150
200
250
300
2-ΔΔC
T
S1/S1 S2/S1 S3/S1 S4/S1 S5/S1 S6/S1 S7/S1
Stage Comparison
alidation Data Using
Quantatitive RTPCR
TAG FA FA
A-CoA
Lipid Body Glyoxysome
Phosphoglycolate Glycolate Glyoxylate Glycine
A
SerineHydroxypiruvate
GlycerateP-glycerate
Calvin Cycle
ChloroplastPeroxisome
Functional Transition
Li
B C
H2O2 H2O + O2
β-Oxidation
5
MalateGlyoxylate
Isocitrate
Citrate
OxaloacetateSuccinate
Glyoxylate Cycle2
3
6
4
1
DC
Succinate
Mitochondria
Glycine
Serine
Mitochondria
pid Mobilization Yellow Cotyledons
Photorespiration Green Cotyledons
Yellow and Green Cotyledons
NADHCO2NH3
Fumarate
TCA Cycle
Malate
Oxaloacetate
Malate
PEP
Gluconeogenesis
p-coumaroyl-C
Proanthocyanidins An
UG
Dihydroflavonols Isof
Chalcones
Aurones
Biflavonoids
Nar
Flavonols Flavan-3,4-diols
DFRFLS
F3H, F3'H, F3'5'H
The Flavonoid Pathway Produces Compand to Nutritional Content and Bioac
L-phenylalanine PAL oA 3 Malonyl-CoA
CHS
nthocyanins
GFGT
flavonoids Flavones
Naringenin
CHI
ingenin chalcone
IFS FS
pounds of Significance to Plant Defense ctive Compounds for Health Benefits
Applications of NanotechnologyPlant Geno
• Extensive genomics and transfofor applying novel nanotechnolgfollowing:
• Photonic crystals for increased sgene expression (ongoing with B
• Microfluidics to extract DNA, RN
• Novel plant transformation methapproaches
y to Gene Expression and omics
rmation expertise available y approaches as the
sensitivity detection of Brian Cunningham)
A from one or a few cells
ods using nanotechnology