Agricultural and Pharmaceutical Nanotechnology Plate Stackers Allow Printing up to 20,000 DNA Oligo...

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

Automated Plate Stackers Allow Prinnting up to 20,000 DNA Oligo per Slide

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




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


Intensity


75-100 vs. 100-200

1.02.03.04.05.06.07.08.0 Normalized

Intensity

set6 (410 gen

75-100 vs. 100-200


Intensity


75-100 vs. 100-200

1.02.03.04.05.06.07.08.0 Normalized

Intensity

set5 (247 gen

75-100 vs. 100-200


Intensity


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


Intensity


RNA Condition


75-100 vs. 100-200


Intensity


RNA Condition


75-100 vs. 100-200


Intensity


RNA Condition


75-100 vs. 100-200


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



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





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

Transformation of Soybeaby Bomb

an Embryogenic Cultures bardment

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

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