Development of cdna microarrays for gene expression research in floriculture crops
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Development of cDNA Microarrays for Gene Expression Research in Floriculture Crops. Dave Clark University of Florida Environmental Horticulture Dept. UF Floriculture Biotech Lab (est. 1995). Our main goal: To bridge the gap between basic molecular biology and applied floriculture

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Development of cDNA Microarrays for Gene Expression Research in Floriculture Crops

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Development of cdna microarrays for gene expression research in floriculture crops

Development of cDNA Microarrays for Gene Expression Research in Floriculture Crops

Dave Clark

University of Florida

Environmental Horticulture Dept.


Uf floriculture biotech lab est 1995

UF Floriculture Biotech Lab(est. 1995)

  • Our main goal:

  • To bridge the gap between basic molecular biology and applied floriculture

    • Florida produces 20% of US floriculture crops


Uf floriculture biotech

UF Floriculture Biotech

Development:

  • Phase 1 – 1995-2000 (then)

    • Biotechnology

  • Phase 2 – 2000-2005 (now)

    • Functional Genomics

  • Phase 3 – 2005-2010 (next)

    • Gene Function


Phase 1 1995 2000

Phase 1 – 1995-2000

  • Establish a biotech system

    • Petunia hybrida

  • Proof of concept research

    • Expansion of available technology

  • Build critical personnel mass

    • Recruit good people

    • Make strong collaborations


Phase 1 1995 20001

Phase 1 – 1995-2000

  • Transgenic Plants - hormones

  • Gibberellins

    • Dwarf plants

  • Cytokinins

    • “Stay Green” leaves

  • Ethylene

    • Long lasting flowers


Internal growth regulators

Internal Growth Regulators

AtGA2ox7

Makes dwarf tobacco

Schomburg et al. 2003


Internal growth regulators1

Internal Growth Regulators

AtGA2ox7 - Makes dwarf petunia


Stay green leaves

Stay Green Leaves

sag-IPT

Makes leaves stay green longer in tobacco

Gan & Amasino 1995


Sag ipt

sag-IPT

Makes leaves stay greener longer in petunia

Clark et al. in press


Sag ipt drought tolerance

sag-IPT – Drought Tolerance


Development of cdna microarrays for gene expression research in floriculture crops

Long Lasting Flowers

Etr1-1

ETR1-1 - Makes plants ethylene insensitive


Development of cdna microarrays for gene expression research in floriculture crops

Long Lasting Flowers

Control

Etr1-1


Problems with 35s etr1 1

Problems with 35S::etr1-1

  • etr1-1 petunias – commercially limited

rooting

pathogens – etr1-1

‘MD’

etr1-1


Conclusions phase 1

Conclusions – Phase 1

  • We have a good experimental system

  • We have good people in the lab

  • We have good collaborators

  • We needdifferent genes

  • We needbetter promoters


Phase 2 2000 2005

Phase 2 – 2000-2005

  • Functional Genomics

    • New tools for genetics research

  • Exploration for new technologies

    • New and novel genes

  • Controlling gene expression

    • Petal specific promoters


Functional genomics

Functional Genomics

  • Tools that help discover the biological function of genes

  • Uncovers how groups of genes work together in a biological process

  • Encompasses many traditional biological and genetic approaches


Development of cdna microarrays for gene expression research in floriculture crops

Petunia EST Project

(EST = Expressed Sequence Tags)

mRNA extraction

Tissue collection

Bioinformatics and Microarrays

library synthesis

EST sequencing


Petunia flower genes

Petunia Flower Genes

  • Petunia gene sequencing:

    Floral development library 2774

    C2H4-treated flower library 3264

    Pollinated flower library 1824

    Other libraries 1046

  • Total genes sequenced 8908


Petunia est database

Petunia EST Database


Development of cdna microarrays for gene expression research in floriculture crops

Microarray Construction

4200 unique petunia genes


Development of cdna microarrays for gene expression research in floriculture crops

Microarray analysis – Ethylene Regulated Genes

Green A>B

Yellow A=B

Red A<B

Control

(A)

Ethylene

(B)

A+B


Ethylene regulated genes

Ethylene Regulated Genes

  • We have isolated approximately 50 genes that are regulated by ethylene

  • Some of these genes are thought to be involved in making floral scent volatiles

    • Benzoic acid carboxyl methyltransferase


Phase 2 still going

Phase 2 – Still going

  • We still bridge the gap between basic molecular biology and applied floriculture

    • We have a petunia EST database

    • Microarrays are working well

    • We have many new and novel genes

    • We have good promoters


Phase 3 2005 2010

Phase 3 – 2005-2010

  • Gene Function

    • We are ahead of schedule:

    • Experimental system (phase 1)

      • Adopted by several groups

    • New genetic tools (phase 2)

      • Available to floriculture researchers

    • Understanding gene function


Measuring floral scent

Measuring Floral Scent


Flower scent composition petunia

Flower Scent Composition - Petunia

Benzoic acid carboxyl methyltransferase (BAMT) - makes methyl benzoate in petunia


Stop and smell the roses

Stop and Smell the Roses!

US consumers bought almost 130 million roses on Valentine’s Day 2003

US consumers spent over $7.5B on cut flowers in 2002


Opportunity knocks

Opportunity Knocks!

  • Commercial cut rose varieties don’t have much fragrance


Development of cdna microarrays for gene expression research in floriculture crops

Flower Scent Composition - Rose


The future

The Future

  • We plan to maintain and grow the genetics database

  • We plan to make transgenic plants expressing new genes

  • We plan to knock out 1 gene a week – functional analysis

  • We plan to continue making new collaborations


Who supports us

Who supports us?

  • Intellectually

    • Harry Klee – UF

    • Bill Farmerie – UF

    • Don McCarty – UF

    • Michelle Jones – OSU

    • Jim Giovannoni – USDA/Cornell

    • EU PetNet Group

  • Financially

    • USDA Floriculture Initiative

    • American Floral Endowment

    • Fred Gloeckner Foundation

    • Florida FIRST

    • The Scotts Company


Uf floriculture biotechnology 2002 2003

UF Floriculture Biotechnology2002-2003


Development of cdna microarrays for gene expression research in floriculture crops

Questions?


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