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Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie; Jonathan Cohn; Mark D'Ascenzo; Jeffrey S. Gordon; Jocelyn K. C. Rose; Gregory.

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Presentation on theme: "Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie; Jonathan Cohn; Mark D'Ascenzo; Jeffrey S. Gordon; Jocelyn K. C. Rose; Gregory."— Presentation transcript:

1 Rob Alba; Zhangjun Fei; Paxton Payton; Yang Liu; Shanna L. Moore; Paul Debbie; Jonathan Cohn; Mark D'Ascenzo; Jeffrey S. Gordon; Jocelyn K. C. Rose; Gregory Martin; Steven D. Tanksley; Mondher Bouzayen; Molly M. Jahn; Jim Giovannoni The Plant JournalThe Plant Journal, Volume 39, Number 5, September 2004, pp (18) ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development

2 = transcribed portion of a genome

3 expression profiling RNA gel blot (=northern) differential display cDNA-AFLP sequencing cDNA libraries (ESTs) SAGE microarrays ONE GENE AT A TIME NOT QUANTITATIVE, difficult to confirm CHEAP! little genomic data required EXPENSIVE AND LABOR-INTENSIVE NOT SENSITIVE TO LOW-ABUNDANCE TRANSCRIPTS many genes at once, semi-quantitative expensive

4 low- abundance transcripts sequence errors 2  structure impairs RT human error gene discovery “transcriptom e activity” expressio n arrays mapping, coding regions EXPRESSED SEQUENCE TAGS

5 the TOM1 array  cDNA microarray based on EST library  12,899 features representing 8500 tomato genes  protocols and confirmatory data available at The Tomato Expression Database: differential expression between tomato and pepper pericarp

6 microarray pitfalls cross-hybridization with related sequences non-detection of sequence not included in the array data handling is complex, therefore prone to human error (transformation, normalization, visualization, interpretation) poor replication/experimental design cDNA microarrays: –chimeric clones –inconsistent hybridization due to non-uniformity of microarray features

7 artifacts occur but the pattern is robust 21 RUBISCO homologsphotosynthesis-associated genes

8 data visualization interpretation is problematic

9 expression profiling simultaneously measures as much of the transcriptome as is represented on the chip this provides a valuable resource for studying regulatory and metabolic networks massive quantities of data are generated (and need to be analyzed) high costs and statistical difficulties encourage more focused approaches, but you only find what you’re looking for!

10 Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes Ganga Rao Davuluri, Ageeth van Tuinen, Paul D Fraser, Alessandro Manfredonia, Robert Newman, Diane Burgess, David A Brummell, Stephen R King, Joe Palys, John Uhlig, Peter M Bramley, Henk M J Pennings & Chris Bowler Nature Biotechnology 23, (2005)

11 hydrophobic mevalonic acid pathway hydrophilic acetate-malonate pathway carotenoids flavonoids free radical scavengers/antioxidants enhance vertebrate immune system not synthesized by animals

12 RANK IN NUTRIENT CONTENT RANK IN NUTRIENT CONTRIBUTION TO AVERAGE U.S. DIET modified from C.M. Rick

13 quercetin (flavonoid) flavonoid and carotenoid biosythesis occurs through separate pathways chlorogenic acid (phenylpropanoid) B-carotene (carotenoid) lycopene (carotenoid) naringenin-chalcone (flavonoid)

14 attempts at increasing phenolic/carotenoid production via expression of biosynthetic enzymes/transcription factors Ye et al. 2000: production of β-carotene in rice endosperm via transformation with biosynthetic enzymes from daffodil, Erwinia Fraser et al. 2002: fruit- specific expression of Erwinia phytoene synthase increases carotenoid production in tomato Ducreux 2005: enhanced carotenoid production in potato via heterologous expression of Erwinia phytoene synthase Niggeweg 2004: overexpression of HQA to increase CGA production in tomato Muir 2001: overexpression of petunia chalcone- isomerase increases flavonol tomato Bovy 2002: increased flavonol production through heterologous expression of maize transcription factor

15 Phenotype of the tomato high pigment-2 mutant is caused by a mutation in the tomato homolog of DEETIOLATED1. A C Mustilli, F Fenzi, R Ciliento, F Alfano, and C Bowler Plant Cell February; 11(2): 145–157. de-etiolated 1: in A. thaliana, display light-grown phenotype when grown in the dark tomato hp-2 shows no phenotype in dark, but is hyper-responsive to light and has elevated pigment

16 Ganga Rao Davuluri, Ageeth van Tuinen, Diane Burgess, David A. Brummell, Stephen R. King, Joe Palys, John Uhlig, Henk M. J. Pennings, Chris Bowler, Anna Chiara Mustilli, Alessandro Manfredonia Robert Newman Manipulation of DET1 expression in tomato results in photomorphogenic phenotypes caused by post-transcriptional gene silencing WT HIGH PIGMENT! phenotypes consistent with loss of function, suggesting silencing

17 post-transcriptional gene silencing transcriptional gene silencing transgene- induced silencing gene-specific methylation degradatio n of gene transcripts Davuluri et al. (2005) apply a post-tanscriptional silencing approach (RNAi) under a fruit-specific promoter

18 dimunition of TDET1 transcript in fruit but not other tissues identification of TDET1 degradation products in fruit but not leaves

19 lycopene B-carotene fruit weight brix

20 Arabidopsis genome germplasm resources “functional genomics”: identification of fruit- specific promoters cloning det1 genetic mapping homology to Arabidopsi s genomic libraries expression data


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