Transgenic Research

, Volume 14, Issue 6, pp 887–905 | Cite as

A Microarray-Based Comparative Analysis of Gene Expression Profiles During Grain Development in Transgenic and Wild Type Wheat

  • Per L. GregersenEmail author
  • Henrik Brinch-Pedersen
  • Preben B. Holm


Global, comparative gene expression analysis is potentially a very powerful tool in the safety assessment of transgenic plants since it allows for the detection of differences in gene expression patterns between a transgenic line and the mother variety. In the present study, we compared the gene expression profile in developing seeds of wild type wheat and wheat transformed for endosperm-specific expression of an Aspergillus fumigatus phytase. High-level expression of the phytase gene was ensured by codon modification towards the prevalent codon usage of wheat genes and by using the wheat 1DX5HMW glutenin promoter for driving transgene expression. A 9K wheat unigene cDNA microarray was produced from cDNA libraries prepared mainly from developing wheat seed. The arrays were hybridised to flourescently labelled cDNA prepared from developing seeds of the transgenic wheat line and the mother variety, Bobwhite, at three developmental stages. Comparisons and statistical analyses of the gene expression profiles of the transgenic line vs. that of the mother line revealed only slight differences at the three developmental stages. In the few cases where differential expression was indicated by the statistical analysis it was primarily genes that were strongly expressed over a shorter interval of seed development such as genes encoding storage proteins. Accordingly, we interpret these differences in gene expression levels to result from minor asynchrony in seed development between the transgenic line and the mother line. In support of this, real time PCR validation of results from selected genes at the late developmental stage could not confirm differential expression of these genes. We conclude that the expression of the codon-modified A.␣fumigatus phytase gene in the wheat seed had no significant effects on the overall gene expression patterns in the developing seed.

Key words

cDNA microarrays gene expression phytase safety assessment transgenic wheat 


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  1. Alba, R, Fei, Z, Payton, P, Liu, Y, Moore, SL, Debbie, P, Cohn, J, D’Ascenzo, M, Gordon, JS, Rose, JKC, Martin, G, Tanksley, SD, Bouzayen, M, Jahn, MM, Giovannoni, J 2004ESTs, cDNA microarrays, and gene expression profiling: tools for dissectin plant physiology and developmentPlant J39697714CrossRefPubMedGoogle Scholar
  2. Brinch-Pedersen, H, Galili, G, Knudsen, S, Holm, PB 1996Engineering of the aspartate family biosynthetic pathway in barley (Hordeum vulgare L.) by transformation with heterologous genes encoding feed-back-insensitive aspartate kinase and dihydropicolinate synthasePlant Mol Biol32611620CrossRefPubMedGoogle Scholar
  3. Brinch-Pedersen, H, Olesen, A, Rasmussen, SK, Holm, PB 2000Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytaseMol Breed6195206CrossRefGoogle Scholar
  4. Brinch-Pedersen, H, Sørensen, LD, Holm, PB 2002Engineering crop plants: getting a handle on phosphateTrends Plant Sci7118125CrossRefPubMedGoogle Scholar
  5. Brinch-Pedersen, H, Hatzack, F, Sorensen, LD, Holm, PB 2003Concerted action of endogenous and heterologous phytase on phytic acid degradation in seed of transgenic wheat (Triticum aestivum L.)Transgenic Res12649659CrossRefPubMedGoogle Scholar
  6. Cellini, F, Chesson, A, Colquhoun, I, Constable, A, Davies, HV, Engel, KH, Gatehouse, AM, Kärenlampi, S, Kok, EJ, Leguay, JJ, Lehesranta, S, Noteborn, HP, Pedersen, J, Smith, M 2004Unintended effects and their detection in genetically modified cropsFood Chem Toxicol4210891125PubMedGoogle Scholar
  7. Christensen, AH, Sharrock, RA, Quail, PH 1992Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporationPlant Mol Biol18675689CrossRefPubMedGoogle Scholar
  8. Clarke, BC, Hobbs, M, Skylas, D, Appels, R 2000Genes active in developing wheat endospermFunct Integr Genomics14455CrossRefPubMedGoogle Scholar
  9. Dudoit, S, Yang, YH, Callow, MJ, Speed, TP 2002Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experimentsStat Sinica12111140Google Scholar
  10. Eisen, MM, Brown, PO 1999DNA arrays for analysis of gene expressionMethod Enzymol303179205Google Scholar
  11. Fiehn, O, Kloska, S, Altman, T 2001Integrated studies on plant biology using multiparallel techniquesCurr Opin Biotechnol128286CrossRefPubMedGoogle Scholar
  12. Kok, EJ, Kuiper, HA 2003Comparative safety assessment for biotech cropsTrends Biotechnol21439444CrossRefPubMedGoogle Scholar
  13. Kuiper, HA, Kok, EJ, Engel, K-H 2003Exploitation of molecular profiling techniques for GM food safety assessmentCurr Opin Biotechnol14238243CrossRefPubMedGoogle Scholar
  14. Leader, DJ 2005Transcriptional analysis and functional genomics in wheatJ Cereal Sci41149163CrossRefGoogle Scholar
  15. Lucca, P, Hurrell, R, Potrykus, I 2001Genetic engineering approaches to improve the bioavailability and the level of iron in rice grainsTheor Appl Genet102392397CrossRefGoogle Scholar
  16. Ouakfaoui, SE, Miki, B 2005The stability of the Arabidopsis transcriptome in transgenic plants expressing the marker genes nptII and uidAPlant J41791800CrossRefPubMedGoogle Scholar
  17. Pasamontes, L, Haiker, M, Wyss, M, Tessier, M, Loon, APGM 1997Gene cloning, purification, and characterization of a heat-stable phytase from fungus Aspergillus fumigatusAppl Environ Microbiol6316961700PubMedGoogle Scholar
  18. Quackenbush, J 2002Microarray data normalization and transformationNat Biotechnol Suppl22496501Google Scholar
  19. Rogers, JC, Milliman, C 1983Isolation and sequence analysis of a barley α-amylase cDNA cloneJ Biol Chem25881698174PubMedGoogle Scholar
  20. Shewry, PR, Beaudoin, F, Jenkins, J, Griffiths-Jones, S, Mills, ENC 2002Plant protein families and their relationships to food allergyBiochem Soc Trans30906910PubMedGoogle Scholar
  21. Shewry, PR, Tatham, AS, Halford, NG, Barker, JHA, Hannappel, U, Gallois, P, Thomas, M, Kreis, M 1994Opportunities for manipulating the seed protein composition of wheat and barley in order to improve qualityTransgenic Res3312CrossRefPubMedGoogle Scholar
  22. Simmonds, DH, O’Brien, TP 1981Morphological and biochemical development of the wheat endospermPomeranz, Y eds. Advances in Cereal Science and TechnologyAmerican Society of Cereal Chemists, IncSt. Paul, Minnesota670Google Scholar
  23. Smyth GK (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3(1)Google Scholar
  24. Smyth, GK, Speed, TP 2003Normalization of cDNA microarray dataMethods31265273CrossRefPubMedGoogle Scholar
  25. Smyth, GK, Yang, Y-H, Speed, TP 2003 Statistical issues in microarray data analysisBrownstein, MJKhodursky, AB eds. Functional Genomics: Methods and Protocols, Methods in Molecular BiologyHumana PressTotowa, NJ111136Google Scholar
  26. Thompson, CJ, Novva, NR, Tizard, R, Crameri, R, Davies, JE, Lauwereys, M, Botterman, J 1987Characterization of the herbicide resistance gene bar from Streptomyces hygroscopicusEMBO J625192523Google Scholar
  27. Trethewey, RN 2004Metabolite profiling as an aid to metabolic engineering in plantsCurr Opin Plant Biol7196201CrossRefPubMedGoogle Scholar
  28. Wilson, ID, Barker, GLA, Beswick, RW, Shepherd, SK, Lu, C, Coghill, JA, Edwards, D, Owen, P, Lyons, R, Parker, JS, Lenton, JR, Holdsworth, MJ, Shewry, PR, Edwards, KJ 2004A transcriptomics resource for wheat functional genomicsPlant Biotechnol J2495506Google Scholar
  29. Yang, HY, Dudoit, S, Luu, P, Lin, DM, Peng, V, Ngai, J, Speed, TP 2002Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variationNucleic Acids Res30e15PubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Per L. Gregersen
    • 1
    Email author
  • Henrik Brinch-Pedersen
    • 1
  • Preben B. Holm
    • 1
  1. 1.Department of Genetics and BiotechnologyDanish Institute of Agricultural Sciences, Research Centre FlakkebjergSlagelseDenmark

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