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Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species

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Abstract

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

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Abbreviations

BAC:

Bacterial artificial chromosome

EST:

Expressed sequence tag

GUS:

β-Glucuronidase

LUC:

Firefly luciferase

Nos :

Nopaline synthase

SNP:

Single nucleotide polymorphism

Ubi-1 :

Ubiquitin-1

References

  • Baranowskij N, Frohberg C, Prat S, Willmitzer L (1994) A novel DNA-binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator. EMBO J 13:5383–5392

    PubMed  CAS  Google Scholar 

  • Barow M (2006) Endopolyploidy in seed plants. Bioessays 28:271–281

    Article  PubMed  CAS  Google Scholar 

  • Birch RG (1997) Plant transformation: problems and strategies for practical application. Ann Plant Physiol 48:297–326

    Article  CAS  Google Scholar 

  • Birch RG (2007) Metabolic engineering in sugarcane: assisting the transition to a bio-based economy. In: Verpoorte RA, Alfermann W, Johnson TS (eds) Applications of plant metabolic engineering. Springer, Berlin, pp 249–281

    Chapter  Google Scholar 

  • Bower R, Elliott AR, Potier BAM, Birch RG (1996) High-efficiency, microprojectile-mediated cotransformation of sugarcane, using visible or selectable markers. Mol Breed 2:239–249

    Article  CAS  Google Scholar 

  • Casu RE, Dimmock CM, Chapman SC, Grof CPL, McIntyre CL, Bonnett GD, Manners JM (2004) Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling. Plant Mol Biol 54:503–517

    Article  PubMed  Google Scholar 

  • Chen ZJ, Ni ZF (2006) Mechanisms of genomic rearrangements and gene expression changes in plant polyploids. Bioessays 28:240–252

    Article  PubMed  Google Scholar 

  • Chirgwin JM, Przybyla AE, Macdonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic-acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299

    Article  PubMed  CAS  Google Scholar 

  • Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes—structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689

    Article  PubMed  CAS  Google Scholar 

  • Clarke L, Carbon J (1976) Colony bank containing synthetic Col El hybrid plasmids representative of entire Escherichia coli genome. Cell 9:91–99

    Article  PubMed  CAS  Google Scholar 

  • Cordeiro GM, Taylor GO, Henry RJ (2000) Characterisation of microsatellite markers from sugarcane (Saccharum sp.), a highly polyploid species. Plant Sci 155:161–168

    Article  PubMed  CAS  Google Scholar 

  • Cronn R, Cedroni M, Haselkorn T, Grover C, Wendel JF (2002) PCR-mediated recombination in amplification products derived from polyploid cotton. Theor Appl Genet 104:482–489

    Article  PubMed  CAS  Google Scholar 

  • Cuadrado A, Acevedo R, de la Espina SMD, Jouve N, de la Torre C (2004) Genome remodelling in three modern S-officinarum x S-spontaneum sugarcane cultivars. J Exp Bot 55:847–854

    Article  PubMed  CAS  Google Scholar 

  • D’Hont A, Grivet L, Feldmann P, Rao S, Berding N, Glaszmann JC (1996) Characterisation of the double genome structure of modern sugarcane cultivars (Saccharum spp) by molecular cytogenetics. Mol Gen Genet 250:405–413

    PubMed  CAS  Google Scholar 

  • Edme SJ, Comstock JC, Miller JD, Tai PYP (2005) Determination of DNA content and genome size in sugarcane. J Am Soc Sugar Cane Technol 25:1–16

    Google Scholar 

  • Finnegan J, McElroy D (1994) Transgene inactivation: plants fight back!. Bio/Technol 12:883–888

    Article  Google Scholar 

  • Glaszmann JC, Fautret A, Noyer JL, Feldmann P, Lanaud C (1989) Biochemical genetic-markers in sugarcane. Theor Appl Genet 78:537–543

    Article  CAS  Google Scholar 

  • Graham MW, Mudge SR, Sternes PR, Birch RG (2008) Understanding and avoiding transgene silencing. In: Stewart CN, Touraev A, Citovsky V, Tzfira T (eds) Plant transformation technologies. Wiley-Blackwell, New York, (in Press)

  • Grivet L, Glaszmann JC, Arruda P (2001) Sequence polymorphism from EST data in sugarcane: a fine analysis of 6-phosphogluconate dehydrogenase genes. Genet Mol Biol 24:161–167

    Article  CAS  Google Scholar 

  • Grivet L, Glaszmann JC, Vincentz M, da Silva F, Arruda P (2003) ESTs as a source for sequence polymorphism discovery in sugarcane: example of the Adh genes. Theor Appl Genet 106:190–197

    PubMed  CAS  Google Scholar 

  • Hansom S, Bower R, Zhang L, Potier B, Elliott A, Basnayake S, Cordeiro G, Hogarth DM, Cox M, Berding N, Birch RG (1999) Regulation of transgene expression in sugarcane. In: Proceedings of XXIII International Society Sugar Cane Technologists Congress, New Delhi, India, 22–26 February 1999. vol. 2. pp 278–290

  • Ingelbrecht IL, Irvine JE, Mirkov TE (1999) Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiol 119:1187–1197

    Article  PubMed  CAS  Google Scholar 

  • Jannoo N, Grivet L, Seguin M, Paulet F, Domaingue R, Rao PS, Dookun A, D’Hont A, Glaszmann JC (1999) Molecular investigation of the genetic base of sugarcane cultivars. Theor Appl Genet 99:171–184

    Article  CAS  Google Scholar 

  • Jefferson R (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405

    Article  CAS  Google Scholar 

  • Lu C-A, Ho TD, Ho S-L, Yu SM (2002) Three novel MYB proteins with one DNA binding repeat mediate sugar and hormone regulation of a-amylase gene expression. Plant Cell 14:1963–1980

    Article  PubMed  CAS  Google Scholar 

  • Luehrsen K, Walbot V (1993) Firefly luciferase as a reporter for plant gene expression studies. Promega Notes 44:24–29

    Google Scholar 

  • McIntyre CL, Jackson M, Cordeiro GM, Amouyal O, Hermann S, Aitken KS, Eliott F, Henry RJ, Casu RE, Bonnett GD (2006) The identification and characterisation of alleles of sucrose phosphate synthase gene family III in sugarcane. Mol Breed 18:30–50

    Article  Google Scholar 

  • Milanesi L, Muselli M, Arrigo P (1996) Hamming clustering method for signals prediction in 5′ and 3′ regions of eukaryotic genes. Comput Appl Biosci 12:p399–p404

    Google Scholar 

  • Rae AL, Grof CPL, Casu RE, Bonnett GD (2005) Sucrose accumulation in the sugarcane stem: pathways and control points for transport and compartmentation. Field Crops Res 92:159–168

    Article  Google Scholar 

  • Rogers S, Bendich A (1988) Extraction of DNA from plant tissues. In: Plant molecular biology manual. Kluwer Academic Publishers, Dordrecht, pp 1–10

  • Rubio-Somoza I, Martinez M, Abraham Z, Diaz I, Carbonero P (2006a) Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds. Plant J 47:269–281

    Article  PubMed  CAS  Google Scholar 

  • Rubio-Somoza I, Martinez M, Diaz I, Carbonero P (2006b) HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germination. Plant J 45:17–30

    Article  PubMed  CAS  Google Scholar 

  • Wei HR, Wang ML, Moore PH, Albert HH (2003) Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. J Plant Physiol 160:1241–1251

    Article  PubMed  CAS  Google Scholar 

  • Yang MZ, Bower R, Burow MD, Paterson AH, Mirkov TE (2003) A rapid and direct approach to identify promoters that confer high levels of gene expression in monocots. Crop Sci 43:1805–1813

    CAS  Google Scholar 

  • Yu W, Rusterholtz KJ, Krummel AT, Lehman N (2006) Detection of high levels of recombination generated during PCR amplification of RNA templates. Biotechniques 40:499–507

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was funded by the Australian Sugar Research and Development Corporation. We thank Angelo Fallarino (Botany Department, SIB, UQ) for technical assistance, Stevens Brumbley and BSES for providing the Q200 BAC library, Anne Rae (CSIRO Plant Industry) for providing RNA samples from dissected stem tissues, and Chris Grof (CSIRO Plant Industry) for providing the Q117 Genome Walker libraries. Thanks also to Lynne McIntyre (CSIRO Plant Industry) for helpful discussions.

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Authors and Affiliations

  1. Botany Department, School of Integrative Biology, The University of Queensland, Brisbane, 4072, Australia

    Stephen R. Mudge, Kenji Osabe & Robert G. Birch

  2. CSIRO Plant Industry, Queensland Bioscience Precinct, 306 Carmody Road, St Lucia, 4067, Australia

    Stephen R. Mudge, Rosanne E. Casu, Graham D. Bonnett & John M. Manners

Authors
  1. Stephen R. Mudge
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  2. Kenji Osabe
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  3. Rosanne E. Casu
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  4. Graham D. Bonnett
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  5. John M. Manners
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  6. Robert G. Birch
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Corresponding author

Correspondence to Stephen R. Mudge.

Electronic supplementary material

Below is the link to the electronic supplementary material.

425_2008_852_MOESM1_ESM.doc

Alignment of the genomic sequences of the eight putative ScR1MYB1 alleles identified in Q117, along with the cDNA from EST clone MCSA063B04 (DOC 38 kb)

425_2008_852_MOESM2_ESM.doc

Nucleotide sequences of the Z1 genomic sequence (Z1_gDNA), the fully spliced transcript (Z1_full) and the partially spliced transcript in which intron 1 remains (Z1_no). In rare cases (2/106 RT-PCR products from Q117), an alternative splice acceptor at position 438 on the alignment (sequence TCTGCAG) is used (DOC 34 kb)

425_2008_852_MOESM3_ESM.doc

Alignment of the eight isolated ScR1MYB1 promoter alleles that were functionally tested using reporter gene fusions. The sequence for the upstream Genome Walker adapter and the downstream GSP2 binding site are shown in red. The predicted TATA box is shown in green, and the transcriptional start site determined by 5′RACE is shown in blue (DOC 70 kb)

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Mudge, S.R., Osabe, K., Casu, R.E. et al. Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species. Planta 229, 549–558 (2009). https://doi.org/10.1007/s00425-008-0852-8

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  • DOI: https://doi.org/10.1007/s00425-008-0852-8

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