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

Mudge, Stephen R., Osabe, Kenji, Casu, Rosanne E., Bonnett, Graham D., Manners, John M., Birch, Robert G.
Planta 2009 v.229 no.3 pp. 549-558
Southern blotting, alleles, bacterial artificial chromosomes, clones, crops, gene expression, heterozygosity, polymerase chain reaction, polyploidy, promoter regions, sequence analysis, sugarcane, sustainable engineering, transcription factors, transgenes
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.