Development and application of a mature stem-specific promoter in sugarcane

Kenji Osabe (2010). Development and application of a mature stem-specific promoter in sugarcane PhD Thesis, School of Biological Sciences, The University of Queensland.

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Author Kenji Osabe
Thesis Title Development and application of a mature stem-specific promoter in sugarcane
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2010-05
Thesis type PhD Thesis
Supervisor Professor Robert Birch
Doctor Stephen Mudge
Total pages 184
Total colour pages 20
Total black and white pages 164
Subjects 06 Biological Sciences
Abstract/Summary Developments in gene technology offer the potential to engineer sugarcane as a bio-factory, by converting sucrose into valuable products. Efficient conversion of sucrose in sugarcane is likely to require transgene expression specifically in the mature stems, to avoid interference with plant metabolism at critical stages during development. In principle, this can be achieved by the use of a mature-stem-specific promoter fused to the gene of interest. In a previous study, promoter alleles from ScR1MYB1, a sugarcane gene up-regulated in mature stems, did not drive reporter transgene activity in mature sugarcane stems. It was interpreted that either (1) the isolated promoter alleles lacked the key regulatory elements to drive mature-stem expression, and/or (2) the lack of reporter activity resulted from efficient transgene silencing in sugarcane. To test these possibilities, I decided to (1) test a longer sequence from the promoter region and (2) test sugarcane-derived reporter sequences that might not trigger silencing. To understand the allelic diversity and identify an expressed allele for promoter recovery, the ScR1MYB1 locus was analyzed by sequencing genomic PCR and RT-PCR amplicons, and scoring the occurrence of each allele. In modern sugarcane cultivars Q200 and Q117, seven and eight alleles were identified, respectively. Sequencing and scoring of RT-PCR amplicons revealed at least four of these alleles are expressed in both cultivars, and one (A1 allele) occurred significantly more frequent than other alleles in the mature stems of sugarcane. The A1 allele showed strong mature-stem-specificity, whereas another allele U was up-regulated in roots. This indicates differential allele expression of ScR1MYB1 in sugarcane, and ideal expression pattern of the A1 allele for promoter recovery. An extended (5.7kb) promoter region and a 1.4kb terminator region were cloned from the ScR1MYB1 A1 allele, originating from a sugarcane Q200 BAC library. These control regions were fused to foreign (sucrose isomerase and luciferase) and sugarcane-derived (sugarcane rubisco small subunit and ScR1MYB1) reporter genes to assess mature-stem-specific transgene expression. Foreign genes fused to the promoter and terminator of the A1 allele were not expressed in mature sugarcane, but mature-stem-specific expression of sugarcane-derived reporter gene was shown in three out of six mature transgenic lines. This project demonstrated that it is important to understand the allelic diversity and expression vi pattern, and to select the allele with the desired expression pattern for promoter recovery from sugarcane. Expression of cis-gene (ScR1MYB1) in contrast to foreign genes in equivalent expression cassettes indicates an endogenous gene can avoid the loss of transgene expression. This project also showed transgene silencing could interfere with testing of promoters in sugarcane, and that the 5.7kb A1 promoter and 1.4kb terminator is potentially applicable for sugarcane biotechnology, if used with silencing resistant transgenes. I also assessed the efficacy of hairpin-mediated down-regulation of endogenous genes in sugarcane and demonstrated for the first time that hairpin constructs designed using the EST database can efficiently down-regulate the complete allelic suite of an endogenous gene in sugarcane. This provides a molecular tool for physiological studies and metabolic engineering. I used this method in combination with over-expression using the maize Ubi promoter, to test whether ScR1MYB1 had a key regulatory role in sugar accumulation. The resulting transgenic lines did not show any effect on sucrose level in the mature stems, indicating that this gene does not have a major role in sugar accumulation in sugarcane.
Keyword Allelic diversity
Functional Genomics
Sugarcane (Saccharum)
Tissue-specific Expression
Additional Notes Colour pages: 18, 20, 30, 36, 49, 54, 56, 68, 71, 72, 76, 88, 91, 96, 101, 102, 103, 154, 183 Landscape page: 30

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Created: Tue, 09 Nov 2010, 22:53:23 EST by Mr Kenji Osabe on behalf of Library - Information Access Service