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Type: Thesis
Title: Characterisation of α-expansin genes in Gossypium hirsutum.
Author: Harmer, Sarah Elizabeth
Issue Date: 2003
School/Discipline: School of Molecular and Biomedical Science
Abstract: Cotton fibres arise from single epidermal cells of the ovule and initiate as protrusions on the day of flowering (anthesis). Elongation continues for approximately 20 days, resulting in fibres which are 2-3.5 cm in length. As fibres are single cells of homogenous tissue and their development is synchronous, they are an excellent model system for the analysis of the molecular processes involved in cell growth. One type of cell wall protein, the α-expansins, has been implicated in cell wall modification. α-expansins have been characterised in a number of plant species and are encoded by large gene families (31 members in Arabidopsis), with family members often showing heterogenous patterns of expression (Wu et al., 2001). In some plant species, α-expansins have specifically been implicated in cell elongation, such that a cotton fibre-specific α-expansin is an attractive target for cotton biotechnology. Differential screening of a cotton fibre cDNA library allowed the identification of a fibre-specific α-expansin mRNA (pFS14) of which accumulated at high levels during fibre cell elongation (Orford and Timmis, 1993). The aims of this project focused on the isolation, characterisation and analysis of the fibre-specific α-expansin gene and other cotton α-expansin family members. Using, a combination of genomic library screening and PCR-based approaches six different cotton α-expansin gene family members were isolated (chapter Three). One of the genes, GhExp14.2, was 99% similar to the pFSl4 cDNA, and therefore it was considered to be the genomic representative of pFS14 cDNA. RT-PCR analysis (Chapter Three) showed that five of the six α-expansins have distinctive expression patterns in different cotton tissues, and that the transcripts of four genes were detected in elongating fibres. GhExp14.2 transcripts were detected at high levels in elongating fibres, consistent with the results described by Orford and Timmis (1998), suggesting that GhExp14.2 plays an important role in fibre development. Evolutionary analysis of α-expansin genes from cotton and other plant species (Chapter Four) confirmed previous studies that demonstrate the α-expansin family is divided into four ancient, distinct subclades. Each subclade was further divided into groups on the basis of gene orthology. In some cases 'orthologous groups' within clades contain multiple genes that appeared to be expressed in a similar manner to each other and the evolutionary analysis provides a foundation by which specific orthologous α-expansin genes could be identified and characterised. The development of a transient assay for use in cotton fibres was undertaken (Chapter Five). Various parameters and conditions were tested on tobacco and cotton tissues until an appropriate method was refined, with fibres aged approximately 4 DPA providing the best results. The validity of the transient assay technique was tested using the GhExpl4.2 promoter, with the full available sequence (848 bp up to and including the ATG initiation codon) directing GUS reporter gene expression only in developing fibre cells and not other cotton tissues tested. This work describes an efficient, simple method for the functional dissection of cotton fibre-specific promoters. Known cis-acting consensus elements in the GhExp14.2 fibre-specific promoter were accurately mapped using transcription factor binding site websites (Chapter Six). Detailed comparisons of multiple fibre-specific promoters allowed identification of four new putative fibre-specific promoter elements. A deletion analysis was undertaken to dissect the functional regions of, and putative consensus bindings sites within, the GhExp14.2 promoter. All GhExp14.2 promoter deletions maintained fibre-specific expression of the GUS reporter gene, including the smallest, which consisted of only l49bp. Within the GhExpl4.2 149bp minimal promoter five previously characterised consensus-binding sites (ACGT box, Q-element, RY pyrimidine repeat, an E-box and a GCN4 motif) and one novel binding site (PFE4) were present, and each was suggested to be involved in directing fibre-specific transcription. In Chapter Seven, preparation of four constructs using the fibre-specific cDNA pFS14, under the control of different fibre-specific promoters, is described. These constructs were designed with a view of modifying fibre morphology by altering the temporal expression pattern of the α-expansin gene in transgenic cotton. Results from the transgenic cotton, although not available for incorporation into this thesis, will be used to define the important role that GhExp14.2 plays in fibre elongation, and may also facilitate the production of cotton varieties with improved fibre morphology.
Advisor: Timmis, Jeremy Newman
Orford, Sharon Josephine
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2003
Keywords: α-expansin genes; Gossypium hirsutum; cotton
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exception. If you are the author of this thesis and do not wish it to be made publicly available or If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
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