Approaching an RT-PCR Assay to Analyse Gene Expression in Chilling-Stressed Rhododendron: Partial Cloning of an Ascorbate Peroxidase Gene and Enzyme Activity Studies

Heggie, Laura (1999) Approaching an RT-PCR Assay to Analyse Gene Expression in Chilling-Stressed Rhododendron: Partial Cloning of an Ascorbate Peroxidase Gene and Enzyme Activity Studies. PhD thesis, University of Glasgow.

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Abstract

Production and regulation of active oxygen species are important responses to environmental stress in plant tissues. This study was concerned with development of a competitive RT-PCR assay to study changes in ascorbate peroxidase gene expression in chilled and non-chilled in vitro grown cultures of Rhododendron ponticum, R. hatsugiri and R. impeditum. Oligonucleotides for PCR amplification of ascorbate peroxidase and glutathione reductase DNA sequences were designed using a sequence homology alignment of mRNA/DNA sequences from six distinct plant species. Ligation of PCR products into the pT-Adv plasmid vector and transformation into Escherichia coli, followed by partial sequencing, confirmed fragment identity. The subsequent design of Rhododendron- specific primers, and the construction of a cRNA competitor fragment by in vitro transcription for use in competitive RT-PCR, were also mediated by E. coli cloning. RT-PCR was developed using M-MLV reverse transcriptase and total RNA isolated from R. ponticum. The response of in vitro grown R. ponticum cultures upon exposure to chilling (4 and 2

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Rob Finch
Keywords: Genetics
Date of Award: 1999
Depositing User: Enlighten Team
Unique ID: glathesis:1999-76252
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 16:14
Last Modified: 19 Nov 2019 16:14
URI: http://theses.gla.ac.uk/id/eprint/76252

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