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.

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: https://theses.gla.ac.uk/id/eprint/76252