Isolation of Cytokinin Biosynthesis and Metabolic Genes from White Clover (Trifolium repens L)

Type of content
Theses / Dissertations
Publisher's DOI/URI
Thesis discipline
Cellular and Molecular Biology
Degree name
Master of Science
Publisher
University of Canterbury. School of Biological Science
Journal Title
Journal ISSN
Volume Title
Language
Date
2009
Authors
Evans, Thomas George
Abstract

The factors influencing senescence in white clover (Trifolium repens L.) are of considerable importance to the pastoral sector of New Zealand’s economy. The plant hormones, ethylene and the cytokinins, have been implicated as having opposing influences on senescence. This project focused on the cytokinins. The rate limiting step in cytokinin biosynthesis is catalysed by isopentenyl transferase (IPT) and the primary enzyme in the degradation of cytokinins is cytokinin oxidase/dehydrogenase (CKX). Both IPT and CKX genes are present as multi-gene families. A reduction in the level of active cytokinins either via a decrease in IPT expression, or an increase in CKX expression, or both, would implicate the cytokinins in developmental leaf senescence in white clover. White clover grows in a sequential pattern with leaves at all stages of development making it a good model for studying leaf development and senescence. A decrease in leaf chlorophyll is used as a marker for the onset of senescence. A micro-scale chlorophyll analysis was developed using the NanoDrop™ thus allowing tissue from the same leaflet to be used for gene expression and chlorophyll measurements. The pattern of chlorophyll changes was similar to that shown by Hunter et al.(1999) and Yoo et al.(2003) in white clover stolons used for ethylene research. Reverse transcriptase PCR (RT-PCR) and BLAST analysis was used to identify five putative IPT genes and seven putative CKX genes from white clover. RT-PCR demonstrated the expression of seven of these genes (TrIPT1. TrIPT13, TrIPT15 TrCKX1, TrCKX2, TrCKX6). Analysis with quantitative real-time PCR showed expression of TrCKX2 increased markedly during leaf expansion and was consistently high during senescence, suggesting a potential role for CKX in facilitating the progression of senescence.

Description
Citation
Keywords
Trifolium repens, isopentenyl transferase, cytokinin, cytokinin oxidase/dehydrogenase, quantitative real-time PCR, Gene expression, micro-scale chlorophyll analysis, Leaf development, Senescence.
Ngā upoko tukutuku/Māori subject headings
ANZSRC fields of research
Rights
Copyright Thomas George Evans