Award details

Studies on an Arabidopsis Myb Transcription Factor Controlling Salt Tolerance

Reference	BB/C007654/1
Principal Investigator / Supervisor	Dr Peter Dominy
Co-Investigators / Co-Supervisors	Professor Anna Amtmann, Professor Ari Sadanandom
Institution	University of Glasgow
Department	Institute of Biomedical & Life Sciences
Funding type	Research
Value (£)	313,678
Status	Completed
Type	Research Grant
Start date	01/02/2005
End date	31/08/2008
Duration	43 months

Abstract

[1] Isolation of homozygous, single-insertion myb64 knockout and MYB64 over-expressing Arabidopsis lines. We have generated several transgenic lines of Arabidopsis that are more salt tolerant than WT, and obtained from the SALK T-DNA gene knockout collection mutants that are more salt sensitive than WT. We intend to use standard genetic techniques in conjunction with Southern blot analysis to identify clean lines that are homozygous and carry single insertions. [2] Detailed physiological characterisation of lines from [1] grown in a range of saline conditions. The phenotypes of the above clean lines will be established by monitoring a range of physiological parameters from plants grown in different saline solutions. Measured parameters will include: fresh dry weight: root and shoot length: full nutrient ion content profile by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES): tissue solute potential by vapour pressure osmometry: water potential measurements with a Scholander pressure chamber. These measurements will allow the best lines to be selected for further experiments. [3] Construction of Arabidopsis MYB64 promoter:LUC (luciferase) and MYB64 promoter:GFP reporter lines. We will make reporter lines where the MYB64 promoter drives the expression of the reporter gene. These reporter lines will subsequently be used in whole plant experiments summarised below([4]). [4] Full characterisation of MYB64 expression using reporter lines from [3]. A series of experiments will be undertaken with the LUC reporter lines to establish which elicitors activate MYB64, the dose-dependence of each of these elicitors, and resulting temporal pattern of expression in the plant. Mature rosette plants will be grown in hydroponic solutions, and at the appropriate time sprayed with luciferin and exposed to the elicitor. Plants will be imaged using our Andor photon counting CCD camera. Luminescence will be quantified, and a detailed description of the factors controllingthe MYB64 promoter in vivo will be established. The GFP reporter lines will be treated in the same way and imaged to establish the distribution of MYB64 at the cellular level. [5] Identification of primary MYB64 targets using ChlP technology. Trangenic lines of Arabidopsis will be generated that carry an epitope-tagged MYB64 construct under the control of the MYB64 promoter. Myb64 expression will be induced by salt stress, tissue fixed with formaldehyde, and nuclei isolated. Anti HA (or C-myc) antibody will then be used to precipitate MYB64-protein-DNA complexes, the DNA purified, cloned and sequenced; this will allow us to identify cis-elements (and their corresponding genes) that are directly under the control of MYB64. [6] Identification of down stream MYB64-dependent responses using DNA microarrays. We will use Affymetrix Arabidopsis gene chips to determine the temporal changes in gene expression that result from MYB64 activation. This approach should identify the targets of MYB64, and provide insight into the physiological processes under the control of MYB64.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search