Award details

Cloning and expression of genes involved in gibberellin biosynthesis

Reference	BBS/E/C/00803287
Principal Investigator / Supervisor	Dr Andrew Phillips
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	321,103
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

Gibberellins, such as GA1, control many developmental processes in higher plants, including shoot elongation, flower development, fruit growth and seed germination. Chemical growth regulators that act on the GA biosynthetic pathway are widely used in agriculture to control many aspects of crop development and, hence, GA biosynthesis in plants is a prime target for genetic modification. Such transgenic crops with altered rates of GA biosynthesis would have reduced requirements for synthetic growth regulators, leading to increased efficiency and sustainability. Gibberellins are also known to mediate the effects of environmental factors, such as photoperiod and temperature, on plant development. Such factors may influence both the concentration of gibberellins and the sensitivity of plants to these hormones. There is evidence that the expression of genes for gibberellin-biosynthetic enzymes is modified by environmental signals and also by the action of gibberellins themselves in a type of feed-back regulation. In order to study these processes, it is necessary to isolate cDNA clones for the gibberellin-biosynthetic enzymes, either by screening expression libraries with antibodies raised against purified enzymes or by the use of hybridisation probes prepared by PCR. Full-length clones are being identified by heterologous expression and analysis of protein function. The cDNA probes are being used to study the temporal and spatial distribution of mRNAs for the gibberellin-biosynthetic enzymes and the effects of environmental factors and gibberellin on their accumulation. Genomic clones are being isolated for genes that are transcriptionally regulated and their promoters are being analysed. The cDNA clones will also be used in plant transformation experiments to modify gibberellin production and plant development. Objectives 1996 Sequence fully the GA 20-oxidase cDNA clones from wheat, compare gene expression in tall (rht3) and dwarf (Rht3) genotypes, determine temporal and spatial distribution of expression and prepare antisense constructs for wheat transformation. Isolate and characterise further GA 20-oxidase clones from bean. Isolate other (non-20 oxidase) GA, 2-oxoglutarate dioxygenase cDNAs from Marah macrocarpus. Complete sequencing of GA 20-oxidase genomic clones from Arabidopsis and isolate genomic clones corresponding to the fruit-specific cDNA YAP169 and to GA 3beta-hydroxylase (GA4). Identify the transcription starts of each gene. Prepare reporter gene constructs containing Arabidopsis GA 20-oxidase promoter sequences with the green fluorescent protein open reading frame and initiate Arabidopsis transformation. 1997 The GA20-oxidase genes in wheat will be mapped. Gene-specific probes will be designed to measure expression of the individual 20-oxidase genes in wheat. RT- PCR will also be developed for this purpose. The kinetics for oxidation of GA12 and GA53 by recombinant wheat 20-oxidases, prepared by expression in E coli, will be determined. A GA 2beta-hydroxylase will be purified from developing seeds of French bean and partially sequenced. Cloning of the 2beta- hydroxylase will be attempted from bean or pea seeds, or developing wheat ears using PCR or functional cloning. Copalyl diphosphate synthase (CPS), ent- kaurene synthase (EKS) and GA 3beta-hydroxylase will be cloned from maize.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
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