Analysing the function of existing and novel genetic promoters for tissue-specific expression of transgenes in Zea mays

Objective

Objective: To improve our understanding of the regulation of gene and transgene expression in an important cereal crop by promoter isolation and analysis.
Targets: Promoter motifs conferring tissue specificity on the expression of associated genes will be defined for up to 50 regulatory sequences isolated from maize. The tissue targets are leaf senescence; kernel development; pollen and seedling development; roots and flowers; guard and other leaf epidermis cells.
Approach: Initial characterization of candidate promoters isolated by cDNA/genomic screening or trapping with promoter-less GUS will be followed by transposon mutagenesis to define regions critical for tissue specificity. Promoter fragments thus identified will be further analysed in homologous in-vitro transcription systems and by fusion with reporters and transformation into maize.
Work Package 1. Clone the following genes and isolate their promoter regions: at least 6 senescence-related cDNAs; up to 10 MADS box containing regulatory factors; 20 seedling- and pollen-specific cDNAs; one each guard cell PEP-carboxylase and K channel cDNAs.
Work Package 2. Identify the following new promoters by GUS tagging: at least 4 active in senescence; 4 root/flower-specific; 5 constitutive; 5 leaf cell-specific.
Work Package 3. From segregating mutant stocks evaluated over three successive field seasons, identify Mutator transposon insertions in the following regulatory sequences: at least 2 senescence promoters; at least 3 kernel MADS-boxes; up to 20 seedling/pollen promoters; 4 root/flower promoters; 5 constitutive; at least 2 leaf-cell promoters.
Work Package 4. Analyse the following tissue-specific promoter regions (at least 2 in each case) by the in-vitro transcription initiation procedure: senescence; MADS-box; root; flower; leaf cell; constitutive.
Work Package 5. Produce stable maize transformants with the following genes/promoters: promoter-less GUS trap; senescence-specific promoter driving GUS; at least one each of the following promoters driving GUS or GFP-root, flower, leaf cell, constitutive.

Programme(s)

• FP4-BIOTECH 2 - Specific research, technological development and demonstration programme in the field of biotechnology, 1994-1998

Topic(s)

• 030105 - Gene expression

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

INSTITUTE OF GRASSLAND AND ENVIRONMENTAL RESEARCH

Address

Plas Gogerddan
SY23 3EB Aberystwyth
United Kingdom

Participants (5)

HOECHST SCHERING AgrEvo GmbH

Germany

Address

65926 Frankfurt

ISTITUTO SPERIMENTALE PER LA CEREALICOLTURA

Italy

Address

Via Stezzano 24
24126 Bergamo

MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V.

Germany

Address

Am Muehlenberg 1
14424 Gross Glienicke Potsdam

SES France S.A.

France

Address

60,Route De Blois
41500 Mulsans

UNIVERSITY OF BRISTOL

United Kingdom

Address

Long Ashton Research Station
BS41 9AF Bristol