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Zawartość zarchiwizowana w dniu 2024-05-14

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

Cel



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.

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INSTITUTE OF GRASSLAND AND ENVIRONMENTAL RESEARCH
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SY23 3EB ABERYSTWYTH
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