Transient and stable gene expression in the fungal maize pathogen Cochliobolus heterostrophus after transformation with the β-glucuronidase (GUS) gene
| Autor: | Enrico Mönke, Wilhelm Schäfer |
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| Rok vydání: | 1993 |
| Předmět: |
Genes
Fungal Gene Expression GUS reporter system Biology Cochliobolus heterostrophus Zea mays chemistry.chemical_compound Transformation Genetic Plasmid Ascomycota Gene expression Genetics Promoter Regions Genetic Molecular Biology Gene Glucuronidase Reporter gene Protoplasts fungi food and beverages biology.organism_classification Molecular biology Transformation (genetics) chemistry Hygromycin B |
| Zdroj: | Molecular and General Genetics MGG. :73-80 |
| ISSN: | 1432-1874 0026-8925 |
| Popis: | The bacterial GUS (beta-glucuronidase) gene has been used as a reporter gene in plants and bacteria and was recently expressed in filamentous fungi. Here, we report the application of GUS for the establishment of transient and stable gene expression systems in the phytopathogenic fungus Cochliobolus heterostrophus. The utility of the transient expression system is demonstrated in applications involving promoter analysis and in tests of various parameters of a transformation system, for comparing the rates of stable and transient transformation events using GUS as sole screening marker and for comparing different transformation systems using either GUS or a dominant selection marker. For these purposes two plasmids were constructed harbouring the GUS gene and the hph gene of Escherichia coli which confers resistance to the antibiotic hygromycin B (HygB), ligated either to the P1 or GPD1 (glyceraldehyde 3 phosphate dehydrogenase) promoter of C. heterostrophus. In transient expression studies the first appearance of GUS activity was observed within 2 h after transformation and maximal values were obtained after 7 or 10 h, depending on the promoter fused to the GUS gene. At peak activity, the GPD1 promoter was revealed to be five fold stronger than the P1 promoter. The same difference in promoter strength was observed when the vectors were stably integrated in the fungal genome. Using the GUS gene as a colour selection marker in plate assays, it was possible to detect transformants and monitor the process of transient gene expression visually. Blue transformants obtained by screening for the GUS phenotype were mitotically unstable. Transformants obtained by selecting for HygB resistance were mitotically stable and expressed the beta-glucuronidase gene constitutively.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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