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Development of nuclear gene targeting system for green microalgae

Ziel

Green microalgae like Chlamydomonas reinhardtii are of great value both as model organisms for fundamental biological research and as a resource for the biotechnological industry. The aim of this research project is to develop new tools for functional study of genes in microalgae and to apply them for investigation of photoreceptor function in the green flagellate C. reinhardtii.

Phototaxis in this microorganism and related species occurs via a rhodopsin-mediated sensory system. It combines characteristic features of those found in animals and archaea and represents the only example of such systems so far known in plants. However, unambiguous identification of a gene(s) encoding for Chlamydomonas phototaxis receptor is still pending. One of the reasons that delays solving this problem is a difficulty of producing opsin-knockout mutants in Chlamydomonas. Targeted gene inactivation via homologous recombination occurs in this microorganism at a very low frequency. Therefore, the objectives of this project will be stimulation of nuclear gene targeting by overexpression of bacterial and Chlamydomonas genes involved in homologous recombination, generation of suitable targeting vectors for enrichment of homologous recombinants before screening and, as a powerful complementary approach, creation of vectors for post-transcriptional inactivation of specific genes.

Overexpression of bacterial recA and ruvC genes and Chlamydomonas recA homologs will be used to increase the homologous recombination frequency in the nucleus of Chlamydomonas. Transformants showing the highest level of recombination in the model paromomycin resistance gene previously integrated in the chromosome will be chosen as recipients for generation of targeted mutations in genes of interest. Transformation vectors for gene knockout containing the positive selection marker of paromomycin resistance in the absence of transcriptional regulatory signals and a negative selection marker presented by the synthetic codon-adapted gene of diphtheria toxin A-domain will be created to enrich homologous recombinants before screening. Suitable vector constructs leading to selective degradation of the target RNA will be generated and tested with the isc genes to obtain transformants with different levels of post-transcriptional gene silencing. The developed recipient strains and transformation vectors will be used for directed inactivation of the chlamyopsin cop1/2 and cop3 genes through the disruption of genes and via post-transcriptional silencing. Functional analysis of chlamyopsin knockout mutants and antisense transformants will be carried out by measurement of phototaxis and rhodopsin-mediated photoelectric currents that will enable to establish a role of the gene products in photo signalling.
The expected results will be new molecular tools for functional study of genes in Chlamydomonas, such as C. reinhardtii strains with the increased level of homologous recombination and efficient transformation vectors for generation of targeted mutations and post-transcriptional gene silencing. These new possibilities will facilitate using C. reinhardtii as a model system for solving biological problems of a broad range by methods of molecular biology and genetic engineering. Besides, the expected results on the gene structure and activity of the first identified Chlamydomonas recombinases will greatly open the research field of homologous recombination in green flagellated algae. Studies on the functional role of Chlamydomonas cop genes that takes advantage of the newly developed molecular approaches will provide novel information on the unique rhodopsin-mediated systems involved in photosensing in green flagellate algae, which would help elucidation of the basic principles of light perception and signal transduction and provide a deeper insight into general evolution of the photosensory function.

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Regensburg University
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31 Universitaetsstrasse
93040 Regensburg
Deutschland

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Letzte Aktualisierung: 30 Oktober 2002

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Permalink: https://cordis.europa.eu/project/id/INTAS-2001-00069/de

European Union, 2024