Biotecnología ambiental: desarrollo de estrategias biotecnológicas para la biorremediación de metales pesados
- Autores: Patricia Pérez Palacios
- Directores de la Tesis: Eloísa Pajuelo Domínguez (dir. tes.), Miguel Ángel Caviedes Formento (dir. tes.), Ignacio Rodríguez Llorente (dir. tes.)
- Lectura: En la Universidad de Sevilla ( España ) en 2015
- Idioma: español
- Tribunal Calificador de la Tesis: Javier Vigara Fernández (presid.), Montserrat Argandoña Bertrán (secret.), Marco Betti (voc.), José Javier Pueyo Dabat (voc.), Elizabeth Agostini (voc.)
- Materias:
- Enlaces
- Tesis en acceso abierto en: Idus
- Resumen
1. ABSTRACT Due to the high levels of contamination in the world, it is necessary develop successful strategies to clean the environment avoiding expense physicochemical methods. In this context, bioremediation provides a good cleanup strategy. This research analyzes three different biotechnological tools designed for bioremediation of metal and metalloids in soils or waters.
2. METHODOLOGY These tree tools involve, from whole transgenic plants (A. thaliana) to composite plants of M. truncatula and hairy roots of N. tabacum. The following approaches have been undertaken: a) Generation of transgenic Arabidopsis thaliana by overexpressing the metallothionein gene mt4a in vegetative tissues, in order to increase metal tolerance or accumulation; b) Generation of composite Medicago truncatula plants by heterologous expression of mt4a from A. thaliana in roots, in order to improve tolerance towards metals and nodulation, and enhance metal phytostabilization in soils; c) Generation of transgenic tobacco hairy roots expressing the bacterial genes copC (encoding a copper-binding protein) or arsB (codifying an arsenite efflux pump) in order to accumulate Cu or As, respectively, from polluted waters. In the last case, subcellular targeting to the vacuole was also attempted in order to further increase accumulation or to decrease stress generated by metals. These plants were exposed to different heavy metals, including Zn, Cu and As. Several studies were made: determination of growth and physiological parameters, accumulation profiles, bioconcentration factors, antioxidants assays and analysis of transcriptomic and metabolomic profiles.
3. RESULTS: The results suggest that these plants, expressing different genes, showed improved tolerance towards heavy metal, as deduced from growth and physiological parameters. Concerning metal accumulation, the results were heterogeneous: a) A. thaliana showed a mild increment in Cu accumulation in roots, without increases in Zn content. b) Composite plants of M. truncatula exhibited enhanced tolerance towards Cu, lower oxidative stress and better nodulation, together with increased Cu accumulation in roots, suggesting an application in Cu phytostabilization in soils. c) Hairy roots from N.
tabacum expressing the copC gene achieved maximum accumulation levels of Cu 45,000 ¿g. g-1 without displaying toxicity symptoms, which, to our knowledge, was one of the highest Cu concentrations in a biological sample. These results point to an interesting use of this bio-tool in rhizofiltration. d) On the contrary, hairy roots from N. tabacum expressing the arsB gene showed moderate increases in As accumulation, probably due to a limitation in the entrance of this metalloid through the plasma membrane. Unlocking metal uptake by strategies such as phosphate deprivation or desiccation considerably enhanced metal accumulation, to maximum levels of 10,000 ¿g. g-1 when the ArsB pump was targeted to the tonoplast. However, the oxidative stress and particularly lipid peroxidation leading to membrane damage, was not alleviated. Trancriptomic and metabolomic analyses suggest that in transgenic roots expressing ArsB in the plasma membrane, genes involved in the electron transport chain are highly overexpressed, whereas in those expressing AsrB in the tonoplast, the glutathione/ phytochelatin synthesis pathway was particularly induced.
4. DISCUSSION: In this work, several bio-tools based on transgenic expression of different genes were generated. Genetic engineering has proved to improve the phytoremediation potential of whole plants or hairy roots by increasing metal tolerance and/or accumulation. Transcriptomic and metabolomic analyses led to the knowledge of overexpressed genes and compounds after the exposition of hairy roots to As, being able to find targets for news studies.
