A bioengineering approach to plant-mediated soil bioremediation.

摘要

Extensive anthropogenic activities have resulted in the contamination of large areas of land. Bioremediation is one of several technologies that have been developed to clean up these polluted sites. The purpose of this work was to devise a strategy combining rhizosphere-colonizing fungi, plants and genetic engineering to enhance the effectiveness of soil bioremediation.; A strain of the filamentous fungus Trichoderma harzianum (T.h. 2023), a well-studied biocontrol organism, was investigated for its ability to colonize the root zone (rhizosphere) of plants. We found that the density of T.h. 2023 increased 3 to 5-fold in the rhizosphere of corn seedlings relative to unplanted inoculated soil after 15 days. These results established that a strain of the soil filamentous fungus Trichoderma harzianum , T.h. 2023, was capable of colonizing the rhizosphere of corn seedlings.; For the purpose of increasing the biodegradation potential of T. harzianum, a plasmid containing the organophosphate degrading (opd) gene and other elements necessary for fungal. transformation and expression was constructed. T.h. 2023 was successfully transformed using this plasmid and a stable transformant strongly expressing the opd gene was isolated. When compared with the wild type parent, the transformant exhibited enhanced metabolic characteristics as well as the same level of ability to colonize of the rhizosphere of corn seedlings.; The ability of the transformed T.h. 2023 to degrade paraoxon was investigated in sterile planted and unplanted soil. The rate of paraoxon degradation was 2 to 3-fold greater in planted soil inoculated with the transformed T.h. 2023 relative to the degradation rate of paraoxon observed in unplanted soil inoculated with the transformant or in non-inoculated planted soil. Consistent results were obtained when these experiments were conducted with corn, rapeseed and barley seedlings.; In order to monitor the presence of the transformed fungus after its introduction in the soil environment, specific molecular probes were designed. Soil DNA was extracted from non-sterile, inoculated and non-inoculated rhizosphere soils, after a 15-day growth period. The polymerase chain reaction, using primers specific to the organophosphate degradation gene, was performed on the rhizosphere soil DNA. The presence of the transformed fungus was detected in the inoculated rhizosphere soil.