Phytoremediation of Chlorpyrifos Insecticide: The Use of Woody Plants and Transgenics to Enhance and Understand the Uptake, Translocation, and Transformation of Chlorpyrifos
Lee, Keum Young
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Poplar and willow trees have a significant ability to take up CPS and to translocate/degrade it within the plants and thus, assist in removing it from the hydroponic solution. To our knowledge, the poplar and willow species presented in this study were the first woody plants to be tested for potential phytoremediation applications for CPS. Since poplar and willow are able to efficiently take up CPS, there is the possibility of enhancing the CPS degradative potential by genetic manipulation of metabolism in planta. The results presented here indicate that phytoremediation of CPS and other OP insecticides should be a fertile area for future development. In order to test for this potential, two types of transgenic plants were developed. Transgenic poplar plants (`PON1-34') expressing rabbit pon1 gene, which is known to be involved in OP insecticide degradation pathways, were produced and investigated for increased tolerance and removal of CPS. However, there was no change in phytotoxicity of CPS or uptake of CPS in the transgenic lines. The his-tagged PON1 protein was not detected by the western blot (protein immunoblot), when the his-tag antibody was used to detect his-tagged PON1 protein in bacterial and plant total protein. In order to increase the expression level of PON1 protein in plants, a tobacco plant was transformed with rabbit PON1 using the chloroplast transformation method. The activity of plant-derived PON1 was analyzed and CPS tolerance and uptake were investigated in chloroplast transformed tobacco to assess the function of rabbit PON1 enzyme in plants. However, there was no response in the PON1 transgenic tobacco as compared to wild type tobacco in terms of phytotoxicity, CPS/CPO removal, and PON1 enzyme activity assay. In addition, PON1 protein expressed in E. coli was not detected by the western blot analysis, when the his-tag antibody was used to detect his-tagged PON1 protein in bacterial total protein. Through these two transgenic approaches, three possibilities exist for this failure (i) the PON1 protein is not folding properly and is targeted for destruction, (ii) the addition of the his-tag to the N-terminal did not work for the detection by the western blotting, or (iii) the expression level of the PON1 protein was very low. Since the PON1 enzyme activity assay did give positive results, it suggests that the constructs (pART27-PON1 and pLD2-CtV-PON1) was functional and that the enzyme was active for at least a short period of time under specific conditions.
- Forestry