Remediation of Zinc Contaminated Mine Sites Through Novel Genetically-Engineered Arabidopsis Thaliana
Phyllis Lesnikov
Stratford Hall
Floor Location : S 043 V

Soil is vital to all ecosystems, and plays many roles in supporting life on earth. However, in the past century, industrial activities – such as mining - have resulted in the contamination of soil. Contamination caused by mining is usually in the form of heavy metal contamination, which can be highly toxic to plants and animals, thereby making the contaminated environment uninhabitable. That said, the act of removing contamination is called remediation. This experiment focuses on phytoremediation, a novel biological remediation method that sometimes uses hyperaccumualtors to remediate contaminated mine sites. Hyperaccumulators are plants that are known for their unique ability to uptake and store heavy metals at an accelerated rate in their shoots.

Phytoremediation has the advantage of being much cheaper and more environmentally-friendly than conventional remediation methods. However, there are only 100 or so known species of hyperaccumulators, and they tend to grow very slowly. This prevents them from being used for effective phytoremediation.

This issue, however, can by creating transgenic metal-resistant versions of fast growing plants. “Transgenic” indicates that a plant contains foreign DNA from other organisms. This experiment entailed creating a zinc-resistant version of Arabidopsis thaliana, by over-expressing the zinc transporter AtZIP2. The ZIP family of transporters have not been fully characterized, making this a novel project. Preliminary studies suggest that AtZIP2 is responsible for zinc, manganese, and cadmium uptake, and that it – unlike other ZIP transporters – plays a role in xylem loading and transport to the plant shoot. Because Arabidopsis thaliana is used as a model plant in biology, this experiment not only tests the feasibility of a zinc-resistant hyperaccumutor version of Arabidopsis thaliana but also creates a model for the genetic transfer of ZIP2 into it’s closely-related high-biomass relatives.

In this experiment, AtZIP2 was overexpressed using Agrobacterium tumefaciens - mediated gene transfer.

To perform the experiment, first, DNA from Arabidopsis thaliana was isolated. Second, this DNA sample was used as a template to amplify AtZIP2 gene in the PCR (polymerase chain reaction) using AtZIP2 gene specific primers. Third, a column-purified PCR product and a circular DNA fragment, called plasmid (containing a DNA sequence called cauliflower mosaic virus promoter, driving a very strong gene expression) were cleaved open using the restriction enzymes SalI and BamHI. Fourth, the restriction enzyme digested PCR product and plasmid were column-purified, to remove restriction enzymes from the reaction. Fifth, the connection between the cleaved plasmid and the PCR product was achieved with an enzyme called ligase. The ligation product was transformed into Escherichia coli, and subsequently into Agrobacterium tumefaciens. Finally, Arabidopsis thaliana plants were transformed with the gene construct through dipping flowers in Agrobacterium suspension. The A. thaliana seeds were collected and as the plasmid carries hygromycin resistance, transgenic plants were selected on the agar media plates containing this antibiotic.

No quantitative data has been found at this experiment, but so far the gene has successfully been transferred to Arabidopsis Thaliana. The next step is to plant the transgenic plant in zinc-contaminated soil to test its abilities.