Molecular Cloning and in silico Characterization of the Arabidopsis thaliana Phytochelatin Synthase 1 (AtPCS1) Gene for Potential Phytoremediation Applications

The PCS1 gene encodes phytochelatin synthase 1, a member of the phytochelatin synthase family, which catalyzes the biosynthesis of glutathione-derived peptides known as phytochelatins. These peptides bind heavy metal ions and play a crucial role in heavy metal detoxification and tolerance mechanisms in plants. Due to its central role in metal homeostasis, PCS1 represents an important molecular target for studies related to plant stress responses and phytoremediation. In this study, the PCS1 gene was isolated and cloned from Arabidopsis thaliana. Successful amplification was achieved from A. thaliana using high-fidelity Pfu polymerase. The amplified PCS1 gene was ligated into the plant expression vector pBIN61 using T4 DNA ligase and subsequently transformed into Escherichia coli JM109 by heat shock. Recombinant clones were selected on kanamycin-containing medium and verified by colony PCR and restriction enzyme analysis. Confirmed plasmids harboring the PCS1 gene were isolated and preserved as glycerol stocks at −80 °C. Positive constructs were then transferred into Agrobacterium tumefaciens strain LBA4404 via electroporation. In addition to experimental cloning, in silico analyses were conducted to characterize the physicochemical properties, conserved domain architecture, and sequence conservation of the PCS1 protein, providing complementary insights into its structural and functional features. The resulting Agrobacterium strains carrying the PCS1 gene constitute a valuable resource for future plant transformation studies, and PCS1-expressing transgenic plants may serve as promising tools for the phytoremediation of heavy metal–contaminated environments.