α-Domain of human metallothionein I A can bind to metals in transgenic tobacco plants

With a view to exploring its use as a metal-binding factor in transgenic plants we prepared the α-domain of metallothionein by reconstitution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 with subtilisin. The isolated α-domains were characterised by UV and CD spectroscopy Double-Stranded. DNA encoding the a-domain (106 bp) of the human MTIA was constructed from chemically synthesized oligomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced. A expression construct containing the cloned α-domain was introduced into tobacco cells on a disarmed Agrobacterium tumefaciens Ti-plasmid. Transformed tobacco cells were selected and regenerated on medium containing cadmium and kanamycin. The growth of roots and shoots of transformants was unaffected by up to 100 μM cadmium, whereas control plants showed severe inhibition of root and shoot growth, and chlorosis of leaves on medium containing only 10 μM cadmium. Southern hybridization confirmed the presence of the transgene in the transformed plant tissues. The concentration of human α-domain peptides in transgenic tobacco eaves was determined by the Cd/hemoglobin saturation assay and polarography using the rabbit α-domain as standard. The results indicate that the α-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed efficiently and functions independently in transgenic plant cells.