ALPHA-DOMAIN OF HUMAN METALLOTHIONEIN IA CAN BIND TO METALS IN TRANSGENIC TOBACCO PLANTS

With a view to exploring its use as a metal-binding factor in transgen ic plants we prepared the alpha-domain of metallothionein by reconstit ution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 wi th subtilisin. The isolated alpha-domains were characterised by UV and CD spectroscopy Double-Stranded. DNA encoding the alpha-domain (106 b p) of the human MT-I(A) was constructed from chemically synthesized ol igomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced. A expression construct containing the cloned alpha-doma in was introduced into tobacco cells on a disarmed Agrobacterium tumef aciens Ti-plasmid. Transformed tobacco cells were selected and regener ated on medium containing cadmium and kanamycin. The growth of roots a nd shoots of transformants was unaffected by up to 100 muM cadmium, wh ereas control plants showed severe inhibition of root and shoot growth , and chlorosis of leaves on medium containing only 10 muM cadmium. So uthern hybridization confirmed the presence of the transgene in the tr ansformed plant tissues. The concentration of human alpha-domain pepti des in transgenic tobacco eaves was determined by the Cd/hemoglobin sa turation assay and polarography using the rabbit alpha-domain as stand ard. The results indicate that the alpha-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed effi ciently and functions independently in transgenic plant cells.