ISOLATION AND CHARACTERIZATION OF THE RICE (Oryza sativa L.)METALLOTHIONEIN-LIKE PROTEIN GENES AND EXPRESSION IN ESCHERICHIA COLI.

Cytogenetics Division, National Institute of Agricultural Science and Technology,Suwon 441-707, Korea

We carried out a random cDNA sequencing from immature seed of rice (Oryza sativa L. cv Milyang 23) for expressed sequence tag(EST) cataloguing. In process of analysis, five different metallothionein (MT)-like isoform genes were found as one of low degree redundant clone by BLAST search with GenGank. Although many MT-like genes were isolated in plants, less is known about the putative functions and metal-binding properties of their protein. Here, we describe the characterization of cDNAs specific for five genes (MTa, MTb, MTc, MTd and MTe) encoding metallothionein-like proteins from rice. The isolated clones had central hydrophobic domains flanked by distinct conserved cysteine rich motifs like other plant MT-like genes, and can be divided into three types. MTa has a Cys-X-Cys motif on the N-terminal domain that is a type 1 MT gene. MTb and MTc have a Cys-Cys motif on the N- and C-terminal motif that generally found in type 2 MT gene, but they have one more pair of Cys-X-Cys motif in C-termini when compared with other plant type2a gene. MTd and MTe have similar motif of Ec protein that classified type 2b. Although four isolated genes can be classified into two types, homology identity that between genes in the type 2a and type 2b is low as 54% and 65% in deduced amino acid level, respectably. Molecular weights of isolated type 3 MT clones are lower than that of the others. The theoretical pI of MTd and MTe are acidic (4.9, 4.2), but pI of MTb and MTc are 8.6 and 5.2, respectably. The MTa gene has higher affinity of Cu than Cd and Zn, but the metal-binding affinity is lower then others MT genes. The isolated type 2a MT had a high affinity to Cd and type 2b had a high affinity to Cu.