MOLECULAR-CLONING, CHARACTERIZATION AND EXPRESSION OF MN-SUPEROXIDE DISMUTASE FROM THE RUBBER TREE (HEVEA-BRASILIENSIS)

A genomic clone encoding manganese-containing superoxide dismutase (SO D; EC 1.15.1.1) was isolated from a Hevea brasiliensis genomic library made in A phage EMBL3 by using a heterologous cDNA probe of MnSOD fro m Nicotiana plumbaginifolia. The nucleotide sequence of 4968 bp from t he genomic clone was determined. Based on the putative translation ini tiation codon and stop codon, PCR primers were design ed and utilized for cloning the full-length cDNA from total mRNA. Of the two distinct cDNAs of MnSOD isolated, MnSOD-A has a perfect match with exons of the nuclear gene, while MnSOD-B has a 90.2% homology and is 6 nucleotides longer than MnSOD-A in the putative transit peptide region. The nucle ar gene comprises 6 exons and 5 introns, giving a total length of 3211 bp. The sequences of 1400 bp upstream of the initiation codon and 320 bp downstream of the stop codon were also determined. Southern analys is of genomic DNA from Hevea probed with a genomic fragment indicated there are at least two genes of MnSOD in Hevea. Northern blot analysis showed that MnSOD transcripts were present in all tissues examined (l eaf, petiole, root, latex, callus) with young leaves showing the highe st levels in intact plants. The transcript level in embryogenic callus was nearly 50-fold higher than in mature leaves. In addition, transcr ipts of MnSOD could be induced 3- to 5-fold in response to sucrose, et hephon and Murashige-Skoog salts.