Isolation and characterisation of two divergent type 3 metallothioneins from oil palm, Elaeis guineensis

Abstract

Two distinct class I, type 3 metallothionein-like genes, MT3-A and MT3-B, were isolated from the oil palm, Elaeis guineensis. The MT3-A gene was isolated by subtractive hybridisation from a cDNA library prepared from mesocarp tissue at 15 weeks after anthesis (waa). The MT3-A mRNA made up over 1% of total transcripts and was the most abundant gene product in this ripening fruit tissue. The MT3-B gene was isolated as a genomic clone using an MT3-A-based PCR probe and was shown to contain two introns of 1432 and 306 bp. Southern analysis at low stringency showed at least two MT3 genes in the oil palm genome. Both MT3 genes were expressed in the mesocarp throughout the ripening period with a maximum at 15 waa, although MT3-A transcripts were 50 times more abundant than those of MT3-B. The MT3-B gene was also expressed in roots while the MT3-A gene was induced in senescing leaves. The coding regions of MT3-A and MT3-B are 78% and 70% identical at the nucleotide and protein levels, respectively. The MT3-A gene product was over-expressed in E. coli C41 cells as a carboxyl terminal fusion of glutathione- S-transferase (GST) and its identity was confirmed by amino acid analysis. In media supplemented with 0.25 and 0.50 mM Zn, cells expressing GST/MT3-A accumulated three times more Zn than cells expressing GST only. The purified fusion protein has a strong binding affinity for Zn and its pH of 50% dissociation was estimated as 5.1. The possible roles of these novel metallothioneins in plant development are discussed.