Preliminary studies of the identification and expression of metallothionein-like gene in Festuca rubra

Metallothionein (MT) is a cysteine rich protein related to heavy metal sequestering in animal and fungal cell systems. Festuca rubra cv. "Merlin" is a monocotyledonous grass species isolated from a zinc and lead mine area. Laboratory studies had demonstrated that "Merlin" could tolerate cadmium and copper up to 50 mg/L and 30 mg/L, respectively. On the contrary, just 5 mg/L Cd²⁺ and 2 mg/L Cu²⁺ already inhibited the growth of the sensitive cultivar, Festuca rubra "S59". Molecular understanding on the metal tolerant ability of "Merlin" was studied. Based on the conserved region of plant MT-like genes, a pair of primers was synthesized as follows: 5′-GGAT-CAAGCTGCGGCTGCGGCTCAA-3′ and 5′-GCAGTTGCAAGGGTCGCACTTGCAG-3′. Using PCR, a fragment of 418 bp was amplified with this pair of primers from both the "Merlin" and "S59" genomic DNA. Sequence analysis showed that the MT-like gene of the two cultivars differed by only 5 base pairs, sharing a 98.8 % homology. Amino acid sequence deduced from the amplified fragment also demonstrated a high homology (60% ∼ 75%) to the other known higher plant MT-like proteins and shared the similar structural characteristics of cys-Xaa-cys regions at both end of the sequence. Northern blot analysis on the total RNA showed that the expression of the MT-like gene in the shoots of "Merlin" was enhanced when treated with both 20 mg/L Cu²⁺ and 20 mg/L Cd²⁺ . In shoots of "S59" the expression of the MT-like gene was enhanced by 20 mg/L Cu²⁺ but no effect was observed on the expression upon Cd²⁺ treatment. These evidences suggested that MT-like gene was present in both "Merlin" and "S59" cultivars of Festuca rubra; the expression of the MT-like gene was induced by heavy metal stress, and the expression pattern was different between the tolerant ("Merlin") and the sensitive ("S59") cultivars. Currently, the cloning of the whole gene from the cDNA and genomic library has been under-going. This would probably give a more profound and better understanding of the molecular mechanism on metal tolerance in Festuca