Cloning And Functional Analysis Of HbMT2, A Metallothionein Gene From Heavea Brasiliensis

Metallothioneins widely exist in various plants. Although their physiological functions have not been determined, but most of reports suggests that it may be involved in heavy metal ion transport and detoxification, plant stress response, embryonic development and plant senescence.In order to clarify the mechanisms of ethephon-stimulated production of latex and to identify the related genes, we constructed an ethephon-induced latex suppression subtractive sybridization (SSH) cDNA library from H.brasiliensis, in which an expressed sequence tag (EST)with high homology to MT genes was identified. In this report, a novel metallothionein gene was cloned from H.brasiliensis and its structure, physiological function and expression profile were investigated. The major results were as follows:1. Employing RACE and RT-PCR techniques, a novel metallothionein gene, designated as HbMT2 (GenBank Accession Number:FJ229481), was isolated from H. brasiliensis. Sequence analysis showed that the HbMT2 cDNA has a 382 bp nucleotides with an ORF of 237bp, which encodes 78 amino acid residues. The genomic DNA fragment corresponding to HbMT2 comprises of two intron and three exons (GenBank accession number:GQ292531).2. Bioinformatics analysis showed that the putative HbMT2 contains 14 cysteine residues occurring as C-X-C, C-C andC-X-X-C motifs in the N-terminal, and C-X-C motif in the C-terminal region. The HbMT2 showed high identities of 70.37%, 66.67%,60.76% and 66.95% to those of the metallothionein from the Populus trichocarpa×Populus deltoids (GenBank accession no. AAT02525), alix matsudana (GenBank accession no.ABM21762), Citrullus lanatus (GenBank accession no. BAD26571),and Codonopsis lanceolata(GenBank accession no. BAD18924). Phylogenetic tree analysis confirmed that the putative HbMT2 was identified as a type 2 metallothionein.3. The expression of HbMT2 gene was determined by semi-quantitative RT-PCR analysis, showing that the expression of HbMT2 was constitutively expressed in all tested organs but at different levels. The HbMT2 was strongly expressed in leaves and latex, but weakly in roots and barks. All the mechanical wounding, and ethephon and H2O2 treatments could induce the expression of HbMT2 gene in latex in H.brasiliensis.4. In order to further investigate the physiological functions of HbMT2, the recombinant expression vector PGEX6P-1-HbMT2 was constructed and transformed into E.coli strain Rosetta 2(DE3), and the biologically active recombinant protein was expressed.5. The HbMT2-overexpressing plant expression vector, pCAMBIA1304-HbMT2, was constructed and 22 transgenic tobacco plants were obtained by agrobacterium-mediated leaf disc transformation method. Resistance analysis tests showed that the tolerance of transgenic plants to hydrogen peroxide and ultraviolet irradiation was significantly increased, and the chlorophyll content and the activity of peroxidase and catalase were significantly higher in trangenic plants than the controls in heavy-metal stress condition.Conclusion:The expression of HbMT2 was constitutively expressed in all tested organs but at different levels. The HbMT2 was strongly expressed in leaves and latex, but weakly in roots and barks. The mechanical wounding, and ethephon and H2O2 treatments could induce the expression of HbMT2 gene in latex in H.brasiliensis. In vitro tests confirmed that the HbMT2 can scavenge the reactive oxygen free radicals. The overexpression of HbMT2 improved the viability of E. coli and increased their tolerance to heavy metal ions. The HbMT2-overexpressing transgenic tobacco plants were given the higher tolerance to hydrogen peroxide and ultraviolet irradiation, and their chlorophyll content and activity of peroxidase and catalase were significantly higher than the controls in heavy-metal stress condition.