The Copper Tolerance, And Cloning And Functional Analysis Of Metallothionein Gene In Elsholtzia Haichowensis

Soil and water pollution by toxic heavy metals is one of the most serious environmental problems in many parts of the world. Some heavy metals, such as Cu, Zn and Ni, are essential micronutrients for plant, but are toxic to organisms at high concentrations. Unlike organic contaminants, metals cannot be degraded, when released into the environment, posing a potential human health risk through food cycle. Plants possess a range of potential mechanisms that may be involved in the detoxification of heavy metals and thus tolerance to metal stress. Chelation of metals in the cytosol by high affinity ligands is a very important mechanism of heavy metal detoxification and tolerance. Potential ligands include amino acids, organic acids, and two classes of peptides: the phytochelatin and the metallothionein. In the present study, solution culture experiments were conducted to investigate the effects of excessive Cu on the seed germination, growth of three plant species of the genus Elsholtzia (Elsholtzia haichowensis, Elsholtzia cypriani and Elsholtzia ciliata). Experiments were also conducted to compare Cu uptake and tolerance mechanisms of the three plants. Complete cDNA of EhMT1 gene was isolated from E. haichowensis, and the functions of EhMT1 were analyzed by over-expressing in E.coli and in wild type tobacco.The results showed that treatment with 50μM Cu decreased the hypocotyl and radicle lengths of E. cypriani and E. ciliata during seed germination, and almost completely inhibited root elongations in E. cypriani and E. ciliata seedlings. However, the same Cu treatment had no significant effect on E. haichowensis during germination of their seeds. ? Shoots and roots of E. haichowensis had significantly higher concentrations of Cu than E. cypriani and E. ciliata. The maximum Cu concentrations in the shoots and roots were 217 and 13170 mg/kg DW, respectively, when the plants of E. haichowensis were grown at 100μM Cu supply. The concentrations of soluble proteins were significantly enhanced in the leaves and roots of E. haichowensis under excess Cu supply. No significant increment was found in the leaves and roots of E. cypriani and E. ciliata. The results suggested that E. haichowensis had higher tolerance to excessive Cu than E. cypriani and E. ciliata, and the adaptive Cu tolerance mechanism in E. haichowensis might involve the active participation of proteins.According to five MT sequences (GenBank accession no. L27813, L02306, X95709, X77254, D78491), we designed a pair of degenerate oligonucleotide primers used to amplify MT from Elsholtzia haichowensi by RT-PCR and RACE. A metallothionein gene, EhMT1 (GenBank accession no. DQ059081), was isolated from Elsholtzia haichowensi. The EhMT1 gene contains an open reading frame of 225bp encoding a putative peptide of 74 amino acid residues containing two cysteine-rich domains, which were arranged in CXCXXXCXCXXXCXXC and CXCXXXCXCXXCXC (X are other amino acid; other than cysteine) motifs at amino-terminus and carboxy-terminus, indicating that it was a type 1 MT. This is the first report of the isolation of a type 1 metallothionein gene from E. haichowensis. Sequence alignment of the EhMT1 cDNA with genomic DNA showed that EhMT1 gene contained two introns and three extrons. The sequence comparison between EhMT1 and other MTs showed that EhMT1 shared 75% sequence similarity with Mimulus guttatus. EhMT1 gene expression is higher in roots than that in leaves of E. haichowensis, but is no signal in stems. Semi-reverse transcriptional PCR indicated that the expression of EhMT1 was up regulated by Cu, and induced by heat shock and oxidative stress.The prokaryotic expression vector pET-30a-EhMT1 was constructed and transformed into BL21 (DE3) cells. The growth of recombinants was more prosperous than that of controls under various stresses. The results showed adaptability of recombinant was enhanced by EhMT1, and were tolerant to various stresses. Subcellular localization of EhMT1-GUS fusion proteins suggested that EhMT1 was a cytoplasmic protein. The result agreed with the previous studies. To further study the functions of EhMT1, we generated transgenic tobacco plants over-expressing EhMT1 gene. Here transgenic tobacco plants were more tolerant to copper at higher concentration than wild type, and the average Cu level in transgenic tobacco plants was significant higher than wild type.