Cloning Of Earthworm Metallothionein Encoding Gene And Its Expression In E.coli And Arabidopsis Thaliana

Metallothioneins (MTs) are a family of low molecular weight, cysteine-rich, metal- binding proteins. The cysteine residues constitute nearly one-third of the amino acid residues and MTs contain no disulfide bonds. MTs are present in a wide variety of organisms ranging from prokaryotes, protozoa, plants, yeast, invertebrates and vertebrates. Many studies demonstrated MTs may be involved in regulation of metabolism of trace metals and protection against heavy metal toxicity and oxidative stress, clearance of free-radicals. MTs may also participate in the regulation of hormone and development process, enhance the ability to environmental adaptation to the adverse environment and so on. As a result, MTs have a promising prospect in agriculture, medical care and environmental protection.The 240bp-metallothionein cDNA fragments encoding the Eisenia foetida metallothionein was first amplified by RT-PCR from the Eisenia foetida induced by cadmium chloride. Sequence alignment revealed 95.2% nucleotide sequence homology between the MT sequence of the fragment and the sequence acquired by Gruber, and the identity of the two putative proteins reaches 98.7%. They both contained 21 cysteines, and the cysteines are distributed in six single cysteine residues, three Cys-X-Cys and Cys-X-Cys motifs, and one triple Cys motif. The MT contains no aromatic amino and histidine residues. These results showed that the metallothionein gene had been successfully cloned from Eisenia foetida.The MT gene was introduced to pBI121 and pPZP111 binary vector, generating the constitutive expression vector pBI-efMT and pPZP-efMT which contained CaMV 35S promoter, and then the pBI-efMT and pPZP-efMT were transferred into agrobacterium tumefaciens strains EHA105 and LBA4404 respectively through the assistant plasmid pRK2013/HB101 by tri-parental mating. Results of PCR amplification and restriction digestion showed that the metallothionein gene efMT was introduced into pBI121 and pPZP111, and the expression vector pBI-efMT and pPZP-efMT were transferred into agrobacterium successfully. Arabidopsis thaliana (Columbia ecotype) was transformed by floral dip method mediated by Agrobacterial and T0 seeds have been harvested.