The Accumulation Of Cadmium And Manganese, And Cloning Of Metallothionein Gene And Phytochelatin Synthase Gene In Phytolacca Americana

Heavy metal contamination has received considerable public attention over the last few decades. They are highly toxic nonessential element of particular concern to human health as they can be readily absorbed by plant roots and be concentrated by many cereals, potatoes, vegetables, and fruits.There is an urgent need for efficient and adaptable methods to remediate soils contaminated with heavy metals and other contaminants. Environmental remediation using the ability of endemic plants to accumulate heavy metals has been proposed, termed phytoextraction. It is well recognized that the success of any phytoextraction technique depends upon suitable plant species that can accumulate high concentrations of heavy metals and produce sufficient biomass. Phytoremediation has many advantages such as low cost, no worsening the ecological environment of soils and rivers and no recontamination. Remedying the polluted soil by heavy metal with hyperaccumulator is the most prospective phytoremediation technology. So the present investigation aims to identify Phytolacqa americana that have an ability to successfully grow and reproduce in adversely impacted mining environment, and to asses their metal accumulation capacities, and then to study the mechanisms of metal accumulation in plants. This information would be helpful in determining if those plant species are suitable for ecosystem recovery on mining areas and phytoremediation of metal-contaninated soils and water.To investigate the variation of Cd and Mn accumulation and tolerance of P. americana, samples from three contaminated and one uncontaminated sites were conducted. In addition to these field investigations, seedlings of the two populations:one of them originated from Xiangxi Zn and Mn smelter in Jishou city of Hunan province (Datianwan) and another from uncontaminated site in Nanjing city of Jiangsu province (Zijinshan), were raised under glasshouse conditions to study biomass production and Cd and Mn uptake from nutrient solutions supplemented with different treatments. In general, concentrations of Cd and Mn in the leaves of P. americana and the associated soil from Datianwan were significantly higher than the three others. But significant differences were not found between the two populations of P. americana grown in nutrient culture. All results suggest that high tolerance and hyperaccumulating ability of Cd and Mn in P. americana seemed to be constitutive properties. Furthermore, the plant grows rapidly, produces substantial biomass. P. americana, therefore, offers great potential for use in the phytoremediation of Cd and Mn contaminated soils.To understand Cd adsorption manner by P. americana, hydroponics experiment was conducted. The growth was remarkably inhibited and Cd contents in leaf and root were significantly increased with the Cd concentration increasing in the nutrition solution. ABA, HgCl2 and LaCl3 drastically reduced the extent of Cd bioaccumulation in the shoots and roots of P. americana. ABA induced stomata closure and reduced the stomata transpirations. The present study indicated that transpiration may be involved in Cd accumulation in the shoots of P. americana. Besides, we found that cadmium could permeate through calcium channels.When the plants were exposed to a range of cadmium concentrations, a marked production of non-protein thiol was observed, while the production of glutathionein significantly decreased. GSH serves as a precursor in the phytochelatin biosynthesis. The results suggested that the phytochelatins might be the potential ligands with Cd in P. americana, and involved in the detoxification of heavy metals.We designed a pair of degenerate oligonucleotide primers used to amplify PaMT2 and PaPCS fragments from P. americana by RT-PCR. The full-size cDNA of PaMT2 was cloned by RACE, the sequence was analysis and function of the gene was predicted. The PaMT2 gene contains an open reading frame of 243bp encoding a putative peptide of 80 amino acid residues containing two cysteine-rich domains. The sequence comparison between PaMT2 and other MTs showed that PaMT2 shared 76% sequence similarity with Mesembryanthemum crystallinum. PaMT2 gene expressed in roots, stems and leaves. Semi-reverse transcriptional PCR indicated that the expression of PaMT2 and PaPCS were induced by cadmium. Effects of constitutive PaMT2 expression were examined in Escherichia coli. Cadmium tolerance of E.coli cells expressing recombinant PaMT2 protein was significantly greater than in control cells. To further study the functions of PaMT2, we generated transgenic tobacco plants over-expressing PaMT2 gene. Transgenic tobacco plants were more tolerant to cadmium at higher concentration than wild type.