Studies On The Molecular Mechanisms Of Plant Acclimatizing To The Heavy Metal Stress: Arsenic And Cadmium

Toxic heavy metal pollution has become serious with the development of modern industry. There are many reports about the environmental and hygeian problem raised by heavy metals. In China, the pollution of heavy metals is serious, too. The heavy metals from soils and water can be passed to various organisms through the food chains and lead to various diseases in humans, such as inducing cancer and oaf.Phytoremediation is a cost-effective and environment-friendly technology which can remove or reduce the pollution of heavy metals or organic pollutant by the plants. The utilization of the technology is based on the biological mechanism of phytoremediation. At present, short-term advances are on selecting the hyperaccumulator, improving the soil condition and so on. But long-term improvements in phytoremediation rely on identifying the key genes which confer to the heavy metal resistance or oganic pollutants degradation and creating new plants by transgenic technology, such as the plants which can hyperaccumulate heavy metals fastly with big biomass or the crops which can reject the absorption of heavy metals.In this thesis, some progresses about the mechanism of arsenic phytoremediation with the arsenic hyperaccumulator-Pteris vittata are as follows:1. Differential expression cDNA fragments from Pteris Vittata under arsenic stresss were isolated by suppression subtractive hybridization. One cDNA fragment of them has high homology with ABC transporters (ATP-binding cassette transporter). The full-size cDNA was cloned by RACE, the sequence was analysis and the function of the gene was predicted. The results shown that the cDNA is 2165 bp long, the ORF has 1791 bp, and has 597 deduced amino acids. The result of NCBI conserved domain search shown the cDNA has the ABC transporter domain and no the transmembrane domain. So the cDNA was named PvABCT1 (Accession No. AY496966) .2. The expression pattern of PvABCTl has studied. RT-PCR analysis indicated that the expression of PvABCTl was induced by arsenic. PvABCTl — GFP fusion studies revealed PvABCTl is located in cytoplasm.3. Functional complementation studies using the Saccharomyces cerevisiae ACR3 deletion mutant FD236-6A (arsenic sensitive) confirmed the functional nature of this PvABCTlgene in arsenic resistance. In order to testing whether the PvABCT1 was functional, plasmid containing the whole ORF of PvABCTl was transformed to the S. cerevisiae mutant FD236-6A. The results showed that the PvABCTl cDNA was not only functionally complementary to the deficiency of ACR3 gene of FD236-6A, but also the strain transformed PvABCTl became more sensitive to arsenic and absorbed more arsenic than FD236-6A. These results suggested PvABCTl is related to the arsenic hypperaccumulation in Pteris Vittata.In order to find a way to decrease the heavy metal accumulation in plants, Lactuca sativa, a kind of leaf edible vegetable, was selected as the material to study how calcium ion and lanthanum ion affect its cadmium tolerance and what effects Ca2+ and La3+ have on the cadmium absorption in plants and the expression of phytochelatin synthase (PCS) gene and its catalyzing products-PCs. The results are as follows:1. The study on the effects of Ca2+ and La3+ on the seeds germination and plants growth of Lactuca sativa treated with Cd2+suggested 4 mM CaCl2 or 0.04 mg/L La(NO3)3 can enhance the Cd tolerance in Lactuca sativa.2. The cadmium concentration of leaves and roots under the conditions of 0.5 mM CdCl2, and adding 4 mM CaCl2 or 0.04mg/L La(NO3)3 were measured. The results suggest the accumulation of Cd was more in Cd+Ca treatment, but less in Cd+La treatment comparing with that of Cd treatment alone.3. To understand the interactions between cadmium absorption, the expression of phytochelatin synthase gene and its catalyzing products-phytochelatins (PCs), PCS gene was cloned. Then RT-PCR analysis and phytochelatins determination were manipulated. The results suggested that Ca2+ increased the expression of PC synthase gene under Cd2+ stress, which in turn enhances t...