Phytoremediative potential of Brassica napus L. - heavy metal tolerance and accumulation, phytochelatin system response and γ-glutamylcysteine synthetase genetic background

摘要

Worldwide heavy metal contamination causes severe hazard to ecological balance of ecosystems and to human health.Plants are more and more often used for decontamination of soils from heavy metals (HM) through phytoremediation processes what initiates a demand for heavy metals highly tolerant and hiperaccumulating plants. Those abilities are underlain by a variety of defense mechanisms activated by the plant in response to stress. Phytochelatin system's non-protein thiols (NPT): glutathione (GSH) and phytochelatins (PCs) participate in heavy metals ions chelation and sequestration. During phytochelatin system's response to HM stress PCs - short cysteine rich ions binding peptides - are nonribosomally synthesised from GSH with γ-glutamylcysteine synthetase (γ-ECS) acting as a rate limiting enzyme of the pathway. Brassica napus line DH O-120 phytoremediative potential has been studied through examination of metal accumulation and phytochelatin system performance in response to leadstress. Lead content of 44375.2 mg/kg d.w. in case of 1 mM and 48316.77 mg/kg d.w. in case of 3 mM Pb(NO3)2applied, exceeding lethal values in experiments with other plants proved the high HM tolerance of Brassica napus. However, as 99.8% of metal was located in the roots, lead translocation to aboveground parts appeared to be low. The translocation barrier has been overcome by ED TA addition increasing lead content in the shoot up to 18% m case of 3 mM Pb(NO3)2. The dynamic PCs and GSH biosynthesis has been observed in Brassica napus seedlings and roots with a maximum NPT content in 24th hour of exposition propotional to lead concentration. RT-PCR and real time PCR examined γ-ECS gene (GSH1) transcription level increased in both roots and shoots and was well correlated with the enzyme activity increase. Brassica napus γ-ECS gene sequence has been determined. 15 exons locations within the gene have been predicted basing on A. thaliana GSH1 structure and the sequence similar in 74% to B. juncea y-ECS gene promoter has been determined as putative promoter region. Since Brassicaceae family is known for diversity within GSH1 gene the first exon area has been RT-PCR studied and showed the variability of 9 nucleotide deletion presence indicating at least two distinct alleles of γ-ECS gene occurrence in Brassica napus genome. Relatively high biomass production, heavy metals tolerance and accumulation of oilseed rape as well as proven commercial phytoremediation performance of its relative species B. juncea makes the plant a good phytoremediation prospect.Close relationship to A. thaliana facilitating molecular analysis enable the transgenic approach in producing plants for phytoremediation. Optimal cultivars could be obtained through genetic modifications within phytochelatins biosynthetic pathway,e.g. γ-ECS overexpression.