Functional Analysis Of Two Zinc Tansporters From The Zn/Cd Hyperaccumulator Sedum Alfredii Hance

Study on the physiological and biochemical process, the molecular mechanisms of metal hyperaccumulation in hyperaccumulators, which is an international research concern, is beneficial not only to the devebpment and promotion of phytoremediation, but also to enrich plant nutrition theory. Sedum alfredii Hance, a native zinc(Zn) and cadmium(Cd) hyperaccumulator which has high tolerance and accumulation to Zn and Cd, is a good research material for phytoremediation Investigation of the molecular mechanisms of Sedum alfredii Hance should benefit to clone related genes with independent intellectual property rights.Transporters play an important role on the hyperaccumulation mechanism, including participation of heavy-metal efficient absorption, transport and detoxification. The ZIP family is considered to be the transporters playing an important role on root absorption, xylem transporting and aboveground steady-state. The ZIP family which widely exists in bacteria, fungi, animals, plants and other eukaryotes, takes its name from the first members to be identified "ZRT, IRT-like Protein", they work together to transport Zn, Fe, Mn, Cd, Cu and so on, which makes themselves necessary transporters to maintain metal homeostasis in plantPrevious study of our lab had cloned several ZIP genes from Sedum alfredii Hance previously, but their function is not clear yet, in this paper, a comprehensive study of ZIP2 and ZIP3 genes is conducted by functional complementation in yeast, subcellular localization in onion, q-RT-PCR, and overexpression in Arabidopsis. The main results are as follows:1. Chose yeast strains DY1457 (wild type), DY4743 (wild type), △zrcl (Zn and Cd sensitive mutant), ZHY3 (Zn uptake-defective mutant), △smfl (Mn uptake-defective mutant), DDY4 (Fe uptake-defective mutant), used the lithium acetate transformation method to explore whether the two genes can transport Cd, Zn, Mn or Fe. Both genes made the △zrcl cannot grow in the plate contain Cd, but without improving the mutant’s Cd accumulation, it suggested that ZIP2 and ZIPS gene are plasma membrane not tonopkst transporters in yeast. ZIP2 and ZIP3 gene restored the Zn absorption facility of ZHY3, by which the mutant’s Zn accumulation increased 63.7% and 251.4% respectively, indicating that both genes can transport Zn in yeast, and ZIP2 gene is greater than ZIP3 gene. Only the ZIP3 gene can restore the Mn absorption facility of △smfl, by which the mutant’s Mn accumulation increased 13.4%, although not reached the WT’s level yet, this suggested that the ZIP3 gene not ZIP2 gene can transport Mn in yeast partly. Both genes weakened the DDY4’s Fe absorption facility without influencing its Fe accumulation, this suggested that ZIP2 and ZIP3 gene maybe transport Fe in yeast Above all, the ZIP2 and ZIP3 gene can transport Cd, Zn and probably Fe in yeast, only the ZIP3 gene can also transport Mn in yeast2. Gateway technology was used to construct GFP fusion vector, particle bombardment was used to introduce the vector to the onion epidermal cells, after 16 hours’culturing in dark, the laser scanning confocal microscope was used to observe the fluorescence. Cells expressing SaZIP2-GFP displayed a punctuate pattern of fluorescence in the cytoplasm, SaZIP3 was shown to be targeted to the plasma membrane and nuclear membrane, suggesting that SaZIP2 is an intracellular membrane protein of unknown nature, SaZIP3 is a plasma and nuclear membrane protein. SaZIP2 locates to which cell organelle membrane and why SaZIP3 locates to nuclear membrane, this need further research.3. Compared and analyzed the ZIP2 and ZIP3 gene expression level and their relationship with accumulation of Zn and Cd in two ectypes (HE and NHE) of Sedum alfredii. Lack of Zn for a longtime could enhance the expression of ZIP2 and ZIP3 in both shoot and root of two ecotypes, while improving the Zn level had an opposite result, this indicated that ZIP2 and ZIP3 are high-affinity transporters in Sedum alfredii. When exposure to 5 μM Cd for 24 hours or 8 days, the ZIP2 gene in NHE was up-regulated 2-fold or 7-fold in shoot, whereas down-regulated 25-fold or 4-fold in root, this revealed that SnZIP2 can transport Zn specificity in shoot, while in root, it can also transport Cd. Only when long exposure to 100 μM Cd, the ZIP2 in shoot of HE was up-regulated significantly, this showed that SaZIP2 take part in long-distance translocation of Cd. When exposure to 5 μM Cd for 24 hours or 8 days, the ZIP3 gene in NHE was up-regulated 5-fold or down-regulated 7-fold in shoot, and up-regulated 6-fold or 8-fold in root, this suggested that SnZIP3 is a high-affinity and specific Zn transporter. ZIP3 in shoot of HE was down-regulated when exposure to Cd, and the higher concentration of Cd, the wider range of down-regulated. ZIP3 in root of HE was up-regulated when exposure to low concentration of Cd, and was down-regulated when exposure to high concentration of Cd for a long time, this showed that SaZIP3 plays a role in absorbing Cd.4. Transgenic lines of A. thaliana (ecotype Columbia) were obtained by Agrobacterium-mediated dip flora transformation, then further functional analysis of transgenics for metal tolerance and accumulation was carried out. Under stressed conditions of Zn or Cd, both shoot and root growth of transgenics and WT were found similar, this disclosed that SaZIP2 and SaZIP3 gene don’t take part in detoxification, in accordance with their non-tonoplast location. There was no difference between Zn concentrations in shoot and root of the two transgenics whose shoot Zn concentrations were 90 times higher than that of WT, whose root Zn concentrations improved more than 25%, and whose transfer coefficient was 7 times higher than that of WT, these indicated that SaZIP2 and SaZIP3 gene play a role in the absorption and translocation of Zn. The transgenic overexpressed SaZIP2 had the highest Cd transfer coefficient, and had a higher but not significant Cd concentration than that of WT, the transgenic overexpressed SaZIP3 had a 1.5 times higher in shoot,3 times higher in root Cd concentration than that of WT, but had the lowest Cd transfer coefficient, these suggested that SaZIP2 takes part in Cd translocation, while SaZIP3 takes part in Cd absorption. The results above are in line with functional complement and gene expression conclusions, namely SaZIP2 and SaZIP3 are not specific in transporting Zn, and they also take part in the absorption and translocation of Cd.