Cloning Of SpHMA2 And Its Transport Ability To Heavy Metal Cadmium

Sedum plumbizincicola is a hyperaccumulators discovered in recent years,which has strong hyperaccumulation capacity on heavy metals such as cadmium,zinc and lead.It can accumulate large amounts of heavy metals on the above-ground tissues without any toxic symptoms.However,due to its small biomass and slow growth rate,its large-scale application in heavy metal contaminated phytoremediation is limited.In this study,a gene encoding the heavy metal uptake protein,SpHMA2,was cloned in Sedum plumbizincicola.And then,the function of SpHMA2 in cadmium uptake and tolerance of plants was analyzed by employing bioinformatics analysis,q RT-PCR,and transgenic plant generation.The results indicate that SpHMA2 has strong transport ability to cadmium.The specific results are as follows:1.We designed SpHMA2 primers by referring to the HMA2 gene sequence from Sedum alfredii,and then cloned the sequence of SpHMA2 with c DNA of Sedum plumbizincicola.SpHMA2 gene is a total of 2910 bp,encoding a protein with 969 amino acids and a molecular weight of about 104.3 k Da.The expression level of SpHMA2 gene in leaves and stems of Sedum plumbizincicola is higher than that in roots without cadmium treatment.After treatment with cadmium,the expression levels of the gene in all the examined tissues are significantly increased except for the old leaves,indicating that the gene mainly functions to cadmium stress response in stems and leaves of the plant.2.Software prediction indicated that SpHMA2 may be a protein localized on the plasma membrane.The results obtained from the instantaneous transformation system in both Arabidopsis protoplasts and tobacco leaf epidermal cells further showed that SpHMA2 is localized on the plasma membrane.3.Further,the function of SpHMA2 in heavy metal transport was preliminarily analyzed by using yeast mutant and E.coli cells.The growth curve measurement and titration experiments of yeast indicated that the growth of yeast expressing the SpHMA2 gene was more severely inhibited than the yeast expressing the empty vector under the cadmium stress treatment.The result obtained in E.coli cells is contrary to that in yeast.We hypothesized that the location of SpHMA2 in yeast is different from that in E.coli.In yeast,SpHMA2 may be erroneously localized on the tonoplast and therefore transport the cadmium stored in the vacuole into the cytoplasm,causing the yeast to be suffered more toxic.However in E.coli,it may be located on the cell membrane and therefore excret the cadmium to the outside of the cells,reducing the toxic effect of cadmium to the cells.The results from the two experiments also indicate that SpHMA2 has a transporting ability to cadmium.4.We obtained five SpHMA2-transgenic Arabidopsis thaliana lines using the agrobacterium-mediated transformation.Three lines with higher expression levels of SpHMA2 were selected for cadmium stress treatment.The results showed that under cadmium stress treatment,the growth of transgenic plants was much better than that of the control items,indicating that overexpression of SpHMA2 enhanced the tolerance of Arabidopsis to cadmium.SpHMA2 transgenic rapeseed(variety: K407)was also created by transgenic method mediated by Agrobacterium,and four transgenic lines were obtained.We exerted cadmium treatment on two strains.Through the measurement of heavy metal cadmium concentration,we found that the cadmium concentrations in the above-ground tissue of SpHMA2 transgenic rapeseed are significantly higher than that of wild type rapeseed,indicating that SpHMA2 participates in the transport process of cadmium to the above-ground tissue of the plants.As a whole,in this study,a gene encoding SpHMA2 was firstly cloned from S.plumbizincicola.SpHMA2 is plasma membrane located and its expression levels in leaves and stems of S.plumbizincicola can be greatly increased by cadmium stress.Overexpression of SpHMA2 in Arabidopsis and rapeseed plants enhanced the plants to cadmium stress tolerance and transport ability of the heavy metal from roots to the above-ground tissues,indicating that SpHMA2 is a good candidate genes for the phytoremediation of heavy metal contaminated soil in the future.