The Study Of AteIF5A-2Insertion Mutant And Coexpression Of Multiple Genes In Arabidopsis To Enhance Heavy Metal Accumulation

Plants can absorb the nutrient from soils and also can absorb and accumulate the toxic heavy metals. So the pollution of heavy metals is a problem of global environmental and agricultural production. Of course, plants also posses homeostatic cellular mechanisms to regulate the concentration of metal ions inside the cell and to minimize the potential damage that could result from the exposure to nonessential metal ions and participate in the detoxidant and tolerant mechanisms to heavy metals, decrease the damage to plants. Eukaryotic translation initiation factor5A (eIF5A) is an essential protein that is ubiquitous and highly conserved among all eukaryotic organisms. In the study, we used a T-DNA insertion mutant of AteIF5A-2to study the role of AteIF5A-2in resistance to Cd toxicity in plants; On the other hand, we used Gateway cloning technology to creat transgenic Arabidopsis thaliana with coexpression of multiple genes involed in metal ion uptake, translocation and detoxification and evaluate the possibilty generate heavy metal super-hyaccumulating plants. The main results are summarized as follows:1. AteIF5A-2influences the absorption, accumulation and resistance of Cd in plantsEukaryotic translation initiation factor5A(eIF5A), as an essential protein that is ubiquitous and highly conserved among all eukaryotic organisms, it is mainly studied with animals and the study with plants are limited. We studied the phenotypic change using the T-DNA insertion mutant of AteIF5A-2fbr12under different Cd treatment. We found that there was no difference between fbr12and Col-0under normal growth conditions. When treated with Cd, the root length of fbr12was significant decreased compared to Col-0, and the difference was more obvious following the Cd concentration enrichment, the biomass of plants was the same as the root length. These all indicated that the loss function of AteIF5A-2in Arabidopsis thaliana can increase the sensitivity to Cd. Further analysis, we founded the accumulation of Cd in roots and shoots in for12were significantly increased compared to Col-0under different Cd treatment using ICP-AES. When treated with the same Cd, the total chlorophyll and the capacity of antioxidant were obviously decreased than Col-0; The content of H2O2and MDA were signigicantly higher than Col-0, all these indicated that the oxidative stress in fbrl2was more seriously. The free Pro and GSH fbr12were obviously decreased than that in Col-0, this demonstrated that the degree of Cd toxicity was more seriously in fbr12. The gene expression analysis found that the relative expression abundance of the different isoforms of ROS scavenging enzymes in fbr12were significantly decreased than that in Col-0, and this is further verified that the capacity of antioxidant in for12was lower that that in Col-O.To analysis the potential metal iron transporters like IRT1, ZIP1, CAX2and Nramp3, we found that its expression in fbr12were lower than that in Col-0, which can result in the more accumulation of Cd in fbrl2. Phytochelatins (PCs) are essential for normal constitutive tolerance to several non-essential metals, particularly Cd, the relative abundance of Phytochelatin synthase gene PCS1and PCS2mfbrl2were notably lower than that in Col-0and this is the possible reason that for12was more sensitive to Cd.2. The cooverexpression of multigenes in Arabidopsis thaliana increases the accumulation of heavy metalsWe use multiple round Gateway LR recombination technology to creat the transgenic lines LR2with overexpression AtCS1and TcZNT1and LR4with overexpression AtCS1, TcZNT1, TcOPT3and AtHMA4in Arabidopsis thaliana. We treated the highest gene expression line LR2-5and LR4-16with Cd, Cu and Zn to evaluate the contribution of overexpression of these genes to hyperaccumulation of heavy metals. We found that the accumulation of Cd in roots of LR2-5and LR4-16was signigicantly higher than that in Col-0under different Cd levels, but there was no difference in the Cd concentration in shoots between Col-0and LR2-5, or even lower in LR4-16compared to Col-0; The accumulation of Cu in roots and shoots of LR4-16was notably increased than that in Col-0, but there was no difference between Col-0 and LR2-5when treated with5μM Cu; The accumulation of Zn in roots and shoots of LR4-16were significantly higher than that in Col-0when treated with250μM Zn. The preliminary results indicate that the overexpression of multiple genes in plants have various effects on different metals. To creat the ideal transgenic hyperaccumulator plants, we still need to select the ideal genes together with suitable promoters.