Overexpression Of The Iron Transporter NtPIC1 In Tobacco Mediates Sensitivity To Lead And Mercury

Heavy metal pollutants reduce the quality of environmentand further damage human metabolism by accumulating in the food chain.Phytoremediation has received increasing attention as an environmental-friendly,economical and effective method for purifying heavy metals.In this study,transgenic Nt PIC1 tobacco was used as material to analyze its function under heavy metal Hg²⁺and Pb²⁺stress,which lay a foundation for further revealing the mechanism of Nt PIC1 in the process of heavy metal accumulation in plants.The main results are as follows:1.Identification of T₃ generation plants of Nt PIC1 transgenic tobaccoThe resistant seedlings were obtained by seed germination experiment on high iron medium and indentified them by PCR and qRT-PCR for identification of T₃ generation plants of transgenic tobacco,which obtained three homozygous lines.2.Response of Nt PIC1 transgenic yeast to heavy metal Hg and PbThe Nt PIC1 gene was successfully transferred into yeast and expressed in yeast by lithium acetate transformation,uracil defect culture medium and PCR identification.The experiments on response of yeast BY4741-pYES2 and BY4741-pYES2-Nt PIC1 to heavy metals Hg and Pb showed that the yeast expressing the Nt PIC1 gene was sensitive to Hg and Pb.3.Response of T₂ generation tobacco seeds with Nt PIC1 gene to heavy metals Hg and PbCompared with WT plants,the seeds germination rate of Nt PIC1-OX plants was lower under Hg stress and the survival rate of seedlings was lower under Pb stress,which indicated that the seeds of Nt PIC1-OX plants were sentivity to heavy metals Hg and Pb.4.Responses of T₃ generation Nt PIC1 transgenic tobacco plants to heavy metals Hg and PbCompared with WT,the root length inhibition of Nt PIC1-OX seedlings was higher under heavy metal stress,which indicated that the roots of Nt PIC1-OX plants were more sensitive to heavy metals Hg and Pb.Under stress,the damage degree of Nt PIC1-OX leaves was more serious than that of WT leaves and Chlorophyll content was also lower than that of WT leaves.To explain the result,metal contents of plants were measured under stress.We found the iron content in roots of Nt PIC1-OX plants was lower than that of WT plants and in shoots was higher than that of WT plants,which indicated that overexpression of Nt PIC1could enhance the ability of Nt PIC1-OX plants to transfer iron from roots to shoots.We also found the mercury and lead contents in roots and shoots of Nt PIC1-OX plants were both higher than that of WT plants,which indicated that Nt PIC1-OX plants could absorb more Hg and Pb.5.Relative expression of iron related genesThe expression levels of iron-related genes in WT and Nt PIC1-OX plants were analyzed under Hg and Pb stress by qRT-PCR.We found the expression levels of Nt PIC1 gene,NtNRAMP1 gene and Nt FER1 gene in roots of Nt PIC1-OX plants were lower than that of WT plants and in shoots were higher than that of WT plants,the expression level of Nt IRT1 gene in roots and shoots of Nt PIC1-OX plants was higher than that of WT plants,which suggested that the ability of Nt PIC1-OX plants to transport iron from roots to shoots maybe depend on the expression of these iron related genes.6.Analysis of physiological parameters of plants under Hg and Pb stressUnder Hg and Pb stress,the soluble protein content,soluble sugar content,proline content and antioxidant enzyme activity of Nt PIC1-OX plants were lower than that of WT plants,while the malondialdehyde content,superoxide anion content and hydrogen peroxide content were higher than that of WT plants,indicating that Nt PIC1-OX plants were highly damaged and may be related to the absorption of more Hg and Pb.