Cloning And Functional Analysis Of Natural Resistance Associated Macrophage Protein 1 From Nicotiana Tabacum

Natural resistance-associated macrophages（NRAMPs）are an important class of metal transporters.In many species,such as Arabidopsis and rice,NRAMP protein has been shown the abilities to transport Fe³⁺,Mn²⁺,Zn²⁺and Cd²⁺.Tobacco is not only an important economic crop but also often be used for the bioremediation of heavy metal contaminated soil.At present,the biological function of tobacco Nt NRAMP is still unclear.In this study,Nt NRAMP1.2 gene was successfully cloned from tobacco based on the sequences of homologous genes,and its biological function in terms of its sequence characteristics,gene expression patterns,metal transport characteristics,and subcellular localization were also studied.The results showed that the Nt NRAMP1.2 gene is 1602 bp in length,which encodes a protein containing 533 amino acids and 12 transmembrane domains.Nt NRAMP1.2 belongs to the subfamily II of the NRAMP gene family,and has the highest sequence identity with Nt NRAMP1.1,and the closest relationship with At NRAMP1 and At NRAMP6 in Arabidopsis.The results of heterologous expression of the Nt NRAMP1.2 gene in wild-type and yeast mutants respectively showed that:Nt NRAMP1.2 can increase the Cd²⁺content and Mn²⁺content in yeast cells respectively,thereby increasing the sensitivity of wild-type yeast to Cd²⁺and improving the growth of smf1 mutant under low Mn²⁺condition.However,Nt NRAMP1.2 does not affect the sensitivity of yeast mutant to low Fe³⁺or low Zn²⁺,indicating that Nt NRAMP1.2 can transport Cd²⁺and Mn²⁺,but not Fe²⁺or Zn²⁺.Gene expression analysis found that the expression of Nt NRAMP1.2 gene has constitutive characteristics,but the expression level in old leaves is higher than that in other tissues;the expression of Nt NRAMP1.2 in roots and leaves could be induced by Mn²⁺deficiency or Cd²⁺treatment.GUS staining of Nt NRAMP1.1::GUS transgenic tobacco showed that Nt NRAMP1.1 was mainly expressed in leaf trichome and taproot,and the expression could be induced by the lack of Mn²⁺and excess Cd²⁺,which was consistent with the expression pattern of the Nt NRAMP1.2 gene.The subcellular localization results showed that Nt NRAMP1.2 was localized in the trans-Golgi network（TGN）of the cell.The Nt NRAMP1.1 overexpression transgenic tobacco was treated with different concentrations of metals,and it was found that the overexpression of Nt NRAMP1.1 could not affect the fresh weight and dry weight of the tobacco.The results of the study laid the foundation for a comprehensive elucidation of the biological functions of tobacco Nt NRAMP1.2.