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In order to enhance the iron accumulation in plants, the primers were designed based on the Arabidopsis thaliana IRT1 sequence in GenBank. The total RNA of Arabidopsis thaliana roots was extracted after iron deficiency treatment for 24h. The cDNA of IRT1 gene was cloned by RT-PCR and then was inserted into the pBluescript II SK+ vector. After confirmed by endonuclease digestion assay and gene sequencing, the IRT1 gene was subcloned into the Sac I and BamH I targeting sites of the vector pBI121, and the plant expression vector pBI121IRT1 was constructed. The pBI121IRT1 vector was transformed into the wild type tobacco by Agrobacterium mediated method. Seventy-six resistant shoots were obtained by Kanamycin selection. 21 were identified by PCR and PCR-Southern detection. Five transformants were selected to perform the physiological assay respectively. The results showed the leaf chlorophyll contents were higher in the transformants than in the control, while the leaf MDA contents were lower in the transformants. It indicated that the IRT1 gene enhanced the iron uptake in the transformants and improved the transformants' resistance to iron deficiency.