Cloning,Subcellular Localization And Functional Analysis Of High Expression OsNramp1 Gene Under Iron Deficiency In Rice

The uptake and translocation of metal nutrients in plants are essential for plants growth, and the nutritional value of plants is central importance to human health. As iron availability can be limited by the low solubility, the most widespread human nutritional problem in the world is iron deficiency (WHO.2003). Therefore, increasing the ability of plants to provide a higher level of minerals, especially iron, will has a dramatic impact on human health.Nramp (nature resistance-associated macrophage protein) is a newly identified family of integral membrane proteins, and play an important role in heavy metal transportation especially in the iron transportation and the metal homeostasis.The results of Real-time PCR suggest that the expression of OsNramp1 is regulated by iron deficiency. Under iron starvation, the transcription level of OsNramp1 is highly induced in roots of Oryza sativa, about fourteen times than these grown under iron-sufficient conditions. It suggests that this gene may be involved in the uptake and transportation of iron. OsNramp1 cDNA has been isolated by traditional RT-PCR from the roots of Oryza sativa which grown for five days under iron deficiency conditions. The open reading frame of OsNramp1 consisting of 1557 nucleotides capable of encoding a 517 amino acids polypeptide.Functional complementation in the yeast mutants are used to further analyze the role of OsNramp1,wondering whether it is involved in metal ion transportation, such as: Fe²⁺,Mn²⁺,Zn²⁺,Cu²⁺,Cd²⁺ and Ni²⁺. We find that OsNramp1 cDNA can encode functional proteins and complement the phenotype of strains deficient for iron and manganese transportation. These results propose that OsNRAMP1 participate in the iron and manganese uptake and transport.Observe the subcellular localization of the OsNRAMP1 can help us know how it play the important role in the iron transportation. With the bioinformatics method, the results inferred that OsNRAMP1 protein might be localized in the plasma membrane. In addition, we use the reporter gene-GFP, the expression of OsNRAMP1 :GFP fusion protein in onion epidermal cells and tobacco (Nicotiana tabacum L.) suspension cells(BY-2 cells) showed that OsNRAMP1 proteins are targeted to the membrane system, including the plasma membrane, vacuolar membrane etc.Using cell membrane fluorescent dyes FM4-64, we can find OsNRAMP1:: GFP colocalization with the maker FM4-64. It furtherly approves that OsNRAMP1 is an integral membrane protein. In addition, cultivated BY-2 cells in different time and different conditions (+Fe or -Fe), we can discover an appearance of the probable novel Nramp transporter body.