Isolation And Characterization Of Mb.nramp1 Gene In Malus Baccata (L.) Borkh

Gene members of the Nramp family encoding of integral membrane proteins, are involved in metal ion transportion, especially regulation of iron metabolism. In order to isolate the Nrampl gene (Natural-resistance-associated macrophage proteinl) from Malus baccata (L.) Borkh, RT-PCR using degenerate primers corresponding to conserved motifs of Nrampl in plant, and combined with RACE. And after that further study on function and subcellular localization was developed in model eukaryono The main results are as follows:1. A 2090 bp cDNA fragment was cloned, which contains a full-length open reading frame of Nrampl gene in Malus baccata (L.) Borkh. It encodes a predicted polypeptide of 551 amino acids with a molecular weight of about 59.7kDa. Because this gene shared high homology of Lenrampl, it is nominated as Mb.nrampl. Its accession number is AY724413. Alignment analysis showed that Mb.NRAMPl has higher similarity with LeNRAMPl (66% identity and 80% similarity) than with OsNRAMPl (63% identity and 68% similarity) and AtNRAMPl (55% identity and 58% similarity). The Mb.NRAMPl protein contains all conserved domains of NRAMP such as putative N-linked glycosylation sites, 12 transmembrane domains (TMs) and a Consensus Transporter Motif (CTM) in the fourth intracellular loop between TMs Ⅷ and Ⅸ.2. Nrampl has one single copy in genome of Malus baccata (L.) Borkh. Expression of Mb.nrampl under nutrient-sufficient conditions is very low in roots and not detectable in shoots. The expression level of Mb.nrampl under Fe-deficiency condition increased significantly in roots. In shoots the expression is detectable after 15days' Fe-deficiency treatments.3. Mb.nrampl was expressed in yeast mutant DEY1453, which reversed the growth defect on iron-depleted media. It grew significantly better than the transgenic yeast with empty vector. Growth of Mb.nrampl transformed yeast cells was the same with wild type at 15 u M BPDS and retarded at 30 μM BPDS as the wild type did. At any concentrations of BPDS, growth of Mb.nrampl transformed yeastcells was significantly better than that of DEY1453 mutant strain transformed with empty vector. Because Mb.NRMPl is able to restore the growth of yeast DEY1453 mutant, it is involved in iron uptake.4. Observation of the subcellular localization of the Mb.NRAMPl proteins showed that Mb.NRAMPl was mainly localized in the whole endocytic vacuole membrane and a few in the multiple vesicles. Subcellular localization patterns of the Mb.NRAMPl protein in yeast indicated that Mb.NRAMPl protein probably takes part in the releasing of Fe in these organelles.The plant iron uptake strategy Ⅲ - endocytic mechanism model was established.