Possible participation of metal-binding germin like protein of tomato roots in the acquisition of soil mineral nutrients

在本研究项目中，我们对番茄根中金属结合蛋白LeGlp-1的功能进行了新的研究。(1)利用金属亲和层析纯化了重组LeGlp-1，并将其作为抗原制备抗体。(2)LeGlp-1以单体和六聚体的形式与金属结合。(3)在转LeGlp-1的烟草叶片中，表达的LeGlp-1具有与番茄根中天然的LeGlp-1相同的草酸氧化酶活性。(4)GUS和GFP定位于已构建的转基因烟草的根部，表达的报告蛋白的N端与LeGlp-1的靶序列相融合。根据显微镜观察，LeGlp-1可能定位于皮层质膜或根中的内胚层细胞，可能不分泌到质外体。(5)通过将Cys残基定点突变到LeGlp 1 N端新发现的金属结合位点区域的Ser，突变后的LeGlp-1不能在转基因烟草中构建六聚体。(6)转LeGlp-1基因烟草叶片中的蛋白质组分对大肠杆菌的生长有抑制作用。由于LeGlp-1的丝氨酸被金属结合的Cys残基取代而降低了LeGlp-1的活性，因此认为LeGlp-1是大肠杆菌质膜上的一种金属离子通道，导致细菌细胞内金属动态平衡的紊乱。

In our research project following novel findings were obtained on the function of a metal-binding protein, LeGlp 1, in tomato root.(1)Recombinant LeGlp 1 was purified by metal affinity chromatography and used as antigen to raise antibodies. Antibodies were very specific to LeGlp 1 which enables to quantitatively detect LeGlp 1.(2)Metal binding of LeGlp 1 was shown for its monomeric and hexameric forms.(3)In the leaves of LeGlp 1 transgenic tobacco LeGlp 1 was expressed with a weak oxalate oxidase activity in a form that is identical with the native LeGlp 1 in tomato roots.(4)GUS and GFP were localized in the roots of transgenic tobacco that had been constructed to express either of the reporter proteins fused their N-terminal with the target sequence of LeGlp 1. According to microscopic observation LeGlp 1 may be targeted to plasma membrane of cortex or endoderm cell in roots and may not be secreted to apoplast.(5)By a site-directed mutagenesis of Cys residues to Ser in the region of newly identified metal binding site at the N-terminal of LeGlp 1, mutated LeGlp 1 was shown to fail in the construction of hexameric natural form of LeGlp 1 in transgenic tobacco leaves.(6)Protein fraction of LeGlp 1 transgenic tobacco leaves suppressed growth of E.coli. Since this activity of LeGlp 1 was lowered by the substitution of metal-binding Cys residues to Ser of LeGlp 1, LeGlp 1 was suggested to be a metal ion channel at the plasma membrane of E.coli which resulted in the disorder of metal homeostasis in the bacterial cells.In conclusion, LeGlp 1 is shown to function at the plasma membrane of cortex or endoderm cells as a metal ion channel in the rout of micronutrient uptake from apoplast to xylem tube.