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

番茄根部的金属结合胚芽蛋白样蛋白可能参与土壤矿质养分的获取

• 批准号: 12460034
• 负责人: TAKAHASHI Masaaki
• 金额: $ 6.14万
• 依托单位: Osaka Prefecture University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460034/
• 关键词: germin-like protein; transgenic plant; green fluorescent protein (GFP); GUS staining; target sequence; metal ion channel; site-directed mutagenesis; oxalate oxidase; トマト; 金属イオン輸送; GFP; germin; 微量必須金属イオン; レステイン; 金属結合; 遺伝子導入; germin-like pro

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.

在我们的研究项目中，关于番茄根中金属结合蛋白LeGlp 1的功能获得了以下新发现。(1)通过金属亲和层析纯化重组LeGlp 1并用作抗原来产生抗体。抗体对 LeGlp 1 非常特异，能够定量检测 LeGlp 1。(2)LeGlp 1 的单体和六聚体形式显示出金属结合。(3)在 LeGlp 1 转基因烟草的叶子中，LeGlp 1 表达具有弱草酸氧化酶活性，其形式与番茄根中的天然 LeGlp 1 相同。(4)GUS 和 GFP 定位于 构建表达任一报告蛋白的转基因烟草的根部，其N端与LeGlp 1的靶序列融合。根据显微镜观察，LeGlp 1可能靶向根部皮层或内胚层细胞的质膜，并且可能不分泌到质外体。(5)通过将新的区域中的Cys残基定点诱变为Ser 鉴定出LeGlp 1 N端的金属结合位点，突变的LeGlp 1在转基因烟叶中无法构建LeGlp 1的六聚体天然形式。(6)LeGlp 1转基因烟叶的蛋白质部分抑制了大肠杆菌的生长。由于LeGlp 1的金属结合Cys残基被LeGlp 1的Ser取代而降低了LeGlp 1的活性，因此LeGlp 1被认为是大肠杆菌质膜上的金属离子通道，导致细菌细胞中金属稳态的紊乱。 总之，LeGlp 1被证明在皮质或内胚层细胞的质膜上作为金属离子通道在细菌细胞内发挥作用。 从质外体到木质部管的微量营养素吸收。