Creation of a reporter plant of Mn toxicity

锰毒性报告植物的创建

• 批准号: 07556086
• 负责人: TAKAHASHI Masaaki
• 金额: $ 0.51万
• 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: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556086/
• 关键词: tomato; cDNA cloning; transgenic plant; oxalate oxidase; manganese; シュウ酸酸化酵素; cDNAクローニング; 土壌微生物; 金属結合タンパク質; 水耕栽培

Tomato plants (Lycopersicon esculentum L.cv Ponderosa), grown for 10 days with complete nutrient after germination, were subjected to Mn^<2+> deficiency for the following 10 days. During the Mn^<2+> deficiency, the roots lost most of their Mn, but this was accompanied by the emergence of high-affinity Mn^<2+> uptake. A metal-binding peptide was isolated from chymotrypsin-digested membrane proteins of Mn-depleted root tissues. A cDNA for the metal-binding protein was amplified by PCR with mRNAs from Mn-depleted tomato roots. A full-length cDNA (Mdip1) was shown to encode a precursor protein having a 27-amino acid leader sequence at the NH_2-terminus. The leader extension targeted GUS to cell surface of transgenic tobacco with chimeric transgene of GUS and the gene encoding N-terminal extension of Mdip1. The metal-binding site is located in the NH_2-terminal region of the 197-amino acid-long mature protein. A northern blot analysis revealed that the expression of Mdip1 was enhanced by Mn deficiency. Transformation of tobacco plant with Mdip1 cDNA conferred an oxalate oxidase activity, indicating that Mdip1 encodes an oxalate oxidase which was synthesized as a response to low bioavailability of soil Mn. Resulting H_2O_2 from oxalate oxidation might be a reductant for insoluble MnO_2 upon Mn^<2+> acquisition.

番茄植物（Lycopersicon esculentum L.cv Ponderosa）在发芽后用完全营养生长10天，在接下来的10天中经受Mn^<2+>缺乏。在Mn^<2+>缺乏期间，根系损失了大部分Mn，但这伴随着高亲和力Mn^<2+>吸收的出现。从胰凝乳蛋白酶消化的锰耗尽的根组织的膜蛋白中分离出金属结合肽。从锰耗竭的番茄根的mRNA通过PCR扩增的金属结合蛋白的cDNA。全长cDNA（Mdip1）编码的前体蛋白在NH_2端有一个27个氨基酸的前导序列。将GUS与编码Mdip1的N端延伸的基因嵌合，将前导延伸的GUS靶向至转基因烟草的细胞表面。金属结合位点位于197个氨基酸的成熟蛋白的NH_2端区域。北方印迹分析显示，锰缺乏增强了Mdip 1的表达。将Mdip1 cDNA转化烟草植株，获得了草酸氧化酶活性，表明Mdip1编码的草酸氧化酶是对土壤锰生物有效性低的响应。草酸盐氧化生成的H_2O_2可能是不溶性MnO_2的还原剂。

项目成果

Kitano,T.,Sasaki,K.,Amako,K.,Takahashi,M.: "Plant Natrition-for sustainable food production and environment" Audo,T., 2 (1997)

Kitano,T.、Sasaki,K.、Amako,K.、Takahashi,M.：“植物营养-促进可持续粮食生产和环境” Audo,T., 2 (1997)

Kitano,T.,Sasaki,K.,Fujimoto,N.,Amako,F.,Takahashi,M.: "Expression of a metal-binding protein as a result of Mu^<2+> deficienccy in tomato roots" Plant Physiology. (投稿中).

Kitano, T.、Sasaki, K.、Fujimoto, N.、Amako, F.、Takahashi, M.：“番茄根部 Mu^<2+> 缺陷导致金属结合蛋白的表达”植物生理学（目前正在发布）。

Mitsukawa,N.,Okumura,S.,Shirano,Y.,Sato,S.,Kato,T.,Harashima,S.,Shibata,O.: "Overexpression of an Aradipsis thaliana high-affinity phosphate transporter gene in tobacco cultured cells enhances grou H.under phosphate-limited conditions" Proceedings of Nati

Mitsukawa，N.，Okumura，S.，Shirano，Y.，Sato，S.，Kato，T.，Harashima，S.，Shibata，O.：“拟南芥高亲和力磷酸转运蛋白基因在烟草培养中的过度表达

Ohtani, K., Kitano, T., Amako, K., Takahashi, M.: Ando, T.Plant Natrition-for sustainable food production and environment 2. (1997)

Ohtani, K.、Kitano, T.、Amako, K.、Takahashi, M.：Ando, T.Plant Natrition - 促进可持续粮食生产和环境 2。（1997 年）

Kitano, T., Sasaki, K., Amako, K., Takahashi, M.: Ando, T.Plant Natrition-for sustainable food production and environment 2. (1997)

Kitano, T.、Sasaki, K.、Amako, K.、Takahashi, M.：Ando, T.Plant Natrition-促进可持续粮食生产和环境 2. (1997)