Biochemical and molecular biological studies of electron partitioning among enzymes involved in the assimilation of inorganic compounds in plants

参与植物无机化合物同化的酶之间电子分配的生化和分子生物学研究

• 批准号: 05454019
• 负责人: HASE Toshiharu
• 金额: $ 4.48万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454019/
• 关键词: Ferredoxin; Glutamate synthase; Sulfite reductase; Photosynthesis; Electron transfer; 光合成電子伝達; フェレドキシン-NADP^+還元酵素

1) Using heterologous expression system in E.coli, maize ferredoxin (Fd) was site-directly mutagenized. The substituted residues were designed surrounding [2Fe-2S] cluster and on the surface of molecule. The redox potential and electron transfer abilities to Fd-dependent enzymes, such as Fd-NADP reductase and sulfite reductase, were investigated and the result showed that Fd had a specific region to interact with the each enzyme.2) Two types of Fd isoproteins, photosynthetic and non-photosynthetic one, were expressed in a cyanobacterium, Plectonema boryanum, whose Fd gene (pet F) had been deleted. The transformant with photosynthetic Fd grew in photoautotrophic condition under light as the wild-type cells, while the transformant was not able to grow heterotrophically under dark. The transformant with non-photosynthetic Fd could not be selected probably due to the inability of this type of Fd to complement the Pet F.3) Genes for two glutamate synthase (GOGAT), Fd-GOGAT and NADH-GOGAT,were cloned from P.boryanum. The two GOGAT genes were separately inactivated by insertion of kanamycin cartridge. The resulting mutants were found to lack the corresponding enzymatic activity. However, the remarkable change f phenotype was not observed except for an increased sensitivity to photoinhibition. The two GOGATs are probably functionally compatible.

1)利用大肠杆菌异源表达系统，对玉米铁氧还蛋白（Fd）进行了定点诱变。取代基设计在[2Fe-2S]簇周围和分子表面。结果表明，Fd与Fd-NADP还原酶和亚硫酸盐还原酶具有特异的相互作用区域。2）在Fd基因缺失的蓝细菌中表达了两种Fd同工酶，即光合型和非光合型。具有光合Fd的拟南芥与野生型细胞一样，在光照下能在光合自养条件下生长，而拟南芥在黑暗下不能异养生长。3）克隆了两个谷氨酸合成酶（GOGAT）基因Fd-GOGAT和NADH-GOGAT。通过插入卡那霉素盒分别灭活两个GOGAT基因。发现所得突变体缺乏相应的酶活性。然而，除了对光抑制的敏感性增加外，没有观察到表型的显著变化。这两个GOGAT在功能上可能是兼容的。

项目成果

Y.Fujita: "Identification of chl B gene and the gene product essential for the light-independent biosynthesis of chlorophyll in the cyanobacterium P.boryanam" Plant Cell Physial.in press (1996)

Y.Fujita：“蓝藻 P.boryanam 中叶绿素的光独立生物合成所必需的 Chl B 基因和基因产物的鉴定”Plant Cell Physial.in press (1996)

T.Ideguchi: "cDNA cloning and expression of ferredoxin-dependentsulfite reductase from maize in E.cole celles" Photosynthesis : from Light to Biosphere (ed.P.Mathis). 2. 713-716 (1995)

T.Ideguchi：“在大肠杆菌细胞中从玉米中克隆铁氧还蛋白依赖性亚硫酸盐还原酶的 cDNA 克隆和表达”光合作用：从光到生物圈（ed.P.Mathis）。

T. Ideguchi: "cDNA cloning and functional expression of ferredoxin-dependent sulfite erductase from maize in E. coli cells." photosynthesis : from Light to Biosphere(ed. P. Mathis). vol. 2. 713-716 (1995)

T. Ideguchi：“玉米铁氧还蛋白依赖性亚硫酸酯酶在大肠杆菌细胞中的 cDNA 克隆和功能表达。”

T.Hase: "Modification of functional characteristics of an electron carrier protein." Production and Technology. 47(1). 47-49 (1995)

T.Hase：“电子载体蛋白功能特征的修饰。”

長谷俊治: "鉄・硫黄電子伝達蛋白質から見た植物細胞の無機物還元同化系" 蛋白質核酸酵素. 40. 282-292 (1995)

Shunji Hase：“从铁和硫电子转移蛋白看出植物细胞中的无机还原和同化系统”蛋白质核酸酶。 40. 282-292 (1995)