Analysis of GEK1 function that is required for ethanol tolerance in higher plants

高等植物乙醇耐受性所需的 GEK1 功能分析

• 批准号: 15570045
• 负责人: HIRAYAMA Takashi
• 金额: $ 2.37万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570045/
• 关键词: Arabidopsis; ethanol hypersensitivity; NMR measurement; archaea; GEK1 interacting protein; subcelular localization; alcohol dehydrogenase; エタノール; アセトアルデヒド; NMR; GFP

1.Phenotypic analysis using gek1 mutants.To investigate the effect of gek1 mutation on the metabolic pathway, we tried to detect the change in metabolites by muti-nuclear NMR measurement combined with ^<13>C, ^<15>N stable isotope labelling. The results showed that gek1 activated the amino acid metabolic pathway in the presence of ethanol more strongly than the wild type. Microarray analysis revealed that several stress inducible genes were upregulated more rapidly by ethanol treatment in the gekl mutant.2.Screening for gek1 suppressor mutants.Putative gek1 suppressor mutants, totally 34 lines, were isolated. We analyzed most of them and found they were adh mutants. We are going to analyze rest of them.3.Identification GEK1-interacting proteins.Three putative GEK1-interacting proteins were identified by yeast two-hybrid analysis. Two of them are Zn-finger proteins (#20,#24). The rest one is NFU2(#76) that is presumed to be involved in the formation of Fe-S cluster in chloroplasts. The subcellular localization and the analyses of disruption mutants (#24,#76) did not offer any clear link between these proteins and GEK1 so far.4.Recombinant GEK1 protein.A recombinant GEK1 protein expressed in E.coli was obtained. We investigated its activity to breakdown acetaldehyde in vitro. We tried several conditions, but failed to detect such activity. We obtained the recombinant protein of the Pyrococcus GEK1 homologus gene that is presumed to be more stable. We tried to detect the same activity but failed again. Therefore, we concluded that GEK1 does not have such activity. We also tried to see any activity to change the metabolite profiling. The extract from ^<13>C,^<15>N stable isotope labeled plants were reacted with the recombinant protein and measured by NMR. However, we could not detect any changes.

1.使用gek1突变体进行表型分析。为了研究gek1突变对代谢途径的影响，我们尝试通过多核NMR测量结合^<13>C、^<15>N稳定同位素标记来检测代谢物的变化。结果表明，在乙醇存在的情况下，gek1 比野生型更强烈地激活氨基酸代谢途径。微阵列分析表明，乙醇处理后gek1突变体中的几个胁迫诱导基因上调较快。2.gek1抑制突变体的筛选。分离出假定的gek1抑制突变体，共34个系。我们分析了其中的大多数，发现它们是 adh 突变体。我们将分析其余的蛋白。3.GEK1 相互作用蛋白的鉴定。通过酵母双杂交分析鉴定了三种假定的GEK1 相互作用蛋白。其中两个是锌指蛋白（#20、#24）。剩下的一个是 NFU2(#76)，推测它参与叶绿体中 Fe-S 簇的形成。迄今为止，亚细胞定位和破坏突变体（#24，#76）的分析并未提供这些蛋白与GEK1之间的任何明确联系。4.重组GEK1蛋白。获得了在大肠杆菌中表达的重组GEK1蛋白。我们研究了它在体外分解乙醛的活性。我们尝试了多种条件，但未能检测到此类活动。我们获得了火球菌GEK1同源基因的重组蛋白，推测该蛋白更加稳定。我们尝试检测相同的活动，但再次失败。因此，我们得出结论GEK1不具有这种活性。我们还尝试观察任何改变代谢物分析的活动。使来自 13 C、 15 N稳定同位素标记植物的提取物与重组蛋白反应并通过NMR测量。然而，我们无法检测到任何变化。

项目成果

細胞工学別冊、植物細胞工学20、植物ホルモンのシグナル伝達

细胞工程特刊，植物细胞工程 20，植物激素信号转导

• 发表时间: 2004
• 作者: Asamizu E;Nakamura Y;Miura K;Fukuzawa H;Fujiwara S;Hirono M;Iwamoto K;Matsuda Y;Minagawa J;Shimogawara K;Takahashi Y;Tabata S;平山 隆志
• 通讯作者: 平山 隆志

Takashi Kuromori: "A collection of 11800 single-copy Ds transposon insertion lines in Arabidopsis"Plant Journal. 印刷中. (2004)

Takashi Kuromori：“拟南芥中 11800 个单拷贝 Ds 转座子插入系的集合”，植物杂志，2004 年出版。

エタノール耐性遺伝子を用いる形質転換植物の選抜方法及

使用乙醇耐受性基因选择转化植物的方法

• 发表时间: 2004

植物の代謝解析方法、ラベル直物の製造方法、な

植物代谢分析方法、标记产品的生产方法等

• 发表时间: 2004

Naoki Takahashi: "Expression and Interaction Analysis of Arabidopsis Skp1-Related Genes"Plant Cell Physiology. 45. 83-91 (2004)

Naoki Takahashi：“拟南芥Skp1相关基因的表达和相互作用分析”植物细胞生理学。