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

1.gek1突变体的表型分析：为了研究gek1突变对代谢途径的影响，我们尝试通过多核核磁共振结合稳定同位素标记来检测代谢产物的变化。结果表明，在乙醇存在下，gek1对氨基酸代谢途径的激活作用比野生型更强。基因芯片分析表明，乙醇处理能更快地上调gek1突变体中几个胁迫诱导基因的表达。2.gek1抑制突变体的筛选。我们分析了它们中的大多数，发现它们都是ADH突变体。3.鉴定与GEK1相互作用的蛋白：通过酵母双杂交分析鉴定了三种可能与GEK1相互作用的蛋白。其中两个是锌指蛋白(#20、#24)。另一个是NFU2(#76)，推测它与叶绿体中Fe-S簇的形成有关。亚细胞定位和中断突变体(#24、#76)的分析没有提供这些蛋白与GEK1之间的任何明显联系。4.重组GEK1蛋白。我们考察了它在体外对乙醛的分解活性。我们尝试了几种情况，但都没有检测到这样的活动。我们获得了可能更稳定的热球菌GEK1同源基因的重组蛋白。我们试图检测到相同的活动，但再次失败。因此，我们得出结论，GEK1没有这种活性。我们还试图观察任何改变代谢物特征的活动。~&lt；13&gt；C，^&lt；15&gt；N稳定同位素标记植物提取物与重组蛋白反应，核磁共振检测。然而，我们没有察觉到任何变化。

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.