Functional analysis of gibberellin 3-oxidase N-terminal

赤霉素3-氧化酶N末端的功能分析

• 批准号: 17570046
• 负责人: KOBAYASHI Masatomo
• 金额: $ 1.47万
• 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: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570046/
• 关键词: Gibberellin; Oxidase; Biosynthesis; Mutant protein; Enzyme activity; Arabidopsis; 生合成酵素; イネ; 蛋白工学; 酸化酵素

Gibberellin 3-oxidase (GA3-ox) catalyzes final activation step in the biosynthesis of gibberellin, a plant hormone that regulates germination, elongation, flowering, and etc. GA3-ox belongs to the 2-oxoglutarate dependent-oxidase family, and C-terminal of GA3-ox includes catalytic center of this enzyme. On the other hand, function of the N-terminal of this enzyme has not been characterized yet, although it contains highly conserved motifs such as MWS/A/YEGY/FT. motif. In this study, the function of MWS/A/YEGY/FT motif in Arabidopsis GA3-ox, AtGA3oxl, was characterized by in vivo and in vitro experiments. The methionin or tryptophan residue in the motif was replaced with alanine to produce mutant proteins through preparation of mutant cDNA by PCR and its expression in E call. The mutant proteins prepared as fusion proteins in E. coli were incubated with gibberellin A_9 to examine their enzyme activities. Surprisingly, both mutant proteins showed equivalent activity with the normal protein indicating that MWS/A/YEGY/FT motif does not concern with catalytic activity of this enzyme. Therefore the mutant cDNA was introduced to Arabidopsis to examine its function by in vivo assay. As a result, T1 plants did not show any difference in their physiological appearance and expression level of GA-induced gene. Further analysis with T2 plants is necessary to confirm this result.

赤霉素3-氧化酶（GA 3-ox）是赤霉素生物合成的最后一步催化剂，赤霉素是一种植物激素，对植物的萌发、伸长、开花等具有调节作用。GA 3-ox属于2-酮戊二酸依赖氧化酶家族，其C-末端含有赤霉素的催化中心。另一方面，该酶的N-末端的功能尚未被表征，尽管它包含高度保守的基序，如MWS/A/YEGY/FT。母题本研究对拟南芥GA 3-ox（AtGA 3 oxl）中MWS/A/YEGY/FT基序的功能进行了体内和体外研究。通过PCR方法制备突变体cDNA并在大肠杆菌中表达，将突变体中的蛋氨酸或色氨酸残基替换为丙氨酸，得到突变体蛋白。在大肠杆菌中以融合蛋白的形式制备了突变蛋白。大肠杆菌与赤霉素A_9共孵育，测定其酶活性。令人惊讶的是，这两种突变蛋白显示出与正常蛋白相当的活性，表明MWS/A/YEGY/FT基序与该酶的催化活性无关。因此，将突变体cDNA导入拟南芥中，通过体内测定来检测其功能。结果表明，T1代植株在生理外观和GA诱导基因的表达水平上没有表现出任何差异。需要对T2植物进行进一步分析以证实这一结果。