Functional analysis of gibberellin 3-oxidase N-terminal

赤霉素3-氧化酶(GA3-OX)催化赤霉素生物合成的最终激活步骤，赤霉素是一种调节种子萌发、伸长、开花等的植物激素。GA3-OX属于2-氧戊二酸依赖的氧化酶家族，GA3-OX的C端包含该酶的催化中心。另一方面，尽管该酶含有高度保守的基序如MWS/A/YEGY/FT，但其N末端的功能尚未确定。Motif。本研究通过体内和体外实验研究了MWS/A/YEGY/FT基序在拟南芥GA3-ox中的功能。通过聚合酶链式反应制备突变基因，并在E Call中表达，将基序中的蛋氨酸或色氨酸残基替换为丙氨酸，产生突变蛋白。将突变蛋白在大肠杆菌中制备为融合蛋白，与赤霉素A9孵育，检测其酶活性。令人惊讶的是，两种突变蛋白的活性与正常蛋白相当，表明MWS/A/YEGY/FT基序与该酶的催化活性无关。因此，我们将该突变体基因导入拟南芥中，通过体内实验检测其功能。结果表明，T1植株在生理形态和GA诱导基因的表达水平上没有表现出任何差异。为了证实这一结果，需要对T2植物进行进一步的分析。

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.