組蛋白H2B的穀胱甘肽化調控文心蘭開花之研究

Abstract

文心蘭的開花調控機制受到維他命C (ascorbate) 及穀胱甘肽 (glutathione) 的氧化還原態 (redox state) 所調控，然而氧化態的GSSG (oxidized form) 如何扮演訊號因子去啟動開花調控基因是一個未知的問題。本實驗利用抗穀胱甘肽抗體 (anti-GSH antiserum) 進行開花假球莖總蛋白質之篩選偵測，得到了一個約20 kDa的蛋白質分子，並證明此蛋白質為組蛋白H2B。試管內反應試驗結果，證實H2B重組蛋白分子可與GSSG進行共價結合，利用液相層析雙質譜儀分析結果也證實H2BCys40可與穀胱甘肽結合。若以1 mM GSSG溶液噴灑文心蘭幼苗期之假球莖，發現可縮短文心蘭抽花梗之時間，且H2B的穀胱甘肽化現象 (glutathionylation) 亦很明顯；利用yeast one-hybrid方法分析，初步顯示穀胱甘肽化的H2B可以和SPL10-2啟動子有親合能力。圓二色光譜 (circular dichroism) 分析也顯示穀胱甘肽化的H2B之構造受到改變。綜合上述，文心蘭的開花機制中有可能是染色質的H2B經過穀胱甘肽化後，影響到開花基因的表現所致。

The flowering regulation of Oncidesa is coordinately regulated by ascorbate and glutathione redox states. However, the role of glutathione disulfide (GSSG；oxidized form) as a signal factor to trigger flowering regulatory genes is still unknown. In this study, the glutathionylated proteins approximate 20 kDa were detected by anti-GSH antiserium, and the histone protein H2B was identified as the glutathionylated protein by LC-MS-MS analysis. The H2B-recombinant protein covalently bound with GSSG in vitro. By LC-MS-MS analysis, we found that the glutathionylation site of H2B was identified at cys40. Oncidesa plants in unsheath stage were treated with 1 mM GSSG. The result demonstrated that the early flowering of Oncidesa and the level of H2B glutathionylation were obviously detected. The glutathionyltion of H2B had affinity on SPL10-2 promoter in yeast one-hybrid assay. Further, the circular dichroism (CD) analysis suggested that glutathionylation of H2B affects its secondary structure. Collectively, we assumed that the glutathionylation of H2B alters the chromatin structure, leading to activation of flowering genes in Oncidesa.