低溫逆境下綠豆CYP90A2基因的表現及油菜類固醇促進上胚軸伸長的蛋白質體研究

Abstract

CYP90A2 cDNA是第一個從綠豆中選殖出來的細胞色素P450基因, 其轉譯的氨基酸序列與阿拉伯芥中合成油菜類固醇 (brassinosteroid, BR) 的CYP90A1/CPD蛋白質有77 % 的高相似度。 CYP90A2基因主要表現在葉子, 表現量隨著幼苗的生長而增加, 呈現週日的變化 (diurnal variation), 並受油菜類固醇回饋抑制其基因的表現。 在低溫10 ℃下, 綠豆幼苗停止生長, 其CYP90A2基因的表現量亦降低; 阿拉伯芥幼苗在低溫下則是持續生長, 且負責合成油菜類固醇的各種CYP基因亦持續表現。 利用油菜類固醇處理綠豆幼苗, 發現其上胚軸在低溫逆境下可以持續伸長, 且回溫至28℃常溫生長箱後有較高的存活率。 針對油菜類固醇促進綠豆上胚軸的伸長進行蛋白質體研究, 共觀察到31個增加表現及24個降低表現的蛋白質。進一步利用MALDI-Q-TOF MS/MS質譜儀及Mascot軟體進行分析與資料庫比對, 共鑑定出13個與細胞生長、光合作用及呼吸作用等相關的蛋白質。 針對這些蛋白質進行相關生理實驗, 發現油菜類固醇促進上胚軸的伸長與甲硫胺酸、V-ATPase及enolase有關, 且在黑暗下油菜類固醇反而抑制上胚軸的伸長。 至於在低溫逆境下油菜類固醇促進綠豆上胚軸伸長的蛋白質體研究, 共觀察到17個受低溫抑制表現, 又可藉由處理油菜類固醇而回復表現的蛋白質。 進一步利用MALDI-Q-TOF MS/MS質譜儀及Mascot軟體進行分析與資料庫比對, 共鑑定出11個與甲硫氨酸、ATP形成及細胞生長有關蛋白質。 針對綠豆幼苗處理甲硫氨酸, 發現受低溫抑制伸長的上胚軸可以繼續伸長。本研究除了在低溫逆境下比較耐寒與不耐寒植物中參與合成油菜類固醇的CYP基因差異表現, 亦建立油菜類固醇促進綠豆上胚軸伸長的表現蛋白質體圖譜 (proteomic mapping)。 根據這些蛋白質的功能, 在未來可以設計出更精準的生理實驗來研究油菜類固醇促進細胞生長的機制。

Mung bean CYP90A2 is a putative brassinosteroid (BR) synthetic gene that shares 77 % identity with the Arabidopsis CPD gene. The transcriptions of CYP90A2 are abundantly detected in leaves that increase following seedlings development and exhibit diurnal variation. CYP90A2 is feedback down regulated in the existence of BR. When exposed to 10 ℃ chilling stress, Arabidopsis shows higher growth than mung bean and the expression of Arabidopsis CYP genes are higher than mung bean CYP90A2. Therefore, chilling-inhibited growth and survival rate could be improved by exogenous treatment of BR. In the proteomic study of BR-promoted epicotyl elongation, BR-treated mung bean epicotyls had 31 up-regulated and 24 down-regulated protein spots. Identifications of these proteins using mass spectrometry (MALDI-Q-TOF MS/MS) analysis suggest that most were involved in cell growth, photosynthesis and respiration. In the physiological assay, BR promotes epicotyl elongation only under high light condition, while inhibits elongation under full darkness condition. With regard to interaction of the BR proteome and chilling stress, 17 chilling down-regulated proteins were re-up-regulated by BR treatment. The up-regulated proteins are involved in methionine assimilation, ATP synthesis, and cell growth. This is consistent with the re-up-regulation of methionine synthase and S-adenosyl-L-methionine synthetase, since chilling-inhibited mung bean epicotyl elongation could be partially recovered by exogenous treatment of DL-methionine. In summary, this is the first proteomic mapping established in mung bean, and the regulation between brassinosteroid and chilling response is investigated, to which, possible mechanisms are revealed.