在低溫逆境下綠豆幼苗Rubisco Activase基因啟動子之順位因子的分析研究

Abstract

綠豆(Vigna radiata L. v.2937)屬於不耐寒植物，本實驗室要了解寒溫逆境(chilling stress)對於不耐寒植物的影響，利用差異式篩選法(differential screening)篩選出十三個會受到10℃明顯抑制的基因。本實驗室針對十三個基因中的Rubisco activase及cytochrome P450做更進一步研究，其中對於Rubisco activase基因表現特性有概日韻律，當受到寒溫逆境10℃處理時，概日韻律會受到改變，基因表現受到抑制，並且寒溫逆境會抑制Rubisco activase基因的轉錄作用。 本實驗室更進一步找出Rubisco activase基因的啟動子，長1,161bp。本論文就是針對此啟動子做進一步研究，利用啟動子刪除分析(promoter deletion assay)及阿拉伯芥轉殖系統，找出-521到-431的啟動子區域和低溫調節有關。並且經由凝膠延滯實驗(gel retardation)，證明低溫會使綠豆產生特殊的細胞核蛋白質和-521到-431的啟動子區域做結合。 除了找出-521到-431的啟動子區域和低溫調節有關以外，也經由阿拉伯芥轉殖系統、凝膠延滯實驗及網路資料庫查詢的結果，推論出Rubisco activase基因的啟動子區域-160到-58也和低溫相關。另外也推論出啟動子區域-251到-151在正常生長下會促進Rubisco activase基因表現，但這種促進的作用在低溫下會被抑制。

The mungbean (Vigna radiata L. v. 2937) is a chilling-sensitive plant. A cDNA library was constructed from poly(A)+ mRNA of 28℃-light-grown leaves for screening cold-suppressed nuclear genes. Rubisco activase(rca) gene was one of the 13 cold-suppressed nuclear genes which obtained by differential screening. Rubisco activase gene has single copy in mungbean (Vigna radiata L. v. 2937) and is light-inducible. Under 10℃ chilling stress, rca mRNA synthesis was repressed and circadian rhythm was interrupted. The upstream region (1,161bp) of rca gene had been cloned from mungbean by genomic walking method. The results of promoter deletion assay, Arabidopsis transformation and gel retardation have defined the promoter region from -521 to -431 which was related to the chilling suppression and also bound by nuclear proteins. Those nuclear proteins were isolated from cold-treated (3 days, 10℃) mungbeans. Using the PLACE website, the promoter region from -521 to -431 has a specific region(DOFCOREZM). In other study, the Dof2 protein of maize bound DOFCOREZM region and suppressed the activity of the C4pepc promoter (Yanagisawa, 2000). Dof analogies of mungbean maybe suppress rca gene expression under chilling stress.