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研究生(外文):Chen Chi-de
論文名稱(外文):Expression of Rubisco activase gene in leaves of mungbean seedlings under chilling stress
指導教授(外文):Chen Yih-ming
外文關鍵詞:chilling stressrubisco activasecircadian rhythmmungbean (Vigna radiata)
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綠豆(Vigna radiata L. v. 2937)屬於不耐寒植物,本實驗室為瞭解低溫逆境對葉片細胞核基因之影響,以28℃照光生長葉片之poly(A)+ mRNAs,建立一cDNA library,再以差異式篩選法(differential screening)篩選受低溫明顯抑制之基因,共篩選出13個不同的基因;本論文即是針對其中一個rubisco activase(rca)基因進行更深入的探討。首先,在南方氏雜合分析(Southern blot analysis)中可以發現,rca在綠豆中為單套基因;利用北方雜合分析及西方雜合分析方法(Northern hybridization and Western blot analysis)分析rca基因的表現情形,在28℃常溫及16小時光照的光週期下,發現其mRNA的表現受到概日韻律之調控;且蛋白質的累積量亦是隨著mRNA表現量上升而增加;而綠豆rca基因概日韻律之控制,在10℃的低溫逆境下即發生改變;在相同的光週期下,除了表現量逐漸下降之外,概日韻律的週期亦逐漸變長,失去一定的規則。但低溫並不會影響其蛋白質生成的轉譯作用,RCA蛋白質之累積即使在低溫下亦隨著其mRNA表現量的增減而隨之改變。
確定低溫下rca基因的確是在轉錄作用受到抑制後,為進一步瞭解此基因在低溫下被調控的可能機制,故利用genome walking的方式選殖出一段位於綠豆rca啟動子區域,長1,231 bp之DNA片段;經序列比對後,發現數種可能調控基因表現的區域,分別為:光誘導、組織專一性表現、概日韻律調控、醣類回饋抑制、缺水及ABA誘導等五類可能存在之調控區。但實驗證明的結果,在ABA及缺水逆境處理下的綠豆幼苗植株,其rca基因並未因上述處理而有表現量大增之情形。
The mungbean (Vigna radiata L. v. 2937) is a chilling-sensitive plant. A cDNA library was constructed from poly(A)+ mRNAs of 28℃-light-grown leaves for screening cold-suppressed nuclear genes. One of the 13 cold-suppressed nuclear genes obtained by differential screening was rubisco activase (rca) gene. Southern blot analysis suggested the presence of a single rca gene in the mungbean genome. The expression of leaf rca mRNA was found to be regulated by circadian rhythm under 28℃ and 16L/8D photoperiod. In addition, the accumulation of RCA protein was positively propotional to the rca mRNA expression level. However, the rhythm in mungbean leaves was interrupted by 10℃ chilling treatments. Under the 16L/8D photoperiod, the rca mRNA expression was not only mistiming, but depressed by chilling stress. Even so, the RCA protein was translated as long as the mRNA existed during the 10℃ chilling treatment.
The promoter region (1,231 bp) of rca gene was cloned by the genome walking method. Six possible regulatory motifs were found in this cis-acting regulatory DNA element. They included light-inducible, tissue-specific, circadian control, sugar repression, sugar starvation, ABA and water stress response elements. But the significant increase of rca gene expression was found in the treatments of ABA and water stress.
第一章 前言------------7
第二章 材料與方法-----16
第三章 結果-----------35
第四章 討論-----------41
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