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標題: | 綠豆幼苗葉片Rubisco Activase基因在低溫逆境下表現之研究 Expression of Rubisco Activase Gene in Leaves of Mungbean Seedlings under Chilling Stress |
作者: | 陳志德 |
出版年 : | 2002 |
學位: | 碩士 |
摘要: | 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元於綠豆rca啟動子區域,長1,231bp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 nterrupted 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75311 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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