阿拉伯芥古氏-蛋白質基因啟動子之酵母菌單雜交實驗

Yeast one-hybrid screening of the Ku promoters in Arabidopsis thaliana.

林其鍇(Qi-Kai Lin)

指導教授 ： 潘榮隆

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2007年)

摘要

古氏-蛋白質是早先發現於自體免疫缺失病人血清中的一種自體抗原。結構含一個質量為70千道頓與一個質量為80千道頓的蛋白質單元次體，以非共價鍵結合而組成異型雙次體。古氏-蛋白質以此種異型雙次體的型態，與去氧核苷酸的末端、斷裂處或是單股轉換到雙股之過度型等結合，並在去氧核苷酸之雙股斷裂的修復上扮演了關鍵性的角色。2002年在高等植物阿拉伯芥中，發現並分離了古氏-蛋白質的同源蛋白。其單元次體(70千道頓古氏-蛋白質次體和80千道頓古氏-蛋白質次體)與其在人類個體中的同源蛋白質相比較，彼此分子量大小接近，而序列相似性則分別為28.6% 和22.5%。相關的研究指出在去氧核甘酸破壞劑的刺激下，阿拉伯芥古氏-蛋白質扮演了以非同源雙股末端結合途徑，修復去氧核甘酸雙股斷裂的角色。阿拉伯芥古氏-蛋白質在植物中的此類功用，與其同源蛋白質在其他物種中的功用非常相似。如果失去了古氏-蛋白質，則會造成染色體末端長度控制的失調。
在先前古氏-蛋白質基因序列上游啟動子的研究中發現，古氏蛋白質可由植物賀爾蒙和逆境因子所調控。這些內在型和外來型的訊號，有的會激發古氏蛋白質的表現，其他的則會減弱其表現。然而至目前為止，對於古氏蛋白在植物中的調控機制，仍然所知有限。因此在這個研究中，我們嘗試以酵母菌單雜合檢測來探究其分子層次的調控機制。一個被稱作ATAF1的蛋白質，在這次的檢測中被分離出來。已知ATAF1蛋白質會被乾燥和離層酸所誘導。而ATAF1蛋白質作為轉錄調控因子能抑制其他逆境反應基因的表現。綜合ATAF1蛋白質在基因轉錄上的抑制性調控和先前植物賀爾蒙與逆境因子對古氏-蛋白質的影響，我們推測ATAF1蛋白質可能在植物賀爾蒙與逆境因子處理下，扮演基因轉錄的抑制者而減弱古氏-蛋白質的表現。

關鍵字

酵母菌單雜交實驗 ； 阿拉伯芥 ； 古氏-蛋白質 ； 基因啟動子

並列摘要

Ku was first identified as an autoantigen in the sera of patients with autoimmune diseases, as a heterodimer composed of subunits Ku70 and Ku80 that binds to the DNA ends, nicks, or single-to-double-strand transition. Ku plays a crucial role for DNA double-strand-break repair. Homologous of Ku70 and Ku80 in Arabidopsis were identified in 2002, with conserved sizes sharing 28.6 and 22.5% sequence identity with the respective human proteins. Further studies indicated that AtKu plays a similar role in the repair of DNA double-strand breaks by non-homologous end joining (NHEJ) pathway in response to DNA damaging agents in plants as in other organisms. Loss of AtKu results in deregulation of telomere length control.
The previous studies on AtKu promoter activity under treatment of plant hormones and stress indicated that AtKu is regulated by various endogenous and exogenous factors. Some of these signals activate and others inhibit the expression of AtKu. Up to now, the mechanism of the regulation of AtKu in plants still is limited to know.
In this study, we prompted to investigate the molecular mechanism of these processes by yeast one-hybrid screening. A cDNA encoding ATAF1 protein was identified. ATAF1 was reported to be induced by dehydration and ABA, as a transcriptional regulator, to negatively regulate the expression of the other stress responsive genes. To combine results on hormones and stress stimulation with that on the repressor activity of ATAF1 on transcriptional regulation, we speculate that ATAF1 may function as a transcriptional repressor to down regulate the expression of the AtKu genes.

並列關鍵字

yeast one-hybrid ； Arabidopsis thaliana ； Ku protein ； promoter

參考文獻

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