Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90095
標題: Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis
阿拉伯芥基因CTF7/ECO1修復DNA有絲分裂和減數分裂的作用
作者: 何伯樂
Pablo Alberto Bolanos Villegas
關鍵字: 有絲分裂
減數分裂
染色分體
mitosis, meiosis, chromosomes
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摘要: 細胞分裂時如何將遺傳訊息-DNA妥切及完整地傳遞給後代子細胞,對於真核生物的存活及繁衍十分重要。當細胞分裂時,被複製的染色分體(姊妹染色體)需要緊緊相連在一起,之後此染色分體會被忠實地分離,直到細胞分裂後期。已知在酵母菌中,姊妹染色體的凝聚發生在S期且需要Establishment of cohesion 1 (Eco1) 蛋白質的參與。 Eco1失去 正常的功能, 會導致正在分裂中細胞內的同源染色體分離失敗, 與使得以同源互換為主 的DNA 修復失去功能,進而導致細胞凋亡。 之前研究室中利用生物資訊學的方式從 阿拉伯芥T-DNA插入突變株資料庫中獲得許多無法產生具同源後代(non-homozygous progeny) 的突變株,本篇研究中,我們針對其中一個類似Eco1 的基因- CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION (CTF7/ECO1) 上的兩株 阿拉伯芥T-DNA插入突變株,我們將之命名為ctf7-1和ctf7-2,並分別利 用細胞學、 遺傳學及分子生物學針對該基因的分子功能及作用機制進行一系列研究。外表型觀察顯示ctf7-1和ctf7-2突變株呈現植株極度矮小、 花藥發育不良及不孕的現象。 利用顯微鏡 觀察 發現突變株花藥內的花粉發育缺失是導致無法產生同源後代的主因。藉由流式細胞儀分析突變株的體細胞後發現營養細胞內多倍套數DNA含量增加;掃描式電子顯微鏡觀察亦發現葉片表皮細胞的表面積顯著增加。進一步利用QPCR分析與細胞分裂及DNA修復相關的基因表現,發現一些與DNA修復相關的基因(例如BRCA1和PARP2)在突變株的營養細胞內表現量顯著增加,但一些與進入endocycle相關基因的表現量則無明顯變化。進一步用 QPCR偵測突變株營養組織中的基因表現,發現跳躍基因COPIA28和soloLTR的表現量增加,推測CTF7/ECO1可能參與基因靜默的調控。綜合以上現象推斷,ctf7-1和ctf7-2突變 株 的 養細胞失去DNA修復及細胞分裂的能力。 對於突變株中花粉母細胞的減數分裂 分析 發現 ,整個染色體的結構發生改變且染色體分離 在細胞分裂過程中發生嚴重缺陷; 除此之外,染色體中心粒凝聚的現象和凝聚於中心粒的染色體接合次單元數皆有減少之趨勢。利用QPCR分析突變株花粉內參與減數分裂相關基因,發現DMC1、RAD51C以及S-phase licensing factor-CDC45 基因的表現量皆顯著增加。 總而言之,本實驗結果證明了 阿拉伯芥 CTF7/ECO1對於維持染色體的完整性及在剪數分裂上扮演很重要的角色。
Proper transmission of DNA in dividing cells is crucial for the survival of eukaryotic organisms. During cell division, faithful segregation of replicated chromosomes requires their tight attachment, known as sister chromatid cohesion, until anaphase. Sister chromatid cohesion is established during S-phase in a process that requires an acetyltransferase that in yeast is known as Establishment of cohesion 1 (Eco1). Inactivation of Eco1 typically disrupts chromosome segregation and homologous recombination dependent DNA repair in dividing cells ultimately resulting in lethality. We report here the isolation and detailed characterization of two homozygous T-DNA insertion mutants for the Arabidopsis thaliana Eco1 homologue, CHROMOSOME TRANSMISSION FIDELITY 7/ ESTABLISHMENT OF COHESION 1 (CTF7/ECO1), called ctf7-1 and ctf7-2. Mutants exhibited dwarfism, poor anther development and sterility. Analysis of somatic tissues by flow cytometry, scanning electron microscopy and QPCR identified defects in DNA repair and cell division, including an increase in the area of leaf epidermal cells, an increase in DNA content, and the upregulation of genes involved in DNA repair including BRCA1 and PARP2. No significant change was observed in the expression of genes that influence entry into the endocycle. Analysis of meiocytes identified changes in chromosome morphology and defective segregation; reduced cohesion at centromeres and a reduction in the abundance of chromosomal-bound cohesion subunits. Transcript levels for several meiotic genes, including the recombinase genes, DMC1 and RAD51C, and the S-phase licensing factor, CDC45 were elevated in mutant anthers. Moreover QPCR in vegetative tissues indicated upregulation of transposable elements COPIA28 and soloLTR, which suggest an additional role for CTF7/ECO1 in gene silencing. Taken together our results demonstrate that Arabidopsis CTF7/ECO1 plays important roles in preservation of genome integrity and meiosis.
URI: http://hdl.handle.net/11455/90095
文章公開時間: 2017-01-22
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