博碩士論文 101224009 詳細資訊




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姓名 許藝儒(Yi-Ju HSU)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 阿拉伯芥HIT4為不同於MOM1的新調節方式調控熱誘導染色質重組並在各個植物生長發育轉換時期表現
(Arabidopsis HIT4 is a MOM1-independent regulator which acts on heat-induced reorganization of chromatin and expressed in developmental transitions)
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摘要(中) 為了適應環境變化以生存,植物需演化出相對應的細胞生理調節機制。熱逆境是植物需面臨的環境因子之一。本實驗室先前以正向式遺傳研究法,從ethyl-methane sulfonate (EMS)處理之阿拉伯芥種子中,篩選出一個對熱逆境敏感的突變植株,命名為heat-intolerant 4-1 (hit4-1)。後續研究發現,HIT4蛋白質的功能,在於調節熱逆境所誘導的大規模染色質構型重組,並使原本轉錄靜默(transcriptional gene silencing, TGS)的基因座再活化。是以hit4突變,熱誘導活化TGS會被抑制。目前已知,熱誘導活化TGS的現象,不是經由DNA序列甲基化程度的改變而引起。而阿拉伯芥morpheus’ molecule 1 (mom1) 突變株,則能在不改變DNA甲基化程度的前提下,釋放特定原本靜默的TGS基因。所以,同樣是無涉於DNA甲基化,但MOM1帶有TGS維持者的角色,HIT4則是在特殊狀態下,推動釋放TGS。為了解HIT4和MOM1的關係,本論文使用hit4-1/mom1雙突變株,與hit4-1及mom1進行比較。結果發現,MOM1與HIT4,是經由兩條不同的路徑,來調節TGS。為進一步瞭解HIT4如何調節熱誘導染色質中心消散,吾人觀察HIT4-GFP轉植株,發現HIT4蛋白質於細胞受熱後,在染色質中心尚未消散之前,即從染色質中心轉移到核仁。熱逆境結束後,HIT4則在染色質中心重新形成後,再從核仁回到染色質中心。此外hit4-1突變蛋白質,在細胞受熱後,保有從染色質中心轉移到核仁的能力。此結果表示,HIT4正確的轉移,不足以促使熱誘導染色質中心消散,並暗示有其他分子的參與。此外,吾人的觀察也指出,HIT4基因,在種子的胚胎發育、種子萌芽、幼苗發育與雄配子體時期,有較高的表現。合理推測HIT4在這些時期,可能也扮演促進大規模染色質構型重組,以利不同發育階段所需不同基因的表現。
摘要(英) Plants have evolved various mechanisms to cope with adverse high temperature stress. To understand these mechanisms and identify the underlying genetic determinants, we used a forward genetic approach to screen for ethyl-methane sulfonate mutagenized Arabidopsis mutants that are more thermosensitive than wild type. One of these mutants, hit4-1 (for heat intolerant 4-1) was therefore isolated. Previous research showed that hit4-1 mutation restricted heat-induced large-scale reorganization of chromatin and release of transcriptional gene silencing (TGS) in a DNA methylation independent manner. On the other hand, Arabidopsis morpheus’ molecule 1 (mom1) mutant is known to promote the release of specific TGS loci without alternation in the level of DNA methylation. To understand the relationship between the function of HIT4 and MOM1 in TGS regulation, hit4-1/mom1 double mutant was made and its TGS status was compared to those of hit4-1 and mom1. The results showed that HIT4 controls a DNA methylation-independent TGS regulatory pathway which is different from that controlled by MOM1. Meanwhile, to understand the regulatory function of HIT4 in heat-induced decondensation of chromocenter, HIT4-GFP transgenic plant was utilized to track the fate of HIT4 under heat stress treatment. Results showed that HIT4 relocated from chromocenter to nucleolus before decondensation of chromocenter occurred. Besides, hit4-1 mutant protein maintained the ability to relocate from chromocenter to nucleolus under heat stress treatment, implying that correct relocalization of HIT4 alone was insufficient to promote heat-induced decondensation of chromocenters. Additionally, HIT4 promoter driven GUS reporter gene analysis revealed that HIT4 was highly expressed in male gametophyte and during the stages of seed maturation, seed germination and post-germinative seedling growth. It is plausible to postulate that HIT4 may involve in large-scale chromatin reconfiguration for genes that are required for these developmental stages to be expressed.
關鍵字(中) ★ 阿拉伯芥
★ 熱逆境
★ 染色質中心消散
★ 轉錄靜默
關鍵字(英) ★ Arabidopsis thaliana
★ heat stress
★ HEAT-INTOLERANT 4 (HIT4)
★ MORPHEUS’ MOLECULE 1 (MOM1)
★ chromocenter decondensation
★ transcriptional gene silencing
★ reorganization of chromatin
論文目次 摘要 I
Abstract II
致謝 III
總目錄 IV
圖目錄 VII
表目錄 VIII
一、緒  論 1
1-1 研究背景與緣起 1
1-2 研究目的與內容 4
二、研究內容與方法 6
2-1 實驗材料製備 6
2-1-1 植物材料來源 6
2-1-2 植物的生長條件 6
2-1-3 螢光轉植株製備 7
2-1-4 構築原生質體轉殖用之質體 9
2-1-5 pDW137- HIT4 promoter轉植株的製備 9
2-2 實驗方法 10
2-2-1 植物的熱逆境條件 10
2-2-2 Quantitative real-time PCR (qPCR)的基因表現分析 11
2-2-3 螢光染劑DAPI (4’, 6-diamidino-2-phenylindole) 染色 12
2-2-4 阿拉伯芥原生質體製備及轉殖 12
2-2-5 報導基因GUS (β-glucuronidase) 染色實驗 13
三、結  果 14
3-1 野生型、hit4-1、mom1、hit4-1/mom1植株熱逆境處理後性狀之觀察 14
3-1-1 熱誘導TGS loci的活化情形 14
3-1-2 觀察熱逆境下的染色質中心 15
3-1-3 先天耐熱性測試 16
3-2 處理不同熱逆境觀察HIT4在細胞內的位置 16
3-2-1 長時間熱逆境 16
3-2-2 熱休克逆境 17
3-2-3 HIT4-GFP進行熱逆境處理時位在核仁 17
3-2-4 突變蛋白hit4-1-GFP與正常蛋白HIT4-GFP加熱時的比較 17
3-3 HIT4在不同熱逆境處理完後的位置及染色質中心回復情形 18
3-3-1 長時間熱逆境加熱12小時後回到室溫生長的實驗結果 18
3-3-2 熱休克逆境加熱15分鐘後回到室溫生長的實驗結果 19
3-3-3 不同長時間熱逆境加熱後回到室溫3天的結果 19
3-3-4 不同熱休克逆境加熱後回到室溫3天的結果 20
3-4 HIT4在開花時期與花粉管內的表現 20
3-5 HIT4在種子發育、成熟、萌芽及幼苗生長階段的表現 21
四、討  論 23
4-1 MOM1與HIT4性狀探討及功能的比較 23
4-2 HIT4在細胞內扮演的角色與蛋白質之功能探討 25
4-3 HIT4在生長階段的各個轉換有所表現 27
4-4 結語 30
五、參考文獻 31
六、附  錄 53
附錄一、hit4-1與hit1-1、hit2、hsp101耐熱性之比較。 53
附錄二、mCherry-HIT4及MOM1-GFP共同轉殖到原生質體之結果 54
附錄三、處理後天耐熱性之熱逆境時染色質中心的消散與HIT4在細胞內的位置 55
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指導教授 吳少傑(Shaw-Jye WU) 審核日期 2015-12-22
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