阿拉伯芥囊泡繫鏈因子HIT1在逆境下維持內膜完整性之探討與ret8之基因定位

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范育珊(Yu-Shan Fan) 查詢紙本館藏

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生命科學系

論文名稱

阿拉伯芥囊泡繫鏈因子HIT1在逆境下維持內膜完整性之探討與ret8之基因定位
(Involvement of Arabidopsis HIT1 in the preservation of endomembrane integrity under stress conditions and the mapping of Arabidopsis ret8 locus)

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摘要(中)

hit1-1 (heat intolerant 1-1) 是對熱具過度敏感性的阿拉伯芥突變株，HIT1蛋白質經過序列比對分析發現，與酵母菌的Vps53p為同源性蛋白質，在酵母菌中Vps53p會與Vps51p、Vps52p和Vps54p共同形成一個囊泡繫鏈複合體，稱為Golgi-associated retrograde protein (GARP) complex，負責調節從內膜體逆行回高基氏體的囊泡運輸，而在阿拉伯芥中，HIT1也會與AtVPS52及AtVPS54形成囊泡繫鏈複合體。因此當HIT1突變後，造成植物對熱的過度敏感性，可能是由於缺乏了有效的囊泡運輸，致使hit1-1突變株面臨高溫環境時，無法及時修補或替換膜組成，抑或是HIT1的突變，使得細胞膜本身的組成就已被改變，造成對熱的不耐受性。繫鏈因子的功能對於囊泡運輸的過程非常重要，所以繫鏈因子的突變，除了會影響細胞膜之外，也可能會影響其他內膜系統，因此，在阿拉伯芥中表達了幾個胞器的螢光標記：endoplasmic reticulum (ER)、Golgi及vacuole接黃螢光蛋白 (YFP)；plasma membrane (PM) 接櫻桃色螢光蛋白 (mCherry) 等，利用這些螢光轉殖株來進行實驗及螢光顯微鏡的觀察。
乙烯是常見的植物賀爾蒙，參與調控植物的生長發育和抵抗環境逆境的機制。為了更詳細了解與乙烯相關的生理反應和調控機制，利用化學遺傳法，使用小分子化合物7303干擾乙烯的反應，其主要的結構為喹唑啉酮，可以有效的抑制ethylene overproducer 1 (eto1) 白化苗的三相反應，因此，利用這個特性來挑選突變株，而突變株的外表型還可以像eto1一樣具有三相反應，所以挑選出來的突變株被命名為revert to eto1 (ret)，在本篇論文中，利用map-based cloning (MBC) 的方法，對挑選出的其中一株突變株revert to eto1 8 (ret8) 進行基因定位的工作，而ret8突變基因在AGI map第五條染色體25870.8 kb~25906.5 kb之間，35.7 kb的範圍內。

摘要(英)

Arabidopsis hit1-1 is a heat-intolerant mutant. The HIT1 gene was found to encode a protein with homology to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex. The tethering complex includes Vps51p, Vps52p and Vps54p, that is involved in retrograde trafficking of vesicles to the Golgi. hit1-1 is hypersensitivity to heat stress may be due to the loss plasma membrane integrity caused by ineffective vesicle recycling. When a tethering factor lost the function not only plasma membrane but also endomembrane may be affected. To test this hypothesis, various transgenic Arabidopsis lines expressing YFP-tagged reporter of the Golgi, endoplasmic reticulum (ER), vacuole and mCherry-tagged reporter of plasma membrane (PM) were generated. These transgenic plants allow us to monitor and compare morphological changes of various endomembrane compartments in both wild type and hit1-1 mutant.
Ethylene is a gaseous phytohormone. It is involved in regulation of many aspects of plant growth and development. To gain more understanding about ethylene biology, we used small molecules (hit compounds) to screen Arabidopsis mutants, named revert to etol (ret), with restoration of the ethylene overproducer 1 (eto1) phenotype (triple response) in the presence of hit compounds. One of the ret mutants, ret8, which was identified from Arabidopsis after ethyl-methane sulfonate (EMS) –mutagenesis. To identify the gene underlying the ret8 phenotype, map-based cloning of RET8 gene is being conducted.

關鍵字(中)

★ 囊泡繫鏈因子
★ 內膜系統
★ 基因定位
★ 阿拉伯芥

關鍵字(英)

★ Vesicle tethering factor
★ map-based cloning
★ Endomembrane
★ Arabidopsis thaliana

論文目次

中文摘要 I
ABSTRACT II
目錄 III
圖表目錄 V
前言 1
一、囊泡繫鏈因子HIT1在逆境下參與維持內膜完整性之探討 1
二、阿拉伯芥ret8之基因定位 6
材料與方法 9
一、囊泡繫鏈因子HIT1在逆境下參與維持內膜完整性之探討 9
1. 植物材料及生長方式 9
1-1. 植物材料 9
1-2. 植物生長培養基 9
1-3. 種子消毒與生長環境 9
2. 建立organelle markers轉基因之植株 9
2-1. 農桿菌勝任細胞之製備 9
2-2. 農桿菌之轉型 10
2-3. 阿拉伯芥轉殖與篩選 11
2-4. 建立hit1-1螢光轉殖株 12
2-5. 雙螢光轉殖株 13
3. 測試熱處理後hit1-1之內膜系統有無受到影響 13
3-1. 幼苗 (Seeding) 13
3-2. 原生質體 (protoplast) 14
4. NaCl對於hit1-1生長表現型之影響測試 16
4-1. 幼苗成熟率 (Seeding maturation) 16
4-2. 根長測量 16
5. 利用質壁分離 (plasmolysis) 的方式觀察Col與hit1-1的細胞膜有無差異 16
二、阿拉伯芥ret8之基因定位 17
1. 植物材料及生長方式 17
1-1. 植物材料 17
1-2. 植物生長培養基 17
1-3. 種子消毒與生長環境 17
2. ret8突變種之位址定位 18
2-1. 挑選定位之突變株 18
2-2. 阿拉伯芥基因體之萃取方法 18
2-3. 以分子標記 (DNA molecular markers) 定位 19
結果 22
一、囊泡繫鏈因子HIT1在逆境下對內膜系統之影響 22
二、阿拉伯芥ret8之基因定位 26
討論 28
一、囊泡繫鏈因子HIT1在逆境下對內膜系統之影響 28
二、ret8之突變基因為CESA6 31
參考文獻 33

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指導教授

吳少傑(Shaw-Jye Wu)

審核日期

2012-1-30

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