博碩士論文 962204016 詳細資訊

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姓名 林冠甫(Kuan-Fu Lin)  查詢紙本館藏   畢業系所 生命科學系
(Structure-function relationship of a rice small heat shock protein OsHsp16.9A)
★ 水稻第三群LEA基因在發育時期與逆境表現之探討★ 第三群LEA蛋白質表現與功能分析
★ 水稻小分子量熱休克蛋白質Oshsp16.9A之N端區域功能性分析★ 植物逆境蛋白質基因啟動子與功能分析
★ 植物受溫度調控之基因的功能與機制分析★ 錯誤褶疊蛋白質誘導之擬熱休克反應機制之探討
★ 受熱與ABA調控水稻基因-OsRZFP1之生理功能分析★ 受熱與ABA調控基因AtRZFP33之生理功能分析
★ 水稻第一族小分子量熱休克蛋白質OsHSP16.9A及OsHSP18.0之生理功能分析★ 植化物紫草素在小鼠皮膚上增加血管通透性之研究
★ 蝴蝶蘭開花相關基因PaCOL2啟動子之特性分析★ 利用水稻HSP17.3啟動子探討阿拉伯芥熱休克因子在逆境下對細胞內蛋白質反應之角色分析
★ 蝴蝶蘭開花相關基因PaCOL1 啟動子之特性分析★ 分析水稻 RING 鋅手指蛋白質 OsRZFP34 與其正向調控蛋白質之交互作用
★ 水稻小分子量熱休克蛋白質- OsHSP16.9A在水稻種子耐熱性之功能分析
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摘要(中) 小分子量熱休克蛋白質可以透過與變性蛋白質結合,避免其在細胞內沈澱而傷害細胞活性,為細胞維持恆定所不可或缺。水稻內有一群在種子發育時期大量累積的小分子量熱休克蛋白質,其中表現量最多的為OsHsp16.9A,過去研究指出OsHsp16.9A可以幫助水稻抵抗熱逆境。OsHsp16.9A的胺基酸序列與小麥內同源蛋白質TaHsp16.9蛋白質胺基酸序列高度相似,卻會形成截然不同的聚合體構型。為了釐清小分子量熱休克蛋白質形成不同構型聚合體的機制,我們利用冷凍電子顯微鏡與小角度X光散射建構OsHsp16.9A聚合體的低解析度結構,並搭配圓二色光譜、蛋白質—受體凝集試驗與濁度試驗來探究N端的功能。我們發現除了N端上不同胺基酸的電荷與疏水性分佈會影響其形成不同聚合體外,N端的長度也是影響聚合體構型一個很重要的因素。刪除N端的OsHsp16.9A除了會喪失伴護功能外,也會影響聚合體的完整度。我們意外地發現OsHsp16.9A可以耐受高達攝氏95度的高溫,並且在溫度降低後維持原有的二級結構。以上發現都表示OsHsp16.9A為一俱有伴護功能的蛋白質,並且在水稻對熱的耐受性上扮演重要的角色。
摘要(英) Small heat shock proteins (sHsps) maintain the protein homeostasis by interacting with the unfolded proteins, preventing them from aggregation under stress conditions. We took an interest in OsHsp16.9A, a cytosolic sHsps crucial for thermo-resistance in rice, which shares similar amino acid sequences with a known structure, wheat TaHsp16.9, but with different N-terminal region. To bring a scheme of the structure-function relationship of OsHsp16.9A and clarify the N-terminal sequence difference, we used cryo-EM and small-angle X-ray scattering to generate a structural model and combined with circular dichroism, aggregation, and chaperone activity assay to further investigate the N-terminal effect. Our data suggest that only ten amino acids deleted from N-terminal of OsHsp16.9A would lead to severe loss of chaperone activity. Longer N-terminal region truncation results in the aggregation itself. Furthermore
, the N-terminal region also affects the thermal sustainability of OsHsp16.9A. The structural rigidity of intact OsHsp16.9A is higher than the N-terminal truncated variants. The secondary structure of intact OsHsp16.9A can even remain unchanged after the incubation at 95 °C. It is revealed that OsHsp16.9A is a thermal stable chaperone protein and N-terminal region also play a vital role in the thermostability.
關鍵字(中) ★ 小分子量熱休克蛋白質
★ 低溫電子顯微鏡
★ 小角度散射
★ X光晶體繞射學
★ 水稻
★ 伴護功能
關鍵字(英) ★ Small heat shock protein
★ Cryo-EM
★ X-ray crystallography
★ Rice
★ Chaperone
論文目次 中文摘要 i
Abstract ii
Acknowledgments iii
Table of Contents iv
List of Figures v
Introduction 1
Material and Methods 6
Cloning, Expression, and Purification of OsHsp16.9A oligomer 6
Aggregation assay and chaperone activity assay 7
Negative EM and Cryo-EM 7
Single Particle Reconstruction 8
Small-angle X-ray Scattering (SAXS) 9
Circular Dichroism (CD) Spectrum 9
Results 11
OsHsp16.9A is a dodecamer 11
Three-dimensional reconstruction of OsHsp16.9A 11
The N-terminal regions are important to chaperone function and oligomerization 14
Thermal durability of OsHsp16.9A variants 17
Discussion 20
OsHsp16.9A forms a tetrahedron-like dodecamer 20
Effect of N-terminal region of OsHsp16.9A 22
Conclusion 25
Bibliographies 39
Appendices 47
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指導教授 葉靖輝 審核日期 2019-1-24
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