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研究生:江志文
研究生(外文):Jung,zu-wen
論文名稱:陽明山高溫紅藻Cyanidiumcaldarium熱休克蛋白質之研究
論文名稱(外文):The studies on heat shock proteins in Cyanidium caldarium
指導教授:陳益明陳益明引用關係
指導教授(外文):Chen,Yih-ming
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:62
中文關鍵詞:熱休克蛋白質紅藻陽明山高溫紅藻
外文關鍵詞:Cyanidium caldariumheat shock proteinalgas
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陽明山高溫紅藻Cyanidium caldarium生長在高溫約50℃,pH值為2的極端環境中,經由穿透式電子顯微鏡切片觀察,它是一種構造相當原始的真核生物,其細胞大小直徑約4 μm內含一個細胞核,一個大型葉綠體,多個粒線體。
將室溫 (28℃) 培養以及熱休克48℃處理的紅藻分別以35S-methionine標定蛋白質,經SDS聚丙醯胺膠體電泳及自動放射顯影,結果發現經熱休克處理後在97 kDa、60 kDa、39 kDa、36 kDa、32 kDa、23 kDa、22 kDa、16 kDa及15 kDa蛋白質有合成累積的情形。用大腸桿菌GroEL、酵母菌HSP60蛋白質及大豆第一族低分子量熱休克蛋白質抗體對紅藻蛋白質作進一步的分析,從免疫轉印分析的結果來看,紅藻的蛋白質不會辨認大腸桿菌GroEL蛋白質及大豆第一族低分子量熱休克蛋白質,而紅藻的60 kDa蛋白質與酵母菌HSP60蛋白質有交叉反應,而且產量會隨熱休克而累積。可見紅藻與較原始真核生物酵母菌有較高的親源性,和原核生物的大腸桿菌及高等植物大豆較疏遠。
把紅藻的genomic DNA分別用BamHInHimdⅢ水解,以酵母菌HSP60的cDNA做為探針,進行南方雜合分析,分別在BamHI水解的DNA偵測到2條雜合帶,長度大小分別是2.1及1.7 kb,將其標示為B2.1及B1.7,在HindⅢ水解的DNA中則偵測到1條雜合帶,片段長度是2.7kb,標示為H2.7,由subgenomic libraries中篩選到各對應的clones。

Cyanidium caldarium live in extreme environments about 50℃ and pH 2. It's a primitive eucaryote from observation using transmission-electron microscope. The alga has a nucleus and a big chloroplast which cell diameter is about 4 μm.
The algas are treated at 48℃ as heat shock and those are cultured at room temperature as control, using 35S-methionine to label their proteins. After running SDS-polyacrylamide gel electrophoresis and autoradiography, some proteins are synthesized and accumulated. Their molucular weight are 97 KDa, 60 KDa, 39 Kda, 36 KDa, 32 KDa, 23 KDa, 22 KDa, 16 KDa and 15 KDa. We use E. coli GroEL antibody, yeast HSP60 antibody and soybean class I low-molecular-weight heat shock proteins antibody to detect this alga's proteins. From result on immunoblotting, the red alga's proteins don't react with E. coli GroEL antibody and soybean class I LMW antibody, but yeast HSP60 antibody have cross-reaction with C. caldarium, The reactive proteins amount accumulate as increasing period of heat shock treatment. So C. caldarium have high homologous with yeast than with E. coli or soybean.
After extracting C. caldarium genomic DNA and digested by BamHI and HindIII, we use yeast hsp60 cDNA fragment as probe for Southern hybridization. There are two hybrid bands in DNA, digested by BamHI, and their size are 2.1and 1.7 Kb, marked as B2.1 and B1.7. Only one hybrid band appear in DNA digested by HindIII, fragment length is 2.7 Kb, marked as H2.7.

中文摘要 ooooooooooooooooooooooo 1
英文摘要 ooooooooooooooooooooooo 2
縮寫表 oooooooooooooooooooooooo 3
第一章 前言 oooooooooooooooooooo 4
1. Cyanidium caldarium 簡介 ooooooooooo 4
2. 熱休克反應 ooooooooooooooooooo5
3. 熱休克蛋白質的種類與功能 oooooooooooo7
第二章 材料與方法 ooooooooooooooooo13
1. 實驗材料 ooooooooooooooooooo13
2. 紅藻液體培養 ooooooooooooooooo13
3. 熱休克處理 oooooooooooooooooo14
4. 放射性同位素標定蛋白質 oooooooooooo15
5. 蛋白質萃取法 ooooooooooooooooo16
6. 蛋白質定量 (Bradford assay) ooooooooo 17
7. 蛋白質樣品放射性同位素強度測定oooooooo 17
8. 蛋白質 SDS-聚丙烯醯胺膠體電泳 (SDS-PAGE) ooo18
9. 放射自動顯像 (autoradiography) oooooooo20
10. 免疫轉印分析 (Immunoblotting) ooooooooo21
11. 萃取總 RNA ooooooooooooooooo 23
12. RNA 洋菜膠體電泳ooooooooooooooo24
13. 北方氏雜合分析 (Northern hybridization) ooo26
14. 紅藻總 DNA的抽取oooooooooooooo 26
15. DNA 膠體電泳分析ooooooooooooooo27
16. 南方氏雜合反應 (Southern hybridization) ooo28
第三章 結果 oooooooooooooooooooo 31
1. 從電子顯微鏡切片觀察陽明山高溫紅藻之細胞型態oo 31
2. Cyanidium caldarium 熱休克處理,蛋白質的變化分析32
2.1 放射性同位素標定oooooooooooooo 32
2.2 蛋白質免疫轉印分析 (Western blot) ooooo 32
3. Cyanidium caldarium 熱休克處理,RNA變化分析
(Northern blot) oooooooooooooooo34
4. 以 Yeast HSP60cDNA 為探針,篩選Cyanidium
caldarium 熱休克蛋白質ooooooooooooo35
第四章 討論 oooooooooooooooooooo 36
參考文獻 ooooooooooooooooooooooo41
圖表與說明 oooooooooooooooooooooo49

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