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標題: | 陽明山高溫紅藻Cyanidium caldarium熱休克蛋白質之研究 The studies on heat shock proteins in Cyanidium caldarium |
作者: | 江志文 |
出版年 : | 1999 |
學位: | 碩士 |
摘要: | 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4μm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97kDa、60kDa、39kDa、36kDa、32kDa、23kDa、22kDa、16kDa及15kDa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60kDa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1.7kb,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 2.1 and 1.7 Kb, marked as B2.1 and B 1.7. Only one hybrid band appear in DNA digested by HindIII, fragment length is 2.7 Kb, marked as H2.7. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75038 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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