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標題: | 陽明山高溫紅藻Cyanidium caldarium的化學鑑定及耐熱生理之探討 The Chemotaxnomy and the Studies on Thermal Physiology of Rhodophyta Cyanidium caldarium |
作者: | 阮雪芬 |
出版年 : | 1994 |
學位: | 碩士 |
摘要: | 藍球藻是一種單細胞嗜酸熱性的?核藻。分析其色素成分,含葉綠素a,胡蘿蔔素,藻藍素(phycocyanin),初步判斷是一種過渡型的藻類。 本實驗改良藍球藻的培養條件,使其在短時間內達到靜止期(stationary phase),以利它生理,生化實驗的進行。 利用HPLC分析藍球藻多元胺的含量,結果發現其多元胺分佈類型與Cyanidium caldarium strain KUSATSU相似,故暫時將此藻命名?Cyanidium caldarium。同時也利用HPLC分析,比較以高溫處理過藍球藻多元胺含量與室溫下培養者的差異。putrescine、norspermidine和norspermine明顯升高,推測此三種多元胺在熱保護上扮演重要角色。 此外,也進行熱休克蛋白質(heat shock proteins)的分析。利用單向電泳分析,發現有三種蛋白質隨溫度升高而量增加,其分子量分別?70KD,60KD,30KD,它們對藍球藻能生活在高溫下有貢獻。藍球藻經28℃,48℃處理後以0-50%,50-70%的硫酸銨分劃沉澱法抽取蛋白質,以雙向電泳來分析(一方向是SDS-PAGE,另一方向是IEF膠體)。經48℃處理過的藍球藻,有些蛋白質的量增加,其中包括了HSP60的兩個不同pI值的蛋白質。這些蛋白質量的增加可能是用來保護藻細胞,避免其受熱傷害。藉由免疫轉印分析(immunoblot)的技術配合Densitometer掃描定量,得知藍球藻60KD的蛋白質與酵母菌HSP60同源性很高,與大腸桿菌(E. coli)GroEL也有同源性,但同源性較低。藍球藻LMW的蛋白質與水稻LMW HSP1沒有交叉反應(cross reaction),由此顯示藍球藻在演化上的地位可能較近於酵母菌,而與高等植物相差較遠。以40℃處理過的藍球藻HSP60?28℃下培養之藍球藻HSP60的1.1倍,而以48℃處理過的藍球藻HSP60?28℃下培養之藍球藻HSP60的1.6倍,顯示在未致死的高溫處理下,藍球藻HSP60的量會隨溫度升高而增加,因而推測HSP60在藍球藻的耐熱性扮演保護的角色。再由雙向免疫轉印分析,發現有兩個pI值分別?5.5和5.9的HSP60,pI值?5.5的蛋白質在48℃經24小時處理後的量?28℃下培養的1.57倍,pI值?5.9的蛋白質在48℃經24小時處理後的量?28℃下培養的5.88倍,由此可知其含量會因高溫處理而增加,表示它在藍球藻的熱保護上扮演重要角色,但其真正保護機制值得更近一步探討。 Cyanidium caldarium is a unicellular thermoacidophilic eukaryotic alga, which contains chlorophyll a, carotenoid, and phycocyanin, so we suggest it as a transitional alga. Cyanidium caldarium grows faster in a modified Ascion's condition than in Liang's. The alga reached the stationary phase after seven days in our incubated condition. It is useful for physiological and biochemical experiments. This alga is name as Cyanidium caldarium, because the HPLC analysis of polyamines of this alga was found to be same as the polyamine distribution of Cyanidium caldarium strain KUSATSU. The quantities of polyamines in the alga changed differently when temperature shifts from 28℃ to 40℃. Putrescine, norspermidine, norspermine increase, so we suggest the three polyamines play an important role in thermal protection. In the analysis of heat shock proteins by one-dimensional and two-dimensional electrophoresis, the quantities of the proteins of 70, 60, and 30 KD are enhanced after heat treatment. These three proteins are supposed to be contributed to thermal tolerance. By immunobloting, we know the 60 KD protein of the alga is highly homologous to yeast HSP60, but less homologous to E. coli GroEL. The 60KD protein has no cross reaction with antibody against saybean LMW HSP1. This demonstrates that the evolutionary position of the alga is close to yeasts, but not high plants. Two-dimensional immunobloting showed that HSP60 has two proteins with pI = 5.5 and 5.9 respectively. So we also suggest these two proteins play an important role in thermal protection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76046 |
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顯示於系所單位: | 植物科學研究所 |
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