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標題: | 第一部分:大腸桿菌細胞表現水稻第一族低分子量熱休克蛋白質之免疫定位與耐熱性探討 第二部分:胺基酸類似物誘導大豆第一族低分子量熱休克蛋白質生合成之免疫定位分析 Immunolocalization of Oryza sativa Class I Low Molecular Mass Heat Shock Protein Expressed in E. coli in Relation to Thermotolerance Immunolocalization of Azetidine Induced Class I Low Molecular Mass Heat Shock Proteins in Soybean |
作者: | 宋維文 |
出版年 : | 2001 |
學位: | 碩士 |
摘要: | 本實驗室先前將大腸桿菌細胞以pUC(僅轉殖pUC8表現載體)、pUC-FL(轉殖完整Oshsp16.9 cDNA)及pUC-C108(轉殖可表現Oshsp16.9蛋白質C端108個胺基酸片段)等三種質體(plasmids)進行轉型(transformed)得到三種轉殖株,並表現出Oshsp16.9蛋白質。在本實驗中,將以電子顯微鏡觀察大腸桿菌在高溫處理下,細胞內部構造變化;再以免疫標定(immunolocalization)技術,觀察Oshsp16.9蛋白質或其片段蛋白質於大腸桿菌細胞內累積之位置及移動情形,並與真核細胞的相關情形進行比較,探討與耐熱性之間的關係。。 在正常環境下所生長的之大腸桿菌,其細胞質均呈現深色外細胞質(ectoplasm)與淺色內細胞質(endoplasm)之外觀。經過IPTG誘導或經熱處理後,不論是未轉殖細胞或轉殖細胞,均會在外細胞質處出現一些深色區塊。經IPTG誘導之pUC-FL轉殖菌株,在高溫50℃下處理再予以回溫37℃ 1小時後,約有27%左右之菌體形態可恢復至正常細胞之狀態,pUC與pUC-C108轉殖細胞則僅有0?8%的恢復比例。此結果亦符合了Oshsp16.9蛋白質所具有的熱保護功能。 由Oshsp16.9蛋白質或其片段蛋白質之大腸桿菌轉殖株免疫標定實驗結果顯示,有無IPTG誘導pUC-FL及pUC-C108細胞Oshsp16.9表現有相當大的影響。。經過不同溫度處理後Oshsp16.9累積位置亦沒有位移現象,但Oshsp16.9表現的免疫標定金粒在深色區塊有較高之分佈密度。 第二部份實驗則針對大豆白化幼苗由胺基酸類似物azetidine誘導所產生之第一族低分子量熱休克蛋白質進行分析,先前本實驗室研究中發現經azetidine誘導之此熱休克蛋白質經次細胞分離分析時會與粒線體一同被分離。在本實驗中同樣以免疫標定技術研究由azetidine所誘導之熱休克蛋白質在大豆細胞內的表現情形。結果發現azetidine對大豆根尖細胞誘導之熱休克蛋白質仍大多存在於細胞質中,僅少數堆積在粒線體之周圍,離開azetidine處理環境後,細胞累積出之熱休克蛋白質仍能穩定存在於細胞中,且較平均分佈於細胞質,若再以40℃處理,其蛋白質分佈位置並沒有明顯之變化。 Escherichia coli cells were transformed with pUC8 (pUC8 vector only), pUC-FL (rice class I low molecular mass heat shock protein Oshsp16.9 cDNA ORF inserted into pUC8), and pUC-C108 plasmids (DNA fragment containing C-terminal 108 amino acids of Oshsp16.9 cDNA ORF inserted into pUC8) previously in our laboratory and the recombinant proteins were expressed. In the present study, the ultrastructural changes of the transformed E. coli cells affected by heat shock were studied with electron microscopy. Meanwhile, immunolocalization and the movement of Oshsp16.9 or its fragment in E. coli cells affected by heat shock were also reported. Escherichia coli grown under normal environment, cells was enclosed by cell wall with dense ectoplasm and more or less light endoplasm, in which nucleoid region located. With IPTG induction or heat treatment, some dark regions were found in ectoplasm. Having been transferred back to 37℃ after heat shock, about 27% of pUC-FL cells pretreated with IPTG induction recovered to normal. Cells without IPTG pretreatment, only had 0?8% of recovery. These results are in agreement with the thermoprotection of Oshsp16.9. Immunolocalization of Oshsp16.9 expressed in E. coli after IPTG induction were found both in pUC-FL and pUC-C108 transformed clones. Quantitative comparison of immuno-gold particles in cells under different treatments showed results as follows: there was a distinct difference in Oshsp16.9 expression of cells with and without IPTG induction; there was no conspicuous movement of Oshsp16.9 in normal or high temperature environment; and the density of gold particles in dark regions was higher than that in other regions. In the second part of this thesis, class I low molecular mass heat shock protein induced by azetidine were precipitated with mitochondria by subcellular fraction were investigated. In this issue, immunolocalization of soybean class I low molecular mass heat shock protein induced by azetidine were performed. Most of the class I low molecular mass heat shock protein accumulated in the cytoplasm and the less around mitochondria of soybean seedlings pretreated by azetidine. When seedlings pretreated with azetidine were transferred back to normal condition and their heat shock proteins would move to cytosol. When soybean seedlings pretreated with azetidine were shifted to heat shock of 40℃, the class I heat shock proteins were observed more abundant in cytosol. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75208 |
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顯示於系所單位: | 植物科學研究所 |
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