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標題: | 胺基酸類似物及酒精逆境對大豆白化幼苗熱休克蛋白質基因表現之影響 Effects of Amino Acid Analogs and Ethanol on Expression of Heat Shock Protein Genes in Soybean Seedlings |
作者: | 蔡瑩霏 |
出版年 : | 1998 |
學位: | 碩士 |
摘要: | 胺基酸類似物L-azetidine-2-carboxylic acid(proline之類似物,簡稱Aze)會誘導大豆白化幼苗合成第一族低分子量熱休克蛋白質。本實驗利用pCE53(大豆,GmHsp17.5E),hsp70(大豆,Gmhsp70A)和hsc-2(蕃茄,hsc-2)三個CDNA當探針,檢測大豆在Aze處理下熱休克蛋白質基因的表現情形,並用大豆第一族低分子量熱休克蛋白質抗體進行免疫轉印分析,檢測大豆第一族低分子量熱休克蛋白質累積的情形。北方雜合反應的結果顯示,各處理組中,這三種熱休克蛋白質mRNA累積量的變化情形大致相同。以10mM Aze處理6小時,即有明顯的熱休克蛋白質mRNA累積,持續處理24小時,其熱休克蛋白質mRNA的累積量可增加至與熱休克處理(40℃,二小時)相當的量。蛋白質累積的情形,在Aze處理6小時,才開始累積低分子量熱休克蛋白質,而後持續增加,到Aze 24小時達到與熱休克處理所誘導的量相當。若在10mM Aze處理6小時之後,換至不含任何胺基酸的振盪緩衝液中持續處理24小時,其熱休克蛋白質mRNA的累積會維持一定的量,低分子量熱休克蛋白質累積的情形則與Aze持續存在時的情形相似。但是若在10mM Aze處理6小時之後,移至含有10mM Pro(proline)的振盪緩衝液中持續處理24小時,其pCE53 mRNA的累積量,從第2小時之後就開始下降,第12小時之後幾乎偵測不到pCE53 mRNA;而hsp70 mRNA與hsc-2 mRNA的累積量,則是從第6小時之後開始下降,第16小時之後幾乎偵測不到hsp70 mRNA與hsc-2 mRNA。低分子量熱休克蛋白質則是在第12小時有最大的累積量,第18小時之後就開始下降。 酒精會誘導一群50?80kDa左右的蛋白質累積。其中,一群60kDa左右的蛋白質累積量相當高;但是酒精處理下,並沒有發現大豆第一族低分子量熱休克蛋白質被誘導產生。利用pCE53,HSP60(酵母菌,60kDa),hsp70以及hsc-2當作探針,檢測大豆在酒精處理下熱休克蛋白質mRNA的累積情形。在非致死酒精處理下,顯示有微量pCE53 mRNA及hsp70 mRNA的累積,但是沒有發現與HSP60相當的熱休克蛋白質mRNA的累積;不過,與hsc-2相當的熱休克蛋白質mRNA卻會被大量誘導,而且累積的情形會隨著處理的濃度與時間的增加而增加。 The proline analog -- L-azetidine-2-carboxylic acid (Aze) is capable of inducing etiolated soybean seedlings to synthesize class I low-molecular-weight heat shock proteins. In this study, we used three HS cDNA (pCE53, hsp70, and hsc-2) as probes and a soybean class I LMW HSP antibody to analyze the effect of the analog on RNA transcription level and HSPs accumulation in etiolated soybean seedlings. From the northern hybridization results, the mRNA accumulation patterns of the three HSPs induced by Aze treatment were almost the same. The three HS transcripts accumulated to a significant level after 6 hours of Aze treatment and after 24 hours of Aze treatment, the accumulation of these transcripts reached to the same level as observed in heat-shocked seedlings. Although the level of pCE53 mRNA induced by 6 hours of Aze treatment was significant, the level of class I LMW HSPs accumulated was low. However, the protein accumulation correlated with the mRNA pattern after 24 hours of treatment. If seedlings treated with 10 mM Aze for 6 hours were transferred to a shaking buffer without amino acids, the HS mRNA transcript remained for 24 hours. Conversely, if 10 mM Aze 6 hours treated seedlings were transferred to a shaking buffer containing 10 mM proline, pCE53 mRNA declined after 2 hours of treatment, and was almost undetected after 12 hours treatment; whereas hsp70 mRNA and hsc-2 mRNA declined after 6 hours of treatment, and almost were not detected after 16 hours treatment. As to the class I LMW HSPs, they accumulated to a climax after 12 hour treatment and declined after 18 hours of treatment. Ethanol induced a group of proteins with the 60-kDa group being the prominent, but it did not induce LMW HSPs of soybean seedlings. We used pCE53, HSP60, hsp70, and hsc-2 as probes to determine the expression of the HS mRNA after ethanol treatment. Under non-lethal conditions, only a trace of pCE53 and hsp70 mRNA was accumulated, but HSP60 mRNA was not detected. Suprisingly, the mRNA transcript of hsc-2 was largely induced and was proportional to the concentration and duration of ethanol treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76385 |
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顯示於系所單位: | 植物科學研究所 |
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