臺灣博碩士論文加值系統

為了解鐵元素對海洋單細胞綠藻Dunaliella tertiolecta 生長的影響，在培養基中加入一定量的鐵離子，再由培養之細胞中抽取出蛋白質，經電泳分析後發現，在加有鐵離子的細胞，其分子量為38 kDa之蛋白質消失，此外葉綠體結構完全改變。為了進一步了解這種鐵離子所造成細胞的變化，是否與鐵離子傳遞的蛋白質有關，本論文利用抗人類的鐵傳遞蛋白質（transferrin）之抗體，進行細胞蛋白質的免疫分析。結果發現缺鐵狀態下，分子量為150 kDa的蛋白質明顯增加。此蛋白質可與人類的鐵傳遞蛋白質之抗體作用，因此稱之為似鐵傳遞蛋白質（transferrin-like protein）。欲知此蛋白質的形成如何受到鐵的調控，筆者再進一步進行似鐵傳遞蛋白質基因之探討。由已知之D. salina 似鐵傳遞蛋白質基因之核 酸序列，設計出三組寡核 酸 ( 230與235 ; 232與238 ; 237與239 ) 做為引子，並以D. tertiolecta 之染色體DNA為模板，進行聚合 鍊反應（polymerase chain reaction；PCR），得到三段分子量約為3.6、4.1和2.7 kb大小之DNA片段，據此分別進行核 酸序列分析，並將三個DNA序列連接，得到一完整的9.5 kb DNA序列，內有由30個exon及29個intron所組成的基因，其開放解讀框（open reading frame）則包含了3825個核甘酸，形成由1274個胺基酸組成之蛋白質分子量150 kDa。此蛋白質之胺基酸排列，與D. salina 之似鐵傳遞蛋白質，極為類似，而與人及雞的鐵傳遞蛋白質也有相同之處，是故稱之為D. tertiolecta 之似鐵傳遞蛋白質，其形成亦受鐵缺乏所誘導。

The role of iron in the growth and physiology of the marine alga Dunaliella tertiolecta was investigated. Comparison of the electrophoretic profiles of total proteins from cells grown under iron-replete or iron-deficient conditions revealed that the abundance of a 38-kDa protein was markedly increased in cells deprived of iron. Electron microscopy also revealed that cells subjected to iron limitation exhibited substantial chlorosis and a defective photosynthetic apparatus. In addition, immunoblot analysis showed that iron deprivation increased the abundance of a 150-kDa protein that reacts with antibodies to human transferrin. With the use of the polymerase chain reaction and primers based on the nucleotide sequence of a gene for a transferrin-like protein in D. salina, a 9.5-kb fragment of D. tertiolecta genomic DNA that encodes a homologous protein was isolated and sequenced. The Dunaliella tertiolecta gene comprises 30 exons and contains an open reading frame of 3825 bp that encodes a protein of 1274 amino acids. The predicted amino acid sequence of the encoded protein shows a high degree of homology to those of various transferrin proteins from other organisms.

封面
中文摘要
英文摘要
縮寫字代號
前言
材料與方法
1.藻種及保存方法
2.藻類和其生長條件
3.蛋白質分析
3.1蛋白質之萃取
3.2蛋白質濃度測定
3.3多聚丙烯醯胺膠體電泳
3.4蛋白質泳膠體硝酸銀染色法
3.5多聚丙烯醯胺電泳膠體之保存
4.穿透式電子顯微鏡的觀察
4.1固定與包埋
4.2超薄切片
4.3雙重染色
5.西氏點墨法
6.基因組DNA的純化
7.DNA酒精沉澱
8.瓊脂醣膠體電泳分析
8.1瓊脂醣膠體電泳
8.2瓊脂醣膠體之染色
9.引子的設計、製造與純化
10.多聚合連鎖反應
11.由瓊脂醣膠回收DNA的片段
12.DNA自動定序分析
13.核酸及胺基酸序列之分析比對
結果
1.微量重金屬離子對D.tertiolecta蛋白質的影響
2.不同濃度下鐵離子Fe+3對D.tertiolecta總蛋白質的影響
3.穿透式電子顯微鏡鐵離子Fe+3對D.tertiolecta細胞賽的影響
4.鐵離子Fe+3對似鐵傳遞蛋白質表現的影響
5.選殖D.tertiolecta似鐵傳遞蛋白質之基因
6.D.tertiolecta之似鐵傳遞蛋白質內部的重覆片段
討論
參考文獻
圖表
圖表說明
附錄(培養基藥品與溶液)

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