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研究生:林麗娟
研究生(外文):Lin Li-Jen
論文名稱:芝麻油體上的油體固醇結合去氫酶
論文名稱(外文):Steroleosins,Sterol-Binding Dehydrogenase in Sesame Seed Oil Bodies.
指導教授:曾志正曾志正引用關係
指導教授(外文):Jason T. C. Tzen
學位類別:博士
校院名稱:國立中興大學
系所名稱:生物科技學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:85
中文關鍵詞:油體固醇結合去氫酶芝麻種子
外文關鍵詞:Oil BodySteroleosinSterol-Binding DehydrogenaseSesameSeed
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中文摘要
植物種子儲存三酸甘油酯(triacylglycerol) 作為發芽所需之能量。三酸甘油酯儲存於一種植物種子特有的胞器稱為油體。目前所提的油體構造模型是一團三酸甘油酯包在一層磷脂質(phospholipid)內,此磷脂質層鑲滿了構造蛋白質叫油體膜蛋白 (oleosin)及一些微量蛋白質。在前幾年,其中一種微量蛋白質的基因(Sop1)已經被選殖了。 由它們序列上的分析以及生化上的研究得知,其會和鈣離子結合,因此命名為油體鈣蛋白(caleosin)。本篇論文主要是研究另外兩個微量蛋白(Sop2 and Sop3)的結構與功能以及它們在種子成熟時期結合油體蛋白的機制。
在2000年,利用免疫檢測法從成熟的芝麻種子篩選出Sop2其對應的基因。根據從基因推出的胺基酸序列作比對,得知此蛋白質在N端具有結合油體的區域,在此區域後為固醇結合的位置,因此我們將此蛋白質命名為油體固醇蛋白。此油體固醇蛋白存在許多不同的油料作物的種子的油體中,並且在不同物種中伴隨著訊息傳遞的功能。利用南方墨點技術得知,在芝麻的基因體中存在一個油體固醇蛋白和許多類似油體固醇蛋白的基因。由此可知,在不同植物組織中存有結合不同固醇的氧化還原酶以作為訊息傳遞的角色。
在2002年,利用PCR的技術成功選殖出成熟芝麻種子已知oil body蛋白質中最後一個且含量最少的微量蛋白質,Sop3。比對Sop2和Sop3的基因序列顯示,這兩個蛋白質為同類的蛋白質,並且具有高度保留區的NADP+結合位置以及不同大小的固醇結合位置,因此將Sop3蛋白質命名為油體固醇蛋白-B,而Sop2則改稱為油體固醇蛋白-A。雖然Sop2和Sop3為同類的蛋白,但兩者之間無法被彼此相對應的抗體所辨認。根據去氫酶活性的測試,發現油體固醇蛋白-B比油體固醇蛋白-A具有較廣的固醇選擇性及對NADP+較高的特異性。
在2003年,證明了油體固醇蛋白-A/油體固醇蛋白-B結合到成熟油體的路徑不同於油體鈣蛋白/油體膜蛋白。 油體固醇蛋白利用N端厭水區域結合油體,而油體鈣蛋白和油體膜蛋白是利用中間厭水區域結合油體,再者,油體固醇蛋白N端第一個胺基酸為Met,但油體鈣蛋白和油體膜蛋白的N端卻被封鎖住。根據實驗結果,油體固醇蛋白傾向到微粒體膜上,而油體鈣蛋白和油體膜蛋白卻較傾向到人造油體膜上。

ABSTRACT
Seed oil bodies are lipid storage organelles that comprise a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with proteins. The oil-body associated proteins include the abundant structural protein, oleosin and at least three minor proteins, Sop1-3. Recently, the gene encoding Sop1 protein was cloned from maturing sesame seeds. The Sop1 sequence analyses and biochemical studies indicated that the protein is calcium-binding, and thus named caleosin. This dissertation intended to study structure-function relationship of Sop2 and Sop3 and their targeting to oil bodies during seed maturation.
In 2000, the Sop2 cDNA sequence and its corresponding genomic sequence from maturing sesame seeds was cloned by immuno-screening. The deduced protein, tentatively named steroleosin, seems to exist in diverse seed oil bodies, and comprises an oil-body anchoring segment preceding a sterol-binding dehydrogenase involved in signal transduction in diverse organisms. Southern hybridization implies that one steroleosin gene and certain steroleosin-like genes may exist in sesame genome. The results suggest that different sterol-binding dehydrogenases/reductases may be present in diverse plant tissues and involved in signal transduction.
In 2002, the Sop3 cDNA sequence was cloned by PCR, the last and the least abundant of the proteins identified in oil bodies of sesame seeds as far. Sequence comparison revealed that Sop2 and Sop3 were homologous proteins, and thus tentatively named steroleosin-A and steroleosin-B. These two steroleosins possessed a conserved NADP+ binding subdomain but a diverse sterol-binding subdomain of different size. Although Sop2 and Sop3 were found homologous, they could not be cross-recognized immunologically with the antibodies prepared in our lab. Dehydrogenase activity detected in their expressed proteins indicated that steroleosin-B might comparably possess a broader sterol selectivity and higher NADP+ specificity than steroleosin-A.
In 2003, I proved that steroleosin-A/steroleosin-B and caleosin/oleosin may target to maturing oil bodies via distinct pathways. The hydrophobic domain responsible for oil-body anchoring is located in the N-terminal region of steroleosin, but in the central region of caleosin or oleosin. Steroleosin possessed a free methionine at its N-terminus while caleosin and oleosin were N-terminally blocked. In contrast with caleosin and oleosin, steroleosin preferentially targeted to microsomal membranes instead of artificial oil emulsions in an in vitro competition experiment.

Table of Contents
縮寫表 P3
List of figures P4
English abstract P8
Chinese abstract P10
Research Background P12
Chapter 1. Steroleosin, A Sterol-Binding Dehydrogenase in Seed Oil Bodies P24
Chapter 2. Two Distinct Steroleosins Are Present in Seed
Oil Bodies P56
Chapter 3. Targeting Pathway and Essential Structural Domain for Directing Steroleosin to Seed Oil Bodies P84
Conclusion P111

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