Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36047
標題: 芝麻油體鈣蛋白相似基因的選殖
Cloning of caleosin-like gene in sesame
作者: 黃世宏
關鍵字: oil body;油體;caleosin;油體鈣蛋白
出版社: 生物科技學研究所
摘要: 
在油料作物的種子裡,植物以油體為能量的儲存胞器並做為萌發時期的能量來源。在芝麻種子的油體表面上除了有大量的結構蛋白:油體膜蛋白(Oleosin)外,尚有三個微量蛋白,分別為油體鈣蛋白(Caleosin)、油體固醇蛋白A(Steroleosin A)以及油體固醇蛋白B (Steroleosin B),此三個微量蛋白的生理功能目前仍然未知。本實驗的目的在於油體鈣蛋白相似基因的選殖,根據報導指出,在阿拉伯芥植株中有其它油體鈣蛋白相似基因存在於不同的組織部位,而在水稻植株中此基因亦受逆境誘導;本實驗室於芝麻種子成熟過程中所選殖的油體鈣蛋白基因為胚專一表現的基因,因此,為探討此基因或此相似基因在芝麻植株的表現情形,以及藉由相似基因的選殖提供油體鈣蛋白在植物細胞內可能扮演的生理功能的線索,本實驗乃針對芝麻植株於逆境處理下進行油體鈣蛋白相似基因的選殖。
由芝麻種子成熟過程中的cDNA 基因庫已成功的選殖出油體鈣蛋白相似基因的部分片段,由北方墨點的偵測結果可發現其表現時期與油體鈣蛋白基因於種子成熟過程中表現情形相似,並可利用西方墨點法測得其位於油體表面之訊號。另外將發芽十五天後的芝麻植株以不同的逆境處理,並以芝麻油體鈣蛋白基因為探針進行北方墨點法,由於組織樣品的取得與萃取total RNA品質上的考量,本實驗先行以葉子為Caleosin相似基因選殖的材料。經與種子成熟過程中的total RNA比對的結果顯示:在逆境處理的樣品中有一個較高的訊號條帶,推測此為Caleosion的相似基因;當以ABA處理並不會影響其表現量,而在滲透逆境下可以誘導此基因大量表現,此外由北方墨點的結果亦顯示此基因為持續性表現的基因。利用RT-PCR的方式進行此基因的選殖,針對Caleosin基因高度保留區段設計數對專一性引子。此外,為偵測蛋白的表現,以糖梯度分離胞器的方式,並以Caleosin抗體進行西方墨點,結果於葉綠体與粒線體的分離層可偵測到約為30 kDa的訊號條帶,為進一步確認此條帶為Caleosin相似蛋白質,擬將回收此條帶,進行MALDI/TOF之蛋白質身份鑑定以及LC/MS/MS之蛋白質定序,希望藉由定序的結果,能有助於此基因的選殖。

In plant seeds, oil bodies are energy reserve organelles, which afford energy for germination and post-germinative growth of seedling. Oleosins are oil-body structure proteins. Besides oleosins, there are three minor proteins embedded in sesame oil bodies: caleosin, steroleosin and sop3. The biological functions of these three minor proteins are still unknown. The aim of my study is to clone caleosin-kike genes in sesame.
According to literature reports, there are 7 homologous genes of caleosin in Arabidopsis genome, and a caleosin-like gene could be induced by stress in rice plant. Therefore, I used 15 day seedling of sesame treated with ABA, CaCl2 and hyper-osmotic conditions to induce caleosin-like gene expression. The Northern blot was carried out with total RNA extracted from leaves after treatment using the total RNA of maturing stage of sesame seeds as the control. The results indicated that a caleosin-like gene was constitutively expressed under different treatments and hyper-osmotic stress. In order to detect the caleosin-like protein localization, organelles of leaves were separated by sucrose density gradient and detected them with caleosin antibody by western blot. A signal which was larger than caleosin could be detected on western blotting in the fractions of choloplast and mitochondria. I will try to recover this protein from SDS-PAGE and analyze by it to MALDI/TOF and LC/MS/MS.
URI: http://hdl.handle.net/11455/36047
Appears in Collections:生物科技學研究所

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