研究生: |
黃偉嘉 Huang, Wei Jia |
---|---|
論文名稱: |
探討阿拉伯芥生長素運輸蛋白中參與吲哚乙酸運輸之重要組胺酸特性 Identification of Essential Histidines Involved in IAA Transport of AUX1 |
指導教授: |
潘榮隆
Pan, Rong Long |
口試委員: |
孫玉珠
Sun, Yuh Ju 林士鳴 Lin, Shih Ming |
學位類別: |
碩士 Master |
系所名稱: |
生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology |
論文出版年: | 2016 |
畢業學年度: | 104 |
語文別: | 中文 |
論文頁數: | 36 |
中文關鍵詞: | 生長素運輸蛋白 、吲哚乙酸 、組胺酸 |
外文關鍵詞: | AUX1, IAA, histidine |
相關次數: | 點閱:1 下載:0 |
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植物的發育生長已知由植物生長素來進行調控,巨觀從開花結果以及各種向性,微觀到細胞胚胎的分化分裂,皆受到生長素的控制。在生長素之中占比例最高的吲哚乙酸 (Indole-3-actic acid,IAA),會於植物的頂芽以及根部合成後,進行極性運輸(Polar auxin transport)至基部,讓需要的細胞進行利用,而在細胞膜上鑲嵌的第一型生長素轉運蛋白(AUX1 transporter; 簡稱AUX1)則利用氫離子濃度梯度差,將吲哚乙酸運輸至所需的細胞內。AUX1 由485個胺基酸組成、擁有11個穿膜區,其分子量的大小為56.8 道爾頓 (kDa),目前在於植物生理上的研究相當踴躍,但仍舊缺乏其生物化學及結構生物學上相關的剖析。
本研究主要是利用分裂酵母(leul-32 h-)、搭配表現載體pREP41,來異體表現AUX1。我們利用同位素標定法測試分裂酵母中AUX1的運輸活性,並且於不同反應時間、以及不同吲哚乙酸濃度下進行活性測量,找出活性表現最佳化的條件。AUX1胺基酸序列中,總共有16個組胺酸(Histidine);同時,吲哚乙酸在正常細胞pH值下為帶負電,且其環狀結構以及羧基結構與組胺酸咪唑官能團(Imidazole)相似,因此,猜測組胺酸有可能與引咄乙酸相互產生吸引力,使成為AUX1中極具潛力的重要胺基酸。本研究主要將組胺酸突變成不帶電且體積小的丙胺酸(Alanine),再測試活性,以找出影響最大的組胺酸殘基。其結果可能提供見解闡明AUX1的結構與功能的關係。
The auxin hormone regulates the growth and development of plants from fruition to various tropisms for macroscopic and from differentiated cells to embryo division for microscopic ways. Indole-3-actic acid (IAA) accounted for the highest proportion of hormone regulation on plants. IAA is transported to the buds and roots of plants by polar movement. AUXIN RESISTANT 1 (AUX1) on the plant cell membrane utilizes the pH and IAA gradients to passively transport IAA to the desired cells. AUX1 is composed of 485 amino acids with 11 transmembrane domains and 56.8 kDa in molecular mass. The physiological roles of AUX1 have been adequately studied but its biochemical and structural analyses are still lacking. This study employed the fission yeast (leul-32h-) with heterologously expressing vector pREP41 for investigating AUX1 transport by using the isotope-labeled IAA (3H-IAA). The optimal condition for transporting IAA was decided. It was shown AUX1 contains totally 16 histidines. The aromatic and the carboxyl structures of IAA are similar to the histidine imidazole moieties and carboxyl group. It is possible the Histidine residue(s) is involved in the transport of IAA. All 16 histidine residues of AUX1 were mutated into uncharged small size alanine and the transport activities of variants measured to identify the crucial histidine residues. The results may provide insights to elucidate the structure-function relationship of AUX1.
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