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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
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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.

    Contents Introduction----------------------------------------------------------------------------------------------1 Auxin Auxin transport AUX1 Materials and Methods--------------------------------------------------------------------------------5 AUX1 modeling Preparation of competent cells and induction of AUX1 expression DNA construction and mutagenesis Protein extraction from fission yeast with TCA Preparation of microsomes from yeast cell Western blot analysis 3H-IAA transport assay Results---------------------------------------------------------------------------------------------------10 Heterologous expression of Arabidopsis AUX1 in yeast Determination of optimal assay conditions Site-specific mutation of histidine in AUX1 3H-IAA transport assay Discussion-----------------------------------------------------------------------------------------------12 Optimal conditions for transport assay 3D-structure involving Histidine residues Tables-----------------------------------------------------------------------------------------------------14 Table 1. YES medium Table 2. EMM medium Table 3. 50x Salt stock Table 4. 1000x Vitamin stock Table 5. 10,000x Mineral stock Table 6. Homogenization buffer Table 7. Washing buffer Table 8. Sample buffer Table 9. Blocking buffer Table 10. PBST Table 11. Immunogen of antibody Figures---------------------------------------------------------------------------------------------------19 Figure 1. The construction of expression vextor Figure 2. The predicted topology of Arbidopsis AUX1 Figure 3. Sid-view AUX1 structure predicted using Phyre2 Figure 4. Top-view AUX1 structure predicted using Phyre2 Figure 5. The clone of pREP41-AUX1 and histidine mutation checked by plasmid extraction, and sequenced Figure 6. Time course of 3H-IAA transport activity Figure 7. Concentration effect on 3H-IAA transport activity Figure 8. The pH profile of 3H-IAA transport activity Figure 9. 3H-IAA transport assay for site-specific mutants under optimal conditions Figure 10. Western blot analysis of whole cell from histidine mutants Figure 11. Western blot analysis of microsomes from histidine mutants Figure 12. Sequence alignment of AUX1 showing highly and moderately conserved histidine residues References-----------------------------------------------------------------------------------------------33

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