利用酵母菌異體系統大量表現及純化阿拉伯芥生長素轉運蛋白

植物生長激素不僅是第一個被確認的植物生長荷爾蒙，且扮演很重要的角色。自然界中的植物生長激素主要有三個種類，包括IAA、4-Cl-IAA 和IBA。其中以IAA為主要存在的種類。植物生長激素是調控植物生長和發育的最重要荷爾蒙，因此維持其平衡的策略很重要；其中包含合成、抑制、分解以及運輸。由於植物生長激素符合傳統荷爾蒙定義¬¬，即其合成處並非其主要的作用部位，因此，運輸部分值得做更的討論。生長素轉運蛋白AUX1 為主要將生長激素運入植物細胞的運輸蛋白。植物生長素轉運蛋白AUX1的分子量為54 kDa，並被預測有11個穿膜區。關於AUX1的探討，以下將著重在以酵母菌異質系統大量表現，作為將來從事蛋白結晶學研究之用。其中以出芽酵母(BJ2168、BY4741)和分裂酵母(leu1-32 h-、avt3)為主，並搭配不同的表現載體pYES2 (BJ2168、BY4741)、pDR196 (BY4741)和pREP41 (avt3、leu1-32 h-)。本論文將測試各種會影響表現的因子，以求得到最大量表現的條件。接著DDM (n-Dodecyl-β-D-maltopyranoside)將用來把目標蛋白從膜上取出，並且將利用金屬螯合層析法來純化目標蛋白。最後，我們期待能利用X-ray結晶繞射來解出生長素轉運蛋白AUX1的3D立體結構。

關鍵字

植物生長素轉運蛋白

並列摘要

Auxin is not only the first-identified plant growth hormone but also the most important hormone in plants. There are three types of natural auxins, including IAA, 4-Cl-IAA and IBA. Especially, IAA is the major type plant hormone. It regulates the growth and development of plants. The tactic used to maintain the homeostasis of auxin is crucial. It includes many aspects, such as synthesis, inactivation, degradation and transport of auxin. Moreover, auxin conforms to the traditional definition of hormone that substance needs to transport from source to sink. Therefore, AUX1, one of the auxin influx transporters, plays a significant role in maintaining the homeostasis of auxin. The molecular mass of AUX1 is about 54 kDa and the number of predicted transmembrane domains is 11. The study here focuses on the overexpression of Arabidopsis AUX1 in the budding yeast Saccharomyces cerevisiae (BJ2168, BY4741), and fission yeast Schizosaccharomyces pombe (leu1-32 h-, avt3). The specific expression systems contain different yeast strains and shuttle vectors, pYES2 (BJ2168, BY4741), pDR196 (BY4741) and pREP41 (avt3, leu1-32 h-). Conditions influencing the expression of protein were explored for the mass production of AUX1. The fractions with highest yield were obtained, followed by the detergent DDM (n-Dodecyl-β-D-maltopyranoside) treatment to isolate the protein from membrane. The metal chelating affinity chromatography was then used to purify the specific protein, AUX1. The 3D structure of AUX1 is expected to be investigated eventually.

並列關鍵字

AUX1

參考文獻

1. Ago, H., Kanaoka, Y., Irikura, D., Lam, B. K., Shimamura, T., Austen, K. F., Miyano, M. (2007). Crystal structure of a human membrane protein involved in cysteinyl leukotriene biosynthesis. Nature 448. 609-612.

3. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72. 248-254.

4. Carrier, D. J., Bakar, N. T., Swarup, R., Callaghan, R., Napier, R. M., Bennett, M. J., Kerr, I. D. (2008). The binding of auxin to the Arabidopsis auxin influx transporter AUX1. Plant Physiol 148. 529-535.

5. Carrier, D. J., Abu Bakar, N. T., Lawler, K., Dorrian, J. M., Haider, A., Bennett, M. J., Kerr, I. D. (2009). Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters. Int J Plant Genomics 2009. 848145.

7. Delbarre, A., Muller, P., Imhoff, V., Guern, J. (1996). Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxy acetic acid, naphthalene-l-acetic acid, and indole-3-acetic acid in suspension-cultured tobacco cells. Planta 198. 532-541.

國際替代計量

利用酵母菌異體系統大量表現及純化阿拉伯芥生長素轉運蛋白

全文下載

主題瀏覽