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。

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.

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.
2. Bennett, M. J., Marchant, A., Green, H. G., May, S. T., Ward, S. P., Millner, P. A., Walker, A. R., Schulz, B., Feldmann, K. A. (1996). Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273. 948-950.
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.
6. Davies, P.J. (2004). Plant hormones: biosynthesis, signal transduction, action! London: Kluwer Academic Publishers. pp. 1-62, 204-220, 282-303 and 451-484.
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.

8. Emmerstorfer, A., Wriessnegger, T., Hirz, M., Pichler, H. (2014). Overexpression of membrane proteins from higher eukaryotes in yeasts. Appl Microbiol Biotechnol 98. 7671-7698.
9. Goldsmith, M. H. M. (1977). The polar transport of auxin. Ann Rev Plant Physiol. 28. 439-478.
10. Hsu, S. H., Pan, R. L. (2004). Expression, purification, and characterization of mung bean his-tagged proton pumping inorganic pyrophosphatase in yeast. M. S. Thesis in Institute of Bioinformatics and Structural Biology, National Tsing Hua University. pp. 9-14.
11. Goyal, P., Kachhwaha, S., Kothari, S. L. (2012). Micropropagation of Pithecellobium dulce (Roxb.) Benth-a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers. Physiol Mol Biol Plants 18. 169-176.
12. Huang L. C., Pan, R. L. (2014). Heterologous expression and characterization of Arabidopsis auxin transporter in E. coli. M. S. Thesis in Institute of Bioinformatics and Structural Biology, National Tsing Hua University. pp. 5-8.
13. Kerr, I. D., Bennett, M. J. (2007). New insight into the biochemical mechanisms regulating auxin transport in plants. Biochem J 401. 613-622.
14. Korasick, D. A., Enders, T. A., Strader, L. C. (2013). Auxin biosynthesis and storage forms. J Exp Bot 64. 2541-2555.
15. Kramer, E. M., Ackelsberg, E. M. (2015). Auxin metabolism rates and implications for plant development. Front Plant Sci 6. 150.
16. Krecek, P., Skupa, P., Libus, J., Naramoto, S., Tejos, R., Friml, J., Zazimalova, E. (200). The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biol 10. 249.

17. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227. 680-685.
18. Lin, S. M., Pan, R. L. (2008). Expression, purification and crystallization of mung bean vacuolar proton-pumping pyrophosphatase. M. S. Thesis in Institute of Bioinformatics and Structural Biology, National Tsing Hua University. pp. 8-20.
19. Lin, S. M., Tsai, J. Y., Hsiao, C. D., Huang, Y. T., Chiu, C. L., Liu, M. H., Tung, J. Y., Liu, T. H., Pan, R. L., Sun, Y. J. (2012). Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature 484. 399-403.
20. Ljung, K., Bhalerao, R. P., Sandberg, G. (2001). Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J. 28. 465-474.
21. Ludwig-Müller, J. (2011). Auxin conjugates: their role for plant development and in the evolution of land plants. J Exp Bot 62. 1757-1773.
22. Maher, E. P., Martindale, S. J. (1980). Mutants of Arabidopsis thaliana with altered responses to auxins and gravity. Biochem Genet 18. 1041-1053.
23. Mano, Y., Nemoto, K. (2012). The pathway of auxin biosynthesis in plants. J Exp Bot 63. 2853-2872.
24. Mravec, J., Skupa, P., Bailly, A., Hoyerova, K., Krecek, P., Bielach, A., Petrasek, J., Zhang, J., Gaykova, V., Stierhof, Y. D., Dobrev, P. I., Schwarzerova, K., Rolcik, J., Seifertova, D., Luschnig, C., Benkova, E., Zazimalova, E., Geisler, M., Friml, J. (2009). Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter. Nature 459. 1136-1140.
25. Ranocha, P., Dima, O., Nagy, R., Felten, J., Corratge-Faillie, C., Novak, O., Morreel, K., Lacombe, B., Martinez, Y., Pfrunder, S., Jin, X., Renou, J. P., Thibaud, J. B., Ljung, K., Fischer, U., Martinoia, E., Boerjan, W., Goffner, D. (2013). Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis. Nat Commun 4. 2625.
26. Rubery, P. H., Sheldrake, A. R. (1974). Carrier-mediated auxin transport. Planta 118. 101-121.
27. Swarup, R., Kargul, J., Marchant, A., Zadik, D., Rahman, A., Mills, R., Yemm, A., May, S., Williams, L., Millner, P., Tsurumi, S., Moore, I., Napier, R., Kerr, I. D., Bennett, M. J. (2004). Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell 16. 3069-3083.
28. Taiz, L., Zeiger, E. (2010). Plant physiology. Sunderland: Sinauer Associates Inc. Publishers. pp. 545-582.
29. Tanaka, H., Dhonukshe, P., Brewer, P. B., Friml, J. (2006). Spatiotemporal symmetric auxin distribution: a means to coordinate plant development. Cell Mol Life Sci 63. 2738-2754.
30. Vanneste, S., Friml, J. (2009). Auxin: a trigger for change in plant development. Cell 136. 1005-1016.
31. Woodward, A. W., Bartel, B. (2005). Auxin: regulation, action, and interaction. Ann Bot 95. 707-735.
32. Yang, H., Murphy, A. S. (2009). Functional expression and characterization of Arabidopsis ABCB, AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe. Plant J 59. 179-191.
33. Yang Y., Hammes U.Z., Taylor C.G., Schachtman D.P., Nielsen E. (2006). High-affinity transporter by AUX1 influx carrier protein. Curr Biol 16. 1123-1127.
34. Zazimalova, E., Murphy A.S., Yang, H., Hoyerova, K., Hosek, P. (2010). Auxin transporters-why so many? Cold Spring Harb Perspect Biol 2010. a001552.