臺灣博碩士論文加值系統

阿拉伯芥的CONSTANS (CO) 是植物特有的轉錄因子，於光週期途徑中開花時間的調控扮演著重要角色。CO基因與16個CONSTANS-Like基因共同組成CO/COL基因家族，在CO/COL基因家族的N端含有一至兩個保守的B-box domain (與蛋白質間交互作用相關)，而C端則有一個保守的CCT domain (與蛋白質入核相關)。前人研究指出AtCOL9/10於阿拉伯芥開花時間調控上扮演抑制子的角色，但於文心蘭中對應到AtCOL9/10的同源基因─OnCOL9/10，卻在開花時間及花藥開裂上扮演著活化子的角色。本研究分別從嘉德麗雅蘭及蝴蝶蘭選殖出CaCOL10及PaCOL9基因進行功能性探討。在胺基酸序列的比對下，CaCOL10及PaCOL9分別與OnCOL10及OnCOL9有著76.8%及77.4%的相同度，推測可能與OnCOL9/10在開花時間的調控及雄蕊發育上具有相似功能。為了進一步探討CaCOL10及PaCOL9的功能，分別將其序列構築於大量表現 (35S promoter)、強烈抑制 (SRDX domain) 及強烈活化 (VP16 domain) 的載體中，並將構築完成的載體轉殖至阿拉伯芥。研究結果顯示，35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16的轉殖株會藉由促進CO、FT及SOC1基因的表現，而產生提早開花之性狀，然而CaCOL10-SRDX及PaCOL9-SRDX的轉殖株則呈現晚開花之性狀。除此之外，35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16的轉殖株會透過抑制與花藥室內壁次級細胞增厚之相關基因─NST1、NST2及Myb85的表現而產生花藥不開裂之性狀，另一方面，CaCOL10-SRDX及PaCOL9-SRDX的轉殖株對於雄蕊發育並沒有影響。由以上實驗結果得知CaCOL10、PaCOL9與OnCOL9/10於開花時間及雄蕊發育的調控具有相似功能，但卻與AtCOL9/10功能呈現相反之情形。由此推論，單子葉蘭花及雙子葉植物中相對應的COL9/10基因經由長久演化，其對於植物開花及花朵發育上的調控方式可能已經產生分歧。

CONSTANS (CO) is a plant-specific transcriptional factor that controls the onset of flowering in response to day-length in photoperiodic pathway. In Arabidopsis CO (AtCO) and 16 CO-like proteins (AtCOLs) contain one or two N-terminal B-box domains involved in protein-protein interaction, and one C-terminal CCT domain for nuclear-localization. Two AtCOLs, AtCOL9/10, functioned as repressors whereas the Oncidium OnCOL9/10 acted as activators to control flowering time and anther dehiscence. In this study, we focused on the functional analysis of two COL homologous genes from Cattleya (CaCOL10) and Phalaenopsis (PaCOL9) orchids. CaCOL10 and PaCOL9 share 76.8% and 77.4% identity of amino acid sequence with that of OnCOL10 and OnCOL9. Thus, CaCOL10 and PaCOL9 could function similarly with OnCOL9/10 in regulating flowering time and anther development. To further characterize the function of CaCOL10 and PaCOL9, we generated three types of Arabidopsis transgenic lines that ectopically express of PaCOL9/CaCOL10 (35S::PaCOL9/35S::CaCOL10), PaCOL9/CaCOL10 fused with a repression domain SRDX (PaCOL9-SRDX/CaCOL10-SRDX), or PaCOL9/CaCOL10 fused with an activation domain VP16 (PaCOL9-VP16/CaCOL10-VP16). 35S::CaCOL10, CaCOL10-VP16, and PaCOL9-VP16 promoted flowering by up-regulating the expression of CO, FT and SOC1. In contrast, CaCOL10-SRDX and 35S::PaCOL9-SRDX delayed flowering in transgenic Arabidopsis. Furthermore, 35S::CaCOL10, CaCOL10-VP16, and PaCOL9-VP16 caused anther indehiscence by down-regulating the expression of NST1, NST2 and Myb85 which are associated with secondary thickening in endothecium. CaCOL10-SRDX and PaCOL9-SRDX didn't affect the anther dehiscence in transgenic Arabidopsis. These results suggest that CaCOL10 and PaCOL9 may share similar function with OnCOL9/10 in promotion of flower time and inhibition of anther dehiscence, which was opposite to that of AtCOL9/10. Our results provide evidence that COL9/10 genes may behave divergently in monocots orchids and eudicots in regulating flowering time and flower development during evolution.

目錄
摘要 i
Abstract ii
前言 1
材料與方法 6
結果 15
一、嘉德麗雅蘭及蝴蝶蘭中CONSTANS-Like基因選殖及序列分析 15
二、嘉德麗雅蘭中CaCOL10基因表現量偵測 15
三、蝴蝶蘭中PaCOL9基因表現量偵測 16
四、35S::CaCOL10/CaCOL10-SRDX/CaCOL10-VP16及35S::PaCOL9/ PaCOL9 -SRDX/PaCOL9-VP16之構築 16
五、35S::CaCOL10/CaCOL10-SRDX/CaCOL10-VP16在阿拉伯芥上的異位表現造成開花時間之差異 17
六、PaCOL9-SRDX/PaCOL9-VP16在阿拉伯芥上的異位表現造成開花時間之差異 18
七、35S::CaCOL10/CaCOL10-VP16/PaCOL9-VP16在阿拉伯芥上的異位表現造成花藥不開裂 19
八、以茉莉酸 (jasmonic acid) 處理35S::CaCOL10/CaCOL10-VP16轉殖株花藥並無法挽救其花藥不開裂之性狀 19
九、35S::CaCOL10/CaCOL10-VP16/PaCOL9-VP16轉殖株內NST1、NST2及Myb85基因表現量下降造成花藥不開裂 20
討論 21
參考文獻 24
圖表 29
表1、本研究中所使用於轉殖之引子 (primer) 序列 29
表2、本研究中所使用於Real-time PCR之引子 (primer) 序列 30
表3、35S::CaCOL10/ 35S::CaCOL10-SRDX/ 35S::CaCOL10-VP16基因轉殖株開花天數與葉片數統計 31
表4、PaCOL9-SRDX/PaCOL9-VP16基因轉殖株開花天數與葉片數統計 32
圖1、嘉德麗雅蘭CaCOL10 cDNA之序列及其相對應胺基酸 33
圖2、蝴蝶蘭PaCOL9 cDNA之序列及其相對應胺基酸 34
圖3、CaCOL10、PaCOL9與CO/COL基因家族蛋白質胺基酸序列演化樹 35
圖4、PaCOL9、CaCOL10與阿拉伯芥及文心蘭COL9/10胺基酸序列比對 36
圖5、嘉德麗雅蘭之營養與生殖器官 37
圖6、嘉德麗雅蘭CaCOL10基因於植株各部位及開花前後花器表現量分析 38
圖7、蝴蝶蘭之營養與生殖器官 40
圖8、蝴蝶蘭PaCOL9基因於植株各部位及開花前後花器表現量分析 42
圖9、嘉德麗雅蘭CaCOL10基因之選殖及35S:: CaCOL10 /CaCOL10-SRDX/ CaCOL10-VP16之構築 44
圖10、蝴蝶蘭PaCOL9基因之選殖及35S::PaCOL9/PaCOL9-SRDX/PaCOL9- VP16之構築 46
圖11、異位表現35S::CaCOL10/CaCOL10-SRDX/CaCOL10-VP16在阿拉伯芥上造成植物花期性狀之分析 48
圖12、異位表現PaCOL9-SRDX/PaCOL9-VP16在阿拉伯芥上造成植物花期性狀之分析 50
圖13、異位表現35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16在阿拉伯芥上造成植物雄蕊及莢果性狀之分析 51
圖14、以亞歷山大染色 (Alexander's stain) 檢測35S::CaCOL10、CaCOL10- VP16及PaCOL9-VP16雄不稔轉殖株之花粉活性 53
圖15、以異花授粉 (cross-pollination) 檢測35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16雄不稔轉殖株之雌蕊生殖活性 55
圖16、以茉莉酸甲酯 (Methyl jasmonate，MeJA) 處理35S::CaCOL10/CaCOL10 -VP16雄不稔轉殖株之花藥及35S::CaCOL10、CaCOL10-VP16及PaCOL9- VP16轉殖株內DAD1之表現量分析 56
圖17、35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16雄不稔轉殖株內之NST1、NST2及Myb85表現情形 58
圖18、以共軛焦顯微鏡 (confocal) 觀察35S::CaCOL10、CaCOL10-VP16及PaCOL9-VP16雄不稔轉殖株之花藥室內壁 60
圖19、AtCOL9/10、OnCOL9/10及CaCOL10基因於阿拉伯芥開花時間與花器發育功能性之假說 62
附錄 63
附圖1、阿拉伯芥開花途徑及調節基因示意圖 63
附圖2、阿拉伯芥中CONSTANS基因調控開花的情形 64
附圖3、阿拉伯芥中與次級細胞壁生合成相關之途徑 66
附圖4、AtCOL9/10與OnCOL9/10基因於阿拉伯芥開花時間及花器發育功能性之假說 67
附圖5、pGEM®-T Easy vector之載體圖譜 (3018 bp) 68
附圖6、pEpyon-32K之載體圖譜 (10.6 kb) 69
附圖7、pEpyon-3aK之載體圖譜 (10 kb) 70
附圖8、pEpyon-3bK之載體圖譜 (10 kb) 71
附圖9、Gen-KB DNA Ladder 72

鐘佩文. 2012. 探討文心蘭中CONSTANS-Like基因調控植物發育及開花時間之特性與功能性分析. 碩士論文.

許哲睿. 2013. 探討阿拉伯芥中CONSTANS-Like基因調控開花時間及植物發育之特性與功能性分析. 碩士論文.

Alabadi, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Mas, P., and Kay S.A. (2001). Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock. Science. 293, 880-883

Alexander, M.P. (1969). Differential staining of aborted and nonaborted pollen. Stain Technol. 44, 117-122.

Blazquez MA. (2005). Plant science. The right time and place for making flowers. Science. 309, 1024-1025.

Cheng, X.F., and Wang, Z.Y. (2005). Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana. Plant J. 43, 758-768.

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.

Datta, S., Hettiarachchi, G.H., Deng, X.W., and Holm, M. (2006). Arabidopsis CONSTANS-LIKE 3 is a positive regulator of red light signaling and root growth. Plant Cell. 18, 70-84.

Galvao, V.C., and Fankhauser, C. (2015). Sensing the light environment in plants: photoreceptors and early signaling steps. Neurobiology. 34, 46-53.

Goldberg, R.B., Beals, P., and Sanders, P.M. (1993). Anther development: Basic principles and practical applications. Plant Cell. 5, 1217-1229.

Hassidim, M., Harir, Y., Yakir, E., and Green, R.M. (2009). Over-expression of CONSTANS-LIKE 5 can induce flowering in short-day grown Arabidopsis. Planta. 230, 481-491.

Hayama, R., and Coupland, G. (2003). Shedding light on the circadian clock and the photoperiodic control of flowering. Curr Opin Plant Biol. 6,13-19.

Higgins, J.A., Bailey, P.C., and Laurie, D.A. (2010). Comparative genomics of flowering time pathways using brachypodium distachyon as a model for the temperate grasses. PLos One. 5, e10065.

Hofgen, R., and Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res. 16, 9877.

Ikeda, M., and Ohme-Takagi, M. (2009). A novel group of transcriptional repressors in Arabidopsis. Plant Cell Physiol. 50, 970-975.

Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., and Okada, K. (2001). The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flowering opening in Arabidopsis. Plant Cell 13, 2191-2209.

Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen, S.K., Nguyen, J.T., Chory, J., Harrison, M.J., and Weigel, D. (1999). Activation tagging of the floral inducer FT. Science. 286, 1962-1965.

Kim Y.Y., Jung, K.W., Jeung, J.U., and Shin, J.S. (2012). A novel F-box protein repress endothecial secondary wall thickening for anther dehiscence in Arabidopsis thaliana. J Plant Physiol. 169, 212-216.

Khanna, R., Kronmiller, B., Maszle, D.R., Coupland, G., Holm, M., Mizuno, T., and Wu, S.H. (2009). The Arabidopsis B-box zinc finger famiy. Plant Cell. 21, 3416-3420.



Koornneef, M., Hanhart, C.J., and van der Veen, J.H. (1991). A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol Gene Genet. 229, 57-66.

Krizek, B.A., and Fletcher, J.C. (2005). Molecular mechanisms of flower development: an armchair guide. Nat Rev Genet 6, 688-698.

Laurie, D.A., Griffiths, S., Dunford, R.P., Christodoulou, V., Taylor, S.A., Cockram, J., Beales, J., and Turner, A. (2004). Comparative genetic approaches to the identification of flowering time genes in temperate cereals. Filed Crops Res. 90, 87-99.

Ledger, S., Strayer, C., Ashton, F., Kay, S.A., and Putterill, J. (2001). Analysis of the function of two circadian-regulated CONSTANS-LIKE genes. Plant J. 26, 15-22.

Lin, C.T. (2000). Photoreceptors and regulation of flowering time. Plant Physiol. 123, 39-50.

Min, J.H., Chung., J.S., Lee, K.H., and Kim, C.S. (2015). The CONSTANS-like 4 transcription factor, AtCOL4, positively regulates abiotic stress tolerance through an abscisic acid-dependent manner in Arabidopsis. J Intergr Plant Biol. 57, 313-324.

Mitsuda, N., Seki, M., Shinozaki, K., and Ohme-Takagi, M. (2005). The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell. 17, 2993-3006.

Quail, P.H. (2002). Phytochrome photosensory signaling networks. Nature Reviews Molecular Cell Biology. 3, 85-93.

Robson, F., Costa, M.M., Hepworth, S.R., Vizir, I., Pineiro, M., Reeves, P.H., Putterill, P.H., and Coupland, G. (2001). Functional importance of conserved domains in the flowering-time gene CONSTANS demonstrated by analysis of mutant alleles and transgenic plant. Plant J. 28, 619-631.

Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C., Lee, P.Y., Truong, M.T., Beals, T.P., and Goldberg, R.B. (1999). Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex. Plant. Reprod. 11, 297–322.

Sanders, P.M., Lee, P.Y., Biesgen, C., Boone, J.D., Beals, T.P., Weiler, E.W., and Goldberg, R.B. (2000). The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell. 12, 1041-1061.

Schultz, T.F., and Kay, S.A. (2003). Circadian clocks in daily and seasonal control of development. Science. 301, 326-328.

Shen, F., Triezenberg, S.J., Hensley, P., Porter, D., and Knutson, J.R. (1996). Transcriptional activation domain of the herpesvirus protein VP16 becomes conformationally constrained upon interaction with basal transcription factors. J Biol Chem. 271, 4827-4837.

Simpson, G.G., and Dean, C. (2002). Arabidopsis, the Rosetta stone of flowering time? Science. 296, 285-289.

Song, Y.H., Shim, J.S., Kinmonth-Schultz, H.A., and Imaizumi, T. (2015). Photoperiodic flowering time measurement mechanisms in leaves. Annu Rev Plant Biol. 66, 441-464.

Srikanth, A., and Schmid, M. (2011). Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci. 68, 2013-2037.

Suarez-Lopez, P., Wheatley, K., Robson, F., Onouchi, H., Valverde, F., and Coupland, G. (2001). CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis. Nature 410, 1116-1120.

Takase, T., Kakikubo, Y., Nakasone, A., Nishiyama, Y., Yasuhara, M., Tokioka-Ono, Y., and Kiyosue, T. (2011). Characterization and transgenic study of CONSTANS-LIKE 8 (COL8) gene in Arabidopsis thaliana: expression of 35S:COL8 delays flowering under long-day conditions. Plant Biotechnol. 28, 439-446.

Valverde, F. (2011). CONSTANS and the evolutionary origin of photoperiodic timing of flowering. J Exp Bot. 62, 2453-2463.

Wang, H., Zhang, Z., Li, H., Zhao, X., Liu, X., Ortiz, M., Lin, C., and Liu, B. (2013). CONSTANS-LIKE 7 regulates branching and shade avoidance response in Arabidopsis. J Exp Bot. 64, 1017-1024.
Yang, C., Xu, Z., Song, J., Conner, K., Vizcay Barrena, G., and Wilson, Z.A. (2007) Arabidopsis MYB26/MALE STERILE35 regulates secondary thickening in the endothecium and is essential for anther dehiscence. Plant Cell. 19, 534-548.

Yano, M., Katayose, Y., Ashikari, M., Yamanouchi, U., Monna, L., Fuse, T., Baba, T., Yamamoto, K., Umehara, Y., Nagamura, Y., and Sasaki, T. (2000). Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS. Plant Cell. 12, 2473-2484.

Zhong, R., and Ye, Z.-H. (2007). Regulation of cell wall biosynthesis. Curr Opin Plant Biol. 10, 564–572.