Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90046
標題: Characterization and Functional Analysis of Orchid(Phalaenopsis and Cattleya) CONSTANS-Like Genes in Regulating Flowering Time and Flower Development
探討蝴蝶蘭及嘉德麗雅蘭中CONSTANS-Like基因調控開花時間及花器發育之特性與功能性分析
作者: 柯玟亘
Wen-Hsuan Ko
關鍵字: 蝴蝶蘭
嘉德麗雅蘭
開花時間
花器發育
Phalaenopsis
Cattleya
flowering time
flower development
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摘要: 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.
URI: http://hdl.handle.net/11455/90046
文章公開時間: 2018-08-25
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