臺灣博碩士論文加值系統

蝴蝶蘭優雅的花型屬於兩側對稱，並具有花瓣高度特化而成的唇瓣，其對稱形式扮演吸引昆蟲授粉的重要功能。前人關於兩側對稱的研究指出CYCLOIDEA基因會透過抑制細胞增生而影響花型的大小，進而改變花瓣的形狀，造成對稱性的差異。CYC基因屬於TCP轉錄因子，而TCP基因具有影響植物生長發育的功能。在金魚草的研究中指出，CYC基因會抑制背側花瓣的大小並且抑制背側花粉的成熟。在苦苣科與菊科的研究顯示，該類CYC/ TB1基因會抑制細胞增生並且與其他轉錄因子結合調控下游基因。本研究由姬蝴蝶蘭中選殖出三個CYC/ TB1相似基因並命名為PeCYC1、PeCYC2和PeCYC3。序列比對分析結果顯示，PeCYC具有TCP轉錄因子典型的TCP domain和R domain。時間與空間的表現分析顯示，PeCYC1與PeCYC2在早期花苞發育時期開始表現，並且高度表現在兩側萼片與唇瓣。此外，PeCYC1與PeCYC2在三唇瓣突變株姬蝴蝶蘭的不同花器表現分析顯示， PeCYC1與PeCYC2除了表現於兩側萼片與唇瓣外，也異位表現於兩側類唇瓣的花瓣中。蛋白質交互作用分析顯示，PeCYC1與PeCYC2可以和B群MADS-box蛋白質PeMADS6形成異源雙聚體。此外，酵母菌雙雜合系統分析也顯示，PeCYC1在缺少R domain的情形下，失去蛋白質交互作用的能力。在轉基因阿拉伯芥中，過量表現PeCYC1造成植株整體變小，包含葉子，根長，與花瓣及萼片。部分長角果莢在轉基因阿拉伯芥中也萎縮與發育不正常，種子數量也相較野生型的長角果莢少。掃描式電子顯微鏡顯示花瓣與葉片的細胞大小也變小。然而類似結果病沒有在過量表現PeCYC2的阿拉伯芥轉植株中發現，推測該基因可能需要和其他轉錄因子結合蘭花轉錄因子結合才能有功能性的表現。以上這些研究結果可以提供我們對於蝴蝶蘭CYC/ TB1基因調控花型發育有更進一步的了解。

關鍵字: 姬蝴蝶蘭、CYCLOIDEA、TCP基因

The Orchidaceae is one of the most species-rich plant families, which are renowned for their spectacular floral diversity. To maintain bilateral symmetry of flower is important for orchid flower to attract pollinator. Previous studies showed that CYCLOIDEA genes can regulate bilateral symmetry by inhibit cell proliferation. CYC genes belong to TCP family, which involved in the development of cell. In Antirrhium majus, CYC gene plays an role on inhibition of the cell proliferation of dorsal petals and stamens. In Asteraceae and Gesneriaceae, previous study showed that CYC/TB1-like proteins can interact with transcription factors to form homodimer or heterodimer. However, study on CYC-like genes in monocots is few. In this study, we identified three CYC-like genes, named as PeCYC1, PeCYC2 and PeCYC3, from Phalaenopsis equestris. Multiple sequence alignment showed PeCYC proteins contain typical TCP domains and R domain. Phylogenetic analysis showed that PeCYC genes belong to class II TCP genes and are closely related to CYC/TB1 genes of monocots rather than eudicots. The expression patterns showed that expression of PeCYC1 and PeCYC2 are restricted in lateral sepals, labellum and column. However, expression of PeCYC1 and PeCYC2 were ectopically expressed into lip-like petals of peloric mutant. Yeast-two hybrid results showed that PeCYC1 may form homodimer. However, PeCYC1 lost its homodimer formation activity when R-domain was deleted. Furthermore, Both PeCYC1 and PeCYC2 has ability to interact with B-class PI-like PeMADS6 protein. Overexpression of PeCYC1 in Arabidopsis thaliana showed delayed flowering, smaller leaves and flowers and shorter roots compared with those of wild type. Scanning Electron Micrographic results showed that, the cells of leaves and petals of transgenic plants were smaller than those of wild-type. These results will shed light on the roles of CYC/TB1 genes played in floral symmetry of monocot plants.

Table of Contents
中文摘要..........i
Abstract........ii
致謝.............iii
List of Tables...vi
List of Figures..vii
1. Introduction................................1
　1.1 Flower symmetry..........................1
　　1.1.1 Diversity of floral symmetry..........1
　　1.1.2 Molecular bases of zygomorphy in model species ...1
　1.2 Study of CYC-like genes in Dicot plants......2
　　1.2.1 Function of CYC-like genes in Asteraceae.....2
　　1.2.2 Function of CYC-like genes in Brassicaceae...2
　　1.2.3 Function of CYC-like genes in Gesneriaceae...3
　　1.2.4 Function of CYC-like genes in Fabaceae ......3
　1.3 Study of CYC-like genes in monocots.............3
　　1.3.1 Function of CYC-like genes in Zea may.......3
　　1.3.2 Function of CYC-like genes in Oryza sativa.... 4
　　1.3.3 Function of CYC-like genes in Commelinaceae.. 4
　1.4 Floral development of orchid...... 4
　　1.4.1 Floral morphology of orchid....4
　　1.4.2 Evolution of lip is associated with the attraction of pollinators......... 4
　　1.4.3 Molecular basis of orchid floral development...5
　1.5 Connections between MADS and TCP transcription factors.................................................6
2. Aim of this study....................................7
3. Materials and Methods................................8
　3.1 Plant materials and growth conditions.............8
　3.2 Arabidopsis transformation ........................8
　3.3 Yeast two-hybrid analysis.........................8
　3.4 Sequence alignments and phylogenetic analysis.....9
　3.5 Scanning electron microscopy......................9
3.6 RNA extraction and RT-PCR........................10
4. Results.............................................11
4.1 Identification of PeCYC genes in P. equestris......11
　4.2 Phylogenetic relationship of PeCYC genes and other TCP genes..................................................11
　4.3 Spatial and temporal expression of PeCYC genes in Phalaenopsis............................................11
　4.4 Analysis of protein behavior of PeCYC by using yeast two-hybrid system.......................................12
4.5 Functional analysis of the PeCYC genes by ectopic expression in Arabidopsis thaliana......................12
5. Discussion..........................................14
5.1 Sequence analysis of PeCYC genes....................14
5.2 PeCYC may be related to zygomorphy of orchid flower.14
5.3 The PeCYC1 protein behavior .......................14
5.4 Ectopic expression of PeCYC1 in Arabidopsis ........15
6. Conclusion...........................................16
7. Reference............................................17

List of Tables
Table. 1. List of primers used in this study...........25
Table. 2. Comparison of seed number between wild type and transgenic plants that ectopic express PeCYC1.....26
Table. 3. Comparison of cell size between wild type and transgenic plants. ……………27
List of Figures
Figure. 1. Alignment of amino acid sequence of PeCYC proteins and TCP protein of Oryza sativa 28
Figure. 2. Phylogenetic analyses of CYC-like genes 29
Figure. 3. Structure of P. equestris flower 30
Figure. 4. Expression patterns of PeCYC1 and PeCYC2. 31
Figure. 5. Analysis of protein-protein interactions between full-length and truncated form of PeCYC proteins by yeast two-hybrid system 32
Figure. 6. Analysis of protein-protein interactions between PeCYC proteins and B class PeMADS proteins by yeast two-hybrid system 33
Figure.7. Phenotypes of leaves and plant size in wild-type and transgenic Arabidopsis overexpress PeCYC1 34
Figure. 8. Phenotypes of flowers and siliques in wild-type and transgenic Arabidopsis overexpress PeCYC1. 35
Figure.9. Phenotypes of leaves and plant size in wild-type and transgenic Arabidopsis overexpress PeCYC2 36
Figure. 10. Scanning Electron Micrographs (SEM) of transgenic Arabidopsis overexpressing PeCYC1 and PeCYC2 . 37

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