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研究生: 陳佑亦
Chen, You-Yi
論文名稱: 蘭科植物YABBY基因全基因組鑑定及蝴蝶蘭DL/CRC基因之功能探討
Genome-wide identification of YABBY genes in Orchidaceae and functional characterization of DL/CRC genes in Phalaenopsis orchids
指導教授: 蔡文杰
Tsai, Wen-Chieh
共同指導: 張松彬
Chang, Song-Bin
學位類別: 博士
Doctor
系所名稱: 生物科學與科技學院 - 生命科學系
Department of Life Sciences
論文出版年: 2021
畢業學年度: 109
語文別: 英文
論文頁數: 130
中文關鍵詞: 合蕊柱YABBY基因蘭花胚珠姬蝴蝶蘭
外文關鍵詞: gynostemium, YABBY genes, orchid, ovule, P equestris
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  • YABBY基因是植物特有的一群小基因家族,屬於鋅指超家族的成員,對於葉片近軸-遠軸極性及生殖組織生長扮演重要角色。蘭花是被子植物中最大的科之一,具有獨特的花部形態、生殖生物學及多樣化的生長方式。在蘭花中關於YABBY基因功能尚不清楚。在本研究中,我們從8個蘭花物種 (深圳擬蘭、鐵皮石斛、天麻、紫金舌唇蘭、廣東舌唇蘭、姬蝴蝶蘭、平葉香莢蘭、深圳香莢蘭)中鑑定出54筆YABBY基因。在親緣演化分析中發現蘭花的YABBY基因主要分為四大類,其中15筆基因屬於DL/CRC基因群,8筆基因屬於INO基因群,17筆基因歸類於YAB2基因群,及14筆基因屬於FIL基因群。基因表現分析結果顯示DL/CRC基因主要表現在花早期發育時期及雄雌蕊癒合形成的合蕊柱中,而INO基因則表現在珠被發育時期,我們也在花早期發育時期檢測到FIL基因的表現。此外,YAB2基因群中的PeYAB2,PeYAB3 及 PeYAB4則表現在營養組織及生殖組織中,其中PeYAB4具有較高的基因表現量。同時我們也對屬於DL/CRC基因群的PeDL1及PeDL2進行功能性分析。研究結果顯示兩筆PeDL基因皆可部分恢復阿拉伯芥crc-1突變株的外表型。而過量短暫表現PeDL基因於蝴蝶蘭中會造成胚珠及蕊柱腔的發育不正常。在基因靜默的實驗也發現胎座上,胚珠始原發育的數量也受到影響。在蛋白質交互作用分析中顯示PeDL2無法與PeCIN8形成異型二聚體,而僅有PeDL1能與PeCIN8形成異型二聚體。這些研究結果顯示蘭花旁系同源的兩筆DL / CRC基因在生殖組織具有重要的新功能。此結果也為蘭科植物中的YABBY基因功能研究開啟新的研究方向。

    The small plant-specific YABBY gene family, belonging to the zinc-finger superfamily, plays important roles in the adaxial-abaxial leaf polarity and reproductive organ development. Orchid is one of the largest families in angiosperm and known for their unique floral morphology, reproductive biology, and diversified lifestyles. However, nothing is known about the role of YABBY genes in orchids. In this study, a total of 54 YABBY genes, including 15 genes in DL/CRC, eight in INO, 17 in YAB2, and 14 in FIL clade, were identified from the eight orchid species (Apostasia shenzhenica, Dendrobium catenatum, Gastrodia elata, Platanthera guangdongensis, Platanthera zijinensis, Phalaenopsis equestris, Vanilla planifolia, Vanilla shenzhenica,). A phylogenetic analysis showed all 54 orchid YABBY genes could be classified into four major clades, including DL/CRC, INO, FIL, and YAB2. An expression analysis of Phalaenopsis YABBY genes revealed that members in the DL/CRC clade have concentrated expressions in the early floral development stage and gynostemium, the fused male and female reproductive organs. The expression of PeINO is consistent with the biological role it played in ovule integument morphogenesis. Transcripts of members in the FIL clade could be obviously detected at the early developmental stage of the flowers. The expression of three genes, PeYAB2, PeYAB3, and PeYAB4, in the YAB2 clade could be revealed both in vegetative and reproductive tissues, and PeYAB4 was transcribed at a relatively higher level than that of PeYAB2 and PeYAB3. Meanwhile, functional characterized of DL/CRC gene PeDL1 and PeDL2 in Phalaenopsis orchids. The results showed that both PeDLs could partially complement the Arabidopsis crc-1 mutant. Transient overexpression of PeDL1 in Phalaenopsis orchids caused abnormal development of ovule and stigmatic cavity of gynostemium. Virus-induced gene silencing (VIGS) of PeDL1 and PeDL2 in Phalaenopsis orchid affects the number of protuberant ovule initials differentiated from the placenta. In the protein-protein interaction analysis, PeDL1, instead of PeDL2, could form a heterodimer with PeCIN8. These results reveal the paralogous of two DL/CRC-like genes in orchids play important roles in orchid reproductive organ innovation. These results provide basic information for understanding the function of the YABBY gene in Orchidaceae.

    Content 中文摘要 i Abstract ii 致謝 iv Chapter 1 Literature survey 1 1.1 Orchidaceae 2 1.1.1 Taxonomy of Orchidaceae 2 1.1.2 Morphology of Phalaenopsis 2 1.2 Reproductive organs in plant kingdom 3 1.2.1 The reproductive organs in angiosperms 3 1.2.2. Evolution of reproductive organs in Orchidaceae 3 1.2.3. Key regulatory event of post-pollination in Phalaenopsis orchid 4 1.2.4 The ovule development in Phalaenopsis 4 1.3. The YABBY transcription factor 5 1.3.1 A small plant specific YABBY gene family 5 1.3.2 The YABBY genes of FIL, YAB2, YAB3, and YAB5 in angiosperms 6 1.3.3 Foundation of the YABBY genes CRC/DL and INO for establishment of carpel and ovule formation in angiosperms 7 1.4 Aims 7 Chapter 2 Genome-wide identification of YABBY genes in Orchidaceae and their expression patterns in Phalaenopsis orchids 2.1 Abstract 10 2.2 Introduction 10 2.3 Materials and methods 13 2.4 Results 15 2.5 Discussion 20 Table (2-1~ 2-3) 24 Figure (2-1~ 2-17) 33 Chapter 3 Ancestral duplicated DL/CRC orthologs display function on orchid reproductive organ innovation 3.1 Abstract 51 3.2 Introduction 51 3.3 Materials and methods 54 3.4 Results 60 3.5 Discussion 70 Table (3-1~ 3-6) 75 Figure (3-1~ 3-23) 94 Chapter 4 Conclusions and perspectives 121 References 123

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