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系統識別號 U0026-2808202015491700
論文名稱(中文) 蘭科植物全基因體比較獨腳金內酯訊息傳導基因及臺灣白花蝴蝶蘭PaD14s功能分析
論文名稱(英文) Genome-wide comparison of strigolactones signaling genes in Orchidaceae and functional characterization of PaD14s in Phalaenopsis aphrodite subsp. formosana
校院名稱 成功大學
系所名稱(中) 熱帶植物與微生物科學研究所
系所名稱(英) Institute of Tropical Plant Sciences and Microbiology
學年度 108
學期 2
出版年 109
研究生(中文) 李竺
研究生(英文) Chu Lee
電子信箱 clheue@gmail.com
學號 Z36064018
學位類別 碩士
語文別 英文
論文頁數 82頁
口試委員 口試委員-陳文輝
口試委員-陳虹樺
口試委員-張文綺
口試委員-蕭郁芸
指導教授-蔡文杰
中文關鍵字 阿拉伯芥  獨腳金內酯  訊息傳導基因  蘭花  蝴蝶蘭 
英文關鍵字 Arabidopsis  orchid  Phalaenopsis  strigolactones  signaling pathway 
學科別分類
中文摘要 蝴蝶蘭不僅開花期長,還具有優雅多變的花形,廣受全球民眾的歡迎。
台灣是蝴蝶蘭的主要出口國之一,在經濟作物中佔有一席之地。借助分子生物學技術,我們可以深入瞭解並研究蝴蝶蘭基因組,利用蝴蝶蘭基因組在模式植物上的表達,研究出蘭花市場喜愛的花形、花色、花香……等等的特徵,以縮短傳統培育新品種蘭花需要的時間,從而促進蝴蝶蘭 產業的發展。
獨腳金內酯(Strigolactones,SLs)是一群從類胡蘿蔔素生合成的植物激素,1995年 被確認是內源性植物訊息傳導化合物。在SL訊息傳導 機制中,SL和接收器α/β水解酶(receptor, D14)結合,結合後會再和具有E3泛素連接蛋白(F-box protein, D3)結合導致轉錄因子抑制蛋白(transcription factor repressor, D53)降解,從而達到下游基因表達的目的。SL在植物發育中具重要作用,包括抑制植物腋芽分支生長、誘導共生菌與植物互利共生和調節植物體營養組織生長作用 。
其中SL經由植物根部釋放到土壤中誘導真菌與植物共生的作用對蘭花來說特別重要,因為蘭花種子極度依賴共生菌的協助才能獲足夠的碳源以利生長;除此之外,有鑑於SL會影響植物分枝數量,若是藉此增加蘭花花梗數量,想必在花卉市場中會更加受歡迎。然而,很少有研究探討SL在蝴蝶蘭中的作用 。本論文主要探討蘭花中獨角金內酯的感知和訊息傳導路徑中相關基因的作用。
首先,我們通過LC-MS / MS檢測了蝴蝶蘭中共有兩種SLs。根據測序的蘭花基因組數據庫,我們在蝴蝶蘭中鑑定出兩個SL接受器基因(PaD14-1,PaD14-2),其餘檢測蘭花,包含石斛、香莢蘭、擬蘭、舌唇蘭、虎舌蘭和天麻…等等所有撿測的蘭花基因租中也都各含有兩個SL接受器基因。在蝴蝶蘭中鑑定出一個轉錄因子抑制基因(PaD53),在其餘蘭花基因組中也各檢測到一個抑制基因,然而香莢蘭中則有兩個抑制基因。在所有蘭花基因組中都有一個F-box蛋白基因,包含蝴蝶蘭中也僅有一個F-box蛋白基因(PaD3),除了紫金舌唇蘭有兩個F-box蛋白基因。通過qRT-PCR分析,我們發現在營養和生殖組織中,PaD14-1的表達遠高於PaD14-2。其中PaD14s, PaD53,PaD3皆是在營養組織中的表達量高於生殖組織中的表達量。進一步利用酵母菌雙雜合系統和雙分子螢光互補作用方法,可以觀測到PaD14s和PaD53間具有蛋白交互作用。藉由在阿拉伯芥中過表達PaD14-1基因來進行基因功能分析,阿拉伯芥PaD14-1OE植物顯示出與野生型Col-0類似的外表型,其中腋芽分枝數量沒有明顯增加,葉片數則是略有增加,幼苗側根與主根長度明顯地縮短。由此可以確認PaD14-1確實會影響阿拉伯芥外表型,並確認PaD14-1是具有功能的SL接受器直系同源基因。
英文摘要 Phalaenopsis orchids have become globally popular for their long-lasting flowers with elegant floral morphology and various flower colors. Taiwan is one of the major exporting countries of Phalaenopsis taking an important role in economic crops. With molecular biology technology, we can have insights into Phalaenopsis genome which can promote the development of floricultural industry.
Strigolactones (SLs), as a family of carotenoid-derived phytohormone, are recently recognized as endogenous plant signaling compounds. SL signal transduction mechanisms employ α/β-hydrolase-derived receptors (D14) that confer E3 ubiquitin ligase-mediated protein (D3) degradation processes which lead transcription factor repressor protein (D53) degradation. SLs play important roles in plant development, including inhibiting plant axillary branching, inducing parasitic interactions and various plant stress responses. After SLs released in the rhizosphere, SLs would be able to attract mycorrhizal fungi to symbiosis with host plants. This SL function is especially important for orchid because orchids seed depends on symbiotic fungi to obtain enough carbon source and grow properly. In addition, SLs can also affect the axillary branching number. If orchids can increase branch numbers by manipulating SLs, I believe orchids will be much more popular.
However, there are seldom researches studying the roles of SL in Phalaenopsis. In this study, we detected two different SLs in Phalaenopsis orchid by LC-MS/MS. Based on the sequenced orchid genome database, we identified two SL receptor genes in all detected orchids and named SL receptor genes in Phalaenopsis aphrodite as PaD14-1 and PaD14-2. There is one transcription factor repressor gene in every detected orchid genome except Vanilla containing two repressor genes. We named the repressor gene in P. aphordite as PaD53. There is one F-box protein in tested orchid genomes except two in Platanthera zijinensis. By qRT-PCR analysis, we found that PaD14-1 expression was obviously higher than PaD14-2 in all detected P. aphordite tissues and that all SL signaling related genes in P. aphordite had higher expression level in vegetative tissues than in reproductive tissues. This result might illustrate to that SLs function mainly affect in vegetative tissue development.
With yeast two hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay, identification of the interaction between α/β hydrolase PaD14s and repressor protein PaD53 was proven in P. aphrodite in the presence of exogenously applied GR24, a synthetic SL analog. Functional analysis was performed by overexpression of PaD14-1 gene in Arabidopsis. The Arabidopsis PaD14-1OE plants displayed a similar phenotype to the wild-type Col-0 with only slightly increased leaf numbers and obviously shortened the length of seedling primary roots and lateral root. Based on this thesis results, SL was confirmed the existence in P. aphrodite and PaD14-1 is confirmed as one of the functional SL receptor genes.
論文目次 中文摘要 I
Abstract III
誌謝 V
Contents VII
List of Tables IX
List of Figures X
List of Appendix Tables XI
List of Appendix Figures XII
Abbreviations XIII
1. Introduction 15
1.1 Orchidaceae 15
1.1.1 The unique features of Orchids 15
1.1.2 Germination of orchid seeds 16
1.1.3 Orchid genome sequencing 17
1.2 The role of strigolactones in plant 18
1.2.1 Strigolactones history and chemistry 18
1.2.2 Strigolactones signaling pathway 19
2. Aim of this study 21
3. Material and Methods 22
3.1 Plant material 22
3.2 Detection of strigolactone in P. aphrodite 22
3.3 RNA preparation 23
3.4 Sequence retrieval for phylogenetic analysis 23
3.5 Real-time quantitative RT-PCR 24
3.6 Bimolecular fluorescence complementation (BiFC) assay 25
3.7 Yeast two-hybrid analysis 26
3.8 Construction of transformed fusions and Arabidopsis transformation 26
4. Results 27
4.1 Strigolactone detection in Phalaenopsis aphrodite subsp. formosana 27
4.2 Identification and analysis of SL signaling genes in the genome sequenced orchids 28
4.3 Expression pattern of receptors and repressor genes in Phalaenopsis 30
4.4 The protein interaction between PaD14 and PaD53 31
4.5 The functional analysis of PaD14-1 receptor gene 32
5. Discussion 34
5.1 Analysis of SL receptor, PaD14s, in P. aphrodite 34
5.2 Analysis of TF repressor protein in P. aphrodite 36
5.3 Protein behaviors between PaD14s and PaD53 37
6. Conclusion and perspectives 38
7. References 39
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