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研究生:謝婷宇
研究生(外文):Ting-Yu Hsieh
論文名稱:阿拉伯芥第一類BPCs調節CYC/TB1家族對花朵對稱性與分枝形成的作用
論文名稱(外文):The regulatory role of Arabidopsis Class I BPCs in floral symmetry and branching formation associated with CYC/TB1 family
指導教授:王俊能蔡皇龍
指導教授(外文):Chun-Neng WangHuang-Lung Tsai
口試委員:余天心林盈仲
口試委員(外文):Tien-Shin YuYing-Chung Jimmy Lin
口試日期:2023-07-19
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生態學與演化生物學研究所
學門:生命科學學門
學類:生態學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
論文頁數:77
中文關鍵詞:BASIC PENTACYSTEINE (BPC)TEOSINTE BRANCHED 1- CYCLOIDEA-PCF(TCP)GAGA 結合因子花對稱性花序分枝
外文關鍵詞:BASIC PENTACYSTEINE (BPC)TEOSINTE BRANCHED1-CYCLOIDEA-PCF(TCP)GAGA-binding factorfloral symmetrybranch
DOI:10.6342/NTU202302755
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花朵的對稱性影響植物繁衍下一代的效率。已知CYCLOIDEA(CYC)為不同植物物種花朵對稱性的關鍵因子。本論文針對植物特有的GAGA序列結合因子,Class I BASIC PENTACYSTEINE(BPC)基因家族對阿拉伯芥CYC及其同源基因調控機制進行研究。本實驗室過往以大岩桐(Sinningia speciosa)SsCYC 基因上游片段進行酵母菌單雜交(yeast one-hybrid)篩選阿拉伯芥轉錄因子基因庫,結果發現阿拉伯芥之BPC1和BPC2能夠結合於篩選用的核酸片段,顯示BPCs可能調控CYC 基因。於是本論文首先檢測阿拉伯芥bpc突變體的花朵對稱性,發現bpc1-1 bpc2(bpc1,2)和bpc1-1 bpc2 bpc3-1(bpc1,2,3)突變體之花朵由輻射對稱(radial symmetry)轉變成兩側對稱(bilateral symmetry)。進一步測試BPC與SsCYC之阿拉伯芥同源基因TEOSINTE BRANCHED 1-CYCLOIDEA-PCF1 (AtTCP1)之關聯性,發現BPC2和BPC3可抑制,而BPC1則可活化TCP1的轉錄,顯示不同的BPC成員對TCP1的作用有差異。由於bpc突變體亦發生了花序結構的改變,因此也檢測了BPC對於花序分枝調節因子TCP12和TCP18的影響。結果發現BPC1、BPC2和BPC3皆可抑制TCP12,反之卻活化TCP18的轉錄。在bpc突變體中TCP18的下調導致了分枝增加,與其已知抑制花序分枝的作用一致。總結本論文研究發現Class I BPC可藉由調節CYC與其同源基因來調控花朵對稱性和花序分枝的途徑,本研究為花朵對稱性和花序分枝發育的調節機制提供了進一步的分子證據。
Flower symmetry is crucial for the reproductive success. The CYCLOIDEA (CYC) gene has been identified as a critical regulator of floral symmetry across different plant species. In this study, I investigated the involvement of Class I BASIC PENTACYSTEINEs (BPCs), plant-specific GAGA-motif binding factors, in the regulation of CYC and its homologs in Arabidopsis. Based on the results of yeast one-hybrid, we found that BPC1 and BPC2, both members of Class I BPC, can bind to the upstream region of CYC in Sinningia speciosa, indicating a potential role in floral symmetry regulation. I therefore examined the floral symmetry of the bpc mutants and observed a remarkable alternation. Flowers of bpc1-1 bpc2 (bpc1,2) and bpc1-1 bpc2 bpc3-1 (bpc1,2,3) mutants are changed from radial symmetry to bilateral symmetry. This prompted me to relate the BPCs to the SsCYC homolog in Arabidopsis, TEOSINTE BRANCHED 1-CYCLOIDEA-PCF1 (AtTCP1). I demonstrated that BPC2 and BPC3 act as inhibitors, while BPC1 enhances the transcript level of TCP1, indicating that various BPC members modulate TCP1 differently in Arabidopsis. As the bpc mutants showed altered architectures of the inflorescence, the impact of BPCs on TCP12 and TCP18, known regulators of inflorescent branching, was investigated. I found that BPC1, BPC2, and BPC3 repressed TCP12 while activated TCP18. The downregulation of TCP18 in the bpc mutants resulted in a significant increase in the number of branches, consistent with its inhibitory role in the inflorescent branching. In summary, my study provides novel insights into the regulatory mechanisms governing floral symmetry and inflorescent branch development. It highlights the potential role of Class I BPCs in modulating the expression of CYC homologs, as well as their impact on the intricate processes shaping floral symmetry and inflorescent branching in Arabidopsis.
口試委員會審定書 I
誌謝 II
摘要 IV
ABSTRACT V
LIST OF TABLE AND FIGURES IX
LIST OF SUPPLEMENTAL FIGURES XI
CHAPTER 1. INTRODUCTION 1
1.1 TCP gene family 1
1.2 CYC/TB1 gene family and floral symmetry 2
1.3 The regulation of floral symmetry in Sinningia speciosa 5
1.4 Class I BPC may regulate TCP1 in Arabidopsis thaliana 6
1.5 Phenotype of Class I BPC mutant lines and related CYC/TB1 genes 7
CHAPTER 2. MATERIALS AND METHODS 9
2.1 Plant material and growth conditions 9
2.2 Cloning of the constructs 10
2.3 Agrobacterium-mediated transformation of Arabidopsis thaliana 10
2.4 RNA extraction and reverse transcription 12
2.5 Polymerase chain reaction ( PCR ) 14
2.6 Quantitative real-time polymerase chain reaction ( qRT-PCR ) 15
2.7 Western blotting 16
2.7.1 Total protein preparation from BPC1, BPC2, and BPC3 inducible lines 16
2.7.2 SDS-PAGE preparation 17
2.7.3 Electrophoresis and blot transfer 17
2.7.4 Blotting and detection 18
2.8 Morphology analyzing 20
2.8.1 Flower petals angle measuring 20
2.8.2 Inflorescence branches counting 21
2.9 Scanning electron microscopy (SEM) analysis of floral organs development 21
2.1 TCP1 expression traced by GUS (GUS staining) 22
2.11 DNA extraction and genotyping 24
CHAPTER 3. RESULTS 26
3.1 TCP1 is a potential target of Class I BPCs 26
3.2 Flowers of bpc1,2 and bpc1,2,3 mutants are zygomorphic 27
3.3 The expression of TCP1 in the inflorescence was regulated by Class I BPCs 28
3.4 bpc mutants have more branches than the wild type does 30
3.5 TCP12 and TCP18 promoters are potential targets of Class I BPCs 31
3.6 TCP12 and TCP18 expression in inflorescence was regulated by Class I BPC. 32
3.7 Class I BPCs differentially regulate the TCP1 promoter 34
CHAPTER 4. DISCUSSION 36
4.1 Class I BPC can regulate CYC/TB1 genes TCP1, TCP12, and TCP18 directly or indirectly. 36
4.2 Loss of Class I BPC function may induce TCP1 expression, affecting petal primordia development. 37
4.3 Regulation of TCP12 and TCP18 by Class I BPC members may involve the participation of auxin. 39
CHAPTER 5. CONCLUSION 42
REFERENCES 43
TABLE 50
FIGURES 51
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