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研究生: 洪靖雯
Hong, Jing-Wun
論文名稱: 驗證 SlSWEET1 在調控番茄植株供源及積儲間糖份分配的角色
Examination of the role of SlSWEET1 in regulating source - sink sugar distribution in tomato plants
指導教授: 郭瑋君
Guo, Woei-Jiun
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 生物科技與產業科學系
Department of Biotechnology and Bioindustry Sciences
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 110
中文關鍵詞: SWEETs糖轉運蛋白供源積儲糖份卸載
外文關鍵詞: SWEETs, sugar transporter, vegetative, source-to-sink, sugar unloading
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  • 營養階段為植物發育時的主要根基,糖類更為生長代謝重要來源。前期研究中發現 SlSWEET1a 及 SlSWEET1c 糖轉運蛋白分別高度表現在幼葉及根,可能參與植物營養階段的糖分配。為驗証此假說,本研究建立 RNAi 轉殖株進行生長功能觀察及糖含量分析,結果發現在 2-3 週營養生長植株中,SlSWEET1a 表達下降對幼葉及成熟葉的糖累積能力並無顯著影響,然而植物進入老齡 (9-10週) 階段時,表達下降的老葉累積顯著較多糖類,推測其角色可能為植物老化時將糖類運往其它積儲器官。在 SlSWEET1c 的功能分析上,發現SlSWEET1c - GFP表現細胞膜上而SlSWEET1c - GUS則高度表現於根部周鞘或是內皮層,顯示SlSWEET1c可能參與從根部糖份的卸載。進一步建立 RNAi 轉殖株進行糖含量分析,發現SlSWEET1c 表達下降並無影響 2 週大植株根的糖份累積,但在生殖階段可能影響糖類從成熟葉送至根部,且植株種子數量減少。為瞭解析供源及積儲之間的糖平衡關係,本研究中更建立 SlSWEET1a - RNAi 及 SlSWEET1c - RNAi 轉殖株的 T1雜交後代,發現以 SlSWEET1a - RNAi 為父本的雜交植株中發現花粉的受粉率較低,導致種子數量明顯減少,推測 SlSWEET1a 亦可能與花粉糖類供給有關,仍待未來研究。

    Vegetative growth determines the productivity of reproductive growth, where sugars are major source of metabolism. Previously, high expression of SlSWEET1a and SlSWEET1c sugar transporters in vegetative leaves and roots, respectively, suggests that these two genes may function in sugar allocation during vegetative growth. To examine the hypothesis, this study established RNAi transgenic tomato plants to examine growth and sugar contents. It was found that reduced expression of SlSWEET1a did not consistently affect sugar contents of young and mature leaves during vegetative ages (2-3 weeks old). Yet, at old stages (9-10 weeks old), reduced expression of SlSWEET1a resulted in sugar accumulation in old leaves, indicating that SlSWEET1a may function to allocate sugars to other sink organs during senescing. In terms of functional assay of SlSWEET1c , localization of GFP in plasma membrane and GUS fusion proteins in pericycle or endodermis cells indicated that SlSWEET1c may participate in sugar unloading to root cells. We further established SlSWEET1c-RNAi transgenic plants to analyze growth phenotypes and sugar contents. Results showed that reduced expression of SlSWEET1c did not affect sugar accumulation during the vegetative stage (2 week old), but affect reproductive growth of seeds. To further understand the sugar homeostasis between source and sink organs, we established F1 seeds of SlSWEET1a-RNAi and SlSWEET1c-RNAi lines. These lines will be investigated in the future.

    目錄 中文摘要 I 英文摘要 II 誌謝 V 目錄 VII 表目錄 XII 圖目錄 XIII 縮寫表 XVI 一、研究背景 1 1-1 植物營養發育的重要性 (The importance of plant vegetative development) 1 1-2 糖類對植物生長的重要性 (The importance of sugar for plant growth) 3 1-3 糖類在植物體的運輸 (Sugar transport in plants and the functional role of SWEET transporters in pathways) 4 1-4 糖轉運蛋白的功能 (Function of sugar transporter) 5 1-5 SWEETs糖轉運蛋白的特性 (Characteristics of SWEETs sugar transporter) 6 1-6 SWEETs糖轉運蛋白的生理功能 (Physiological function of SWEETs sugar transporter) 7 1-7 SWEETs糖轉運蛋白影響植物-微生物互動 (SWEETs sugar transporter affects plant-microbe interaction) 8 1-8 SWEETs糖轉運蛋白在番茄營養生長的角色 (The role of SWEET sugar transporter in tomato vegetative growth) 10 1-9 研究目的 (Aims) 12 二、材料與方法 14 2-1 植物材料與種植方法 (Plant materials and cultivation methods) 14 2-2 RNA萃取與反轉錄 ( RNA extraction and reverse transcription) 17 2-3 即時定量計聚合酶連鎖應 ( Real-time quantitative polymerase chain reaction transcription) 18 2-4 聚合酶連鎖反應 (PCR, Polymerase chain reaction) 19 2-5 DNA 膠體電泳 (DNA gel electrophoresis analysis) 19 2-6 DNA 純化 (DNA purification) 19 2-7 SlSWEET1c-綠色螢光蛋白融合蛋白質體建構 (Construction of SlSWEET1c-green fluorescent protein fusion protein) 20 2-8 原生質體分離以及轉型 (Protoplast isolation and transformation) 21 2-9 共軛焦螢光顯微鏡觀察 (Confocal microscopy for GFP observation) 22 2-10 建構及觀察GUS 報導基因轉殖株 (Construction and observation of transgenic plants expressing GUS reporters) 22 2-11 番茄組織糖類萃取 (Tomato tissue sugar extraction) 27 2-12 以GC-MS 分析糖類組成 (Sugar composition analysis by Gas chromatography-mass spectrometry) 28 2-13 利用酵素分析糖類組成 (Analysis of sugar composition using enzymes) 28 2-14 質體建構方法 (Plasmid construction) 30 2-15 番茄雜交試驗 (Tomato plant hybridization) 31 2-16 幼葉生長速率以及成熟葉面積觀察 (Observation of growth rate of young leaves and mature leaf area) 32 2-17 原生質流追蹤物質螢光染劑觀察 (CFDA ,Observe carboxyfluorescein diacetate) 32 三、結果 34 3-1 SlSWEET1a 在花苞不同階段以及花朵部位的表現 (SlSWEET1a expression at different stages of flower buds and flower parts) 34 3-2 確認 SlSWEET1a 突變轉殖株的基因表現 (Confirm the gene expression of SlSWEET1a in young leaves in SWEET1a RNAi transgenic plants) 34 3-3 SlSWEET1a突變轉殖株營養生長觀察 (Observation on the vegetative growth of SlSWEET1a mutant transgenic plants) 35 3-4 SlSWEET1a 突變轉殖株生殖生長觀察 (Reproductive growth of SlSWEET1a RNAi transgenic plants) 36 3-5 SlSWEET1a 缺失對葉片糖份累積能力的影響 (Effect of SlSWEET1a defects on leaf sugar accumulation capacity) 36 3-6 SlSWEET1a 對糖份運輸速率的影響 (The influence of SlSWEET1a on sugar transportation rate) 40 3-7 SlSWEET1c蛋白在細胞內表現位置 (Subcellular localization of SlSWEET1c proteins) 40 3-8 SlSWEET1c蛋白在組織器官的表現位置 (Tissue-specific expression of SlSWEET1c in tomato plants) 41 3-9 確認 SlSWEET1c-RNAi 轉殖株的基因表現量 (Confirm gene expression in SlSWEET1c-RNAi plants) 44 3-10 SlSWEET1c-RNAi轉殖株表現型觀察 (Phenotypes of SlSWEET1c-RNAi transgenic plants) 45 3-11 SlSWEET1c-RNAi轉殖株的根部生長分析 (Root growth analysis of SlSWEET1c-RNAi mutant) 46 3-12 SlSWEET1c對番茄營養階段根部糖份累積的影響 (Effects of SlSWEET1c on root sugar transport in tomato vegetative stage and reproductive stage) 47 3-13 SlSWEET1c對番茄生殖階段根部糖份累積的影響 (Effects of SlSWEET1c on root sugar transport in tomato reproductive stage and reproductive stage) 47 3-14 確認 SlSWEET1c過表達植株的基因表現量 (Confirm gene expression of SlSWEET1c overexpression plants) 48 3-15 分析SlSWEET1a 和SlSWEET1c 的交互作用 (The interaction between SlSWEET1a and SlSWEET1c) 49 四、討論 50 4-1 SlSWEET1a 在番茄不同生長階段可能扮演著不同 (SlSWEET1a plays different roles in different stages of tomato growth) 50 4-2 SlSWEET1a缺失並不影響幼葉生長發育 (Reduced expression of SlSWEET1a does not affect leaf growth) 52 4-3 SlSWEET1a可能參與花粉發育 (SlSWEET1a may be involved in pollen development) 54 4-4 SlSWEET1c可能作用在細胞膜上 (SlSWEET1c may function on the plasma membrane) 54 4-5 SlSWEET1c可能位於根部周鞘或內皮當中 (SlSWEET1c may be located in the pericycle or Endodermis of the root) 55 4-6 SlSWEET1c可能影響著番茄植株根部的糖份運輸 (SlSWEET1c may affect sugar transport in tomato roots) 56 4-7 SlSWEET1c功能缺失可能影響番茄種子發育 (SlSWEET1c loss of function may affect tomato seed development) 57 4-8 結論 (Conclusion) 57 參考文獻 59 圖表 70 表目錄 表一、用於質體建構引子序列 71 表二、即時定量計聚合酶連鎖反應 (qRT-PCR) 所使用引子 72 圖目錄 圖一、 SlSWEET1a在不同階段花苞以及花朵部位的表現量 73 圖二、 確認 SlSWEET1a-RNAi T2轉殖株當中SlSWEET1a 的 基因表現 74 圖三、 確認 SlSWEET1a-RNAi T2轉殖株當中SlSWEET1a cDNA的基因表現 75 圖四、 SlSWEET1a-RNAi 轉殖株種子列表 76 圖五、 SlSWEET1a-RNAi T3 轉殖株營養生長觀察 77 圖六、 SlSWEET1a-RNAi T3 轉殖株生殖生長觀察 78 圖七、 SlSWEET1a-RNAi T3轉殖株在營養階段糖份含量分析 (ㄧ重複) 79 圖八、 SlSWEET1a-RNAi T3轉殖株在生殖階段糖份含量分析 (ㄧ重複) 80 圖九、 SlSWEET1a-RNAi T3轉殖株在營養階段糖份含量分析 (二重複) 81 圖十、 SlSWEET1a-RNAi T3轉殖株在營養階段糖份含量分析 (三重複) 82 圖十一、 SlSWEET1a-RNAi T2 轉殖株在老齡階段糖份含量分析 (一重複) 83 圖十二、 SlSWEET1a-RNAi T3轉殖株在老齡階段糖份含量分析 (二重複) 84 圖十三、 SlSWEET1a-RNAi T3轉殖株運輸速率觀察 85 圖十四、 建構 SlSWEET1c-GFP 複合蛋白於植物細胞之表現質 體 86 圖十五、 SlSWEET1c-GFP 於植物細胞內表現位置 (共表達細胞 膜複合蛋白) 87 圖十六、 SlSWEET1c-GFP 於植物細胞內表現位置 (共表達液 胞膜複合蛋白) 88 圖十七、 表現 SlSWEET1c-GUS 複合蛋白所使用之表現質體 89 圖十八、 瞬時表達 SlSWEET1c-GUS 複合蛋白於番茄組織 90 圖十九、 SlSWEET1c-GUS 蛋白於番茄組織的專一性表達 91 圖二十、 糖對 SlSWEET1c-GUS 的表現影響 92 圖二十一、 SlSWEET1c cDNA-GUS 蛋白於番茄組織的轉譯表達 93 圖二十二、 SlSWEET1c 蛋白於番茄組織的轉錄表達 94 圖二十三、 SlSWEET1c 蛋白於番茄根部組織切片 95 圖二十四、 SlSWEET1c-RNAi T2轉殖株篩選 (SlSWEET1c-RNAi -2) 96 圖二十五、 SlSWEET1c-RNAi T2轉殖株篩選 (SlSWEET1c-RNAi -6) 97 圖二十六、 SlSWEET1c-RNAi 轉殖株抗性比率統計 98 圖二十七、 確認 SlSWEET1c-RNAi T2轉殖株當中SlSWEET1c的 基因表現 99 圖二十八、 SlSWEET1c 轉殖株種子列表 100 圖二十九、 SlSWEET1c-RNAi T2轉殖株生殖生長觀察 101 圖三十、 SlSWEET1c-RNAi T3轉殖株在不同糖環境下根部的生 長觀察 102 圖三十一、 SlSWEET1c-RNAi T3轉殖株在營養階段糖份含量 分析 103 圖三十二、 SlSWEET1c-RNAi T3轉殖株在生殖階段糖份含量分 析 104 圖三十三、 SlSWEET1c T1過表達轉殖株同源篩選 105 圖三十四、 SlSWEET1c 過表達轉殖株抗性比率統計 106 圖三十五、 確認 SlSWEET1c T2 過表達植株的基因表現 107 圖三十六、 SlSWEET1a-RNAi以及SlSWEET1c-RNAi雜交番茄果 實表現型觀察 108 圖三十七、 SlSWEET1a在營養階段以及老齡階段功能模型 109 圖三十八、 SlSWEET1c在根部的功能模型 110

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