2019-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/691076摘要：苦瓜 (Momordica charantia L.) 是臺灣重要的夏季蔬菜，在冬季為單價最高的蔬果。苦瓜雌雄異花同株，雄花的數量顯著大於雌花，且在較低節位先著生雄花，之後再著生雌花，故其雌花數量及開花時間影響苦瓜產量及產期。本研究擬選用促進苦瓜產生雌花卻促進其他葫蘆科作物產生雄花的激勃素 (Gibberellic acid, GA)，進行苦瓜低溫浸種及幼苗葉面噴施後，抽取總RNA，利用次世代定序 (next-generation sequencing) 技術進行轉錄體分析。由於苦瓜之基因體目前僅有草稿，故本研究採用全新組裝 (de novo assembly) 及基因組參考序列製圖 (reference genome mapping) 兩種方式進行組裝，整合兩組序列所得轉錄物 (transcript) 之序列，與資料庫比對後進行註解後，將從GA差異性表現的基因中找出花性相關基因，如植物荷爾蒙生合成及訊息傳導蛋白基因、各類轉錄因子如NAC、WRKY、MYB、ARF、ERF、MADS等基因、低溫誘導基因、及二級代謝物生合成途徑相關基因，除了以即時定量反轉錄聚合&#37238;連鎖反應 (real-time quantitative reverse transcription-polymerase chain reaction, qRT-PCR)，偵測不同植物荷爾蒙處理之苦瓜內各基因表現，並進行花性調查，藉以瞭解植物荷爾蒙於苦瓜花性分化之相關機制。另針對關鍵基因選取專一性目標序列，利用CRISPR/Cas9系統以花粉電穿孔法進行苦瓜的基因編輯，調查剔除基因對苦瓜花性的影響，期望將來應用該些基因增加單株雌花表現及結果&#63849;，進行苦瓜產期調節及提高產量。本研究之期程為三年，每年之研究項目臚列如下： 1. 苦瓜轉錄體次世代定序 (第一年) (1) 苦瓜幼苗葉面噴施GA處理 (2) 苦瓜低溫及GA浸種處理 (3) 苦瓜總RNA萃取 (4) 次世代定序 (5) 轉錄體序列組裝及註解 2. 差異表現基因分析 (第二年) (1) 植物荷爾蒙生合成蛋白基因 (2) 植物荷爾蒙訊息傳導蛋白基因 (3) 各類轉錄因子如NAC、WRKY、MYB、ARF、ERF、MADS等基因 (4) 低溫誘導基因 (5) 二級代謝物生合成途徑相關基因 3. 生長調節劑處理之苦瓜花性、基因表現及功能分析 (第三年) (1) 植物材料處理及花性調查 (2) qRT-PCR分析 (3) CRISPR/Cas9基因編輯及花性調查<br> Abstract: Bitter gourd (Momordica charantia L.) is an important vegetable in Taiwan during the summer time and with the highest value per fruit during the winter time. The proportion between staminate (male) and pistillate (female) flowers in monoecious plants is quite high and pistillate flowers are generated later than staminate ones. Total RNAs extracted from bitter gourds after foliar spreading or seed soaking with gibberellic acid (GA) are conducting to next-generation sequencing for transcriptomic analysis. Combined with de novo assembly and reference genome mapping all transcripts will be aligned to the sequences in databases for functional annotation. For those genes differentially expressed real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) will be used to confirm gene expression patterns in bitter gourds treated with different plant growth regulators to understand the mechanism of sex determination and flower development in bitter gourd. The candidate genes will be edited by CRISPR/Cas9 and investigate the number of flowers. The identified sex-related genes will be used to increase the amount of pistillate flowers and to regulate their flowering time in the near future. The research items in this 3 year-proposal are as it follows: 1. Next-generation sequencing of transcriptome from bitter gourd (the first year) a. GA and cold treatment on bitter gourd b. RNA extraction and next-generation sequencing c. Read assembly and functional annotation 2. Difference analysis of gene expression (the second year) a. Genes involved in biosynthesis and signal transduction of plant hormones b. Genes encoding transcription factors c. Cold-induced genes d. Genes involved in secondary metabolisms 3. Gene and sex expression analysis in bitter gourd after treatment with plant growth regulators and gene editing by CRISPR/Cas9 (the third year) a. Chemical treatments and investigation of sex expression in bitter gourds b. Gene expression analysis using real-time quantitative reverse transcription-polymerase chain reaction c. Gene editing by CRISPR/Cas9 and investigation of sex expression in bitter gourds雌雄異花同株次世代定序基因表現分析即時定量反轉錄聚合&#37238連鎖反應基因體編輯monoecynext-generation sequencinggene expression analysisreal-time quantitative reverse transcription-polymerase chain reactiongenome editing