激勃素對苦瓜花性之影響及轉錄體分析

Abstract

苦瓜 (Momordica charantia L.) 為熱帶地區的葫蘆科作物，花性為雌雄異花同株，雄雌花數比介於 8 到 25 之間。苦瓜花性分化機制尚未明瞭，本研究以不同的花性誘導處理做為變因，從共差異表現基因推測可能的生理調控機制，期望研究結果能應用於苦瓜栽培育苗產業，透過減少雄雌花數比及降低始花節位，達到提高產量和產期調節之目的。以雌雄異花同株品系‘月華’分別進行六到八葉幼苗期施用 100 mg·L-1 GA3、以 4 ̊C 低溫浸種 15 天的促雌處理；以 4 ̊C 低溫浸種 15 天同時施以 100 mg·L-1 GA3 的抑雌處理，以及僅在葉面噴施蒸餾水的對照組，取六到八葉幼苗期地上部總 RNA，以 Illumina RNA-Seq 定序，新組裝出132,706 條 contig之轉錄體後，比較線上資料庫進行基因註解，搭配線上基因體草稿將覆蓋的讀序數量換算成 TPM (transcript per million) 推估表現量。依據不同的差異表現方向分類共差異表現基因，篩選出與花性有關的組別，進行基因本體論富集分析，觀察到除了細胞訊息傳遞有關的蛋白質磷酸化和脂質反應之生物程序，植物賀爾蒙訊息傳遞及轉錄因子相關的基因也顯著富集。以定量即時聚合酶鏈鎖反應 (quantitative real-time PCR, qRT-PCR) 進行基因表現量分析，驗證植物賀爾蒙生合成及訊息傳遞基因在不同處理組中的調節反應，觀察到相符合的趨勢。由新組裝轉錄體之基因產物預測出 1215 個轉錄因子，經過涵蓋前人研究之基因表現量矩陣綜合評比，篩選出了 5 個分別來自於 HD-ZIP、WRKY、MYB、M-type MADS、以及 bHLH 基因家族的花性分化相關轉錄因子。針對 MADS-box 基因家族進行全基因體識別分析，顯示苦瓜中有 34 個 McMADSs，啟動子區段序列順式作用元件分析顯示激勃素、離層酸、組織特異性、細胞週期等等因子都有可能影響其差異性轉錄。

Bitter gourd (Momordica charantia L., 2n = 2x = 22) is an important tropical and sub-tropical Cucurbitaceae crop. Bitter gourd is a monoecious plant whose male/female flower ratio varies from 8 to 25. Regulation of sex determination could modify the production period and yield. As the first step to understand the sex determination mechanism of bitter gourd the transcriptomes of GA3-spread seedlings at 6-8 leaf stage and seeds soaked with GA3 were compared in aid of next-generation sequencing technology. Pooled reads from four samples were de novo assembled by Trinity. The number of contigs were 132,707 and N50 of the assembly reached 2,921 bp. TPM (transcripts per million) calculated by kallisto were used for gene expression inference. Co-differentially expressed genes were categorized according to their expression pattern in three treatment comparison groups in order to narrow down sex determination related candidate genesd. Gene ontology enrichment analysis results showed that not only cell signal transduction related terms including protein autophosphorylation and lipid response but also biological process terms including plant hormone signal transduction and transcription factors were significantly enriched. Gene expression of plant hormone biosynthesis and signal transduction related genes were detected by quantitative real-time PCR (qRT-PCR) for validation. On top of that, 1,215 transcription factors were predicted from the transcriptome sequences. Five sex determination related transcription factors , from HD-ZIP, WRKY, MYB, M-type MADS, and bHLH gene families, were selected for future research according to a comprehensive gene expression matrix, including expression profiles of different organs and cultivars of bitter gourd obtained from two BioProjects, PRJNA 213805 and PRJNA 319011. MADS-box genes, known to participate in both floral development and GA-signaling, were picked up for genome-wide identification and analysis. Classification of 34 MADS-box proteins in bitter gourd is very similar to that in watermelon. Nearly half of the MADS-box genes were specifically expressed in flower, while the others mainly appeared in leaf and root. Sex determination mechanism may be different between monoecious and gynoecious cultivars. MADS-box genes may be differentially induced by hormones and environmental factors.