暖溫層積處理過程中山櫻花休眠種子的基因調控

The gene regulated in dormant seed of Prunus campanulata during warm stratification

魯建宇(Chien-Yu Lu)

指導教授 ： 林讚標

國立臺灣大學/生命科學院/植物科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.03218

摘要

植物為了選擇一個最合適的發芽時期，透過複雜的機制來調控種子休眠，近幾年來關於休眠方面的研究雖然很多，但是關於休眠的分子調控機制仍然了解得不夠透徹，尤其是關於非模式植物方面的研究了解的更少。因此，本研究以山櫻花種子做為材料，研究休眠種子在打破休眠時的相關基因調控。山櫻花新鮮收穫的種子具有很強的休眠性，需要暖溫處理和隨後的低溫層積處理才能有效的打破休眠，研究顯示，山櫻花種子需要四週的暖溫層積，和隨後八週的低溫層積，才會獲得最高的發芽率，此特殊的機制是阿拉伯芥所不具有的。低溫層積對休眠種子所造成的影響，具有何種生理功能以及基因的調控已有許多的研究；但暖溫層積有何種功效目前還不太清楚，因此將研究重點放在暖溫層積方面，利用分子生物學的抑制性扣減雜交技術，尋找在暖溫層積過程中休眠種子的基因調控模式，探討暖溫層積對於休眠種子的影響。將成熟種子採收洗淨並室內陰乾三天，之後直接進行層積處理，暖溫層積處理在進入第二和第七天分別進行扣減抑制雜合反應，完成兩天和七天暖溫層積處理之扣減式基因庫，先用Reverse Northern確認所篩選到的基因是否確實有差異性表現，再以電泳方式除去基因大小相同的片段後，才將其送去定序。序列分析比對後，成功得到暖溫層積後被大量誘導表現以及被抑制的基因共59個，分別在暖溫層積過程中扮演著各自的生理功能，包含代謝、轉錄和轉譯、訊息傳導、細胞中的過程、生長發育、養分儲存、逆境相關蛋白等。暖溫層積過程中ABA反應負調控者(negative regulator) RACK1和PP2CA的大量表現，以及ABA反應正向調控因子(positive component) 1-phosphatidylinositol-4- phosphate 5-kinase的抑制，具有重要的生理意義，顯示暖溫層積或許透過調控這些基因來降低胚軸對ABA的敏感度，以便為將來種子由休眠狀態轉變為種子發芽狀態做好準備。此外，在暖溫層積進入第七天時，山櫻花種子胚軸內ABA含量也有顯著性的下降情況。綜合研究結果來看，雖然在只有暖溫層積的情況下山櫻花種子不能發芽(GAs含量低)，但暖溫層積會透過降低種子胚軸內的ABA含量，以及透過大量表現ABA反應負調控者和抑制ABA反應正向調控因子來減少種子胚軸對ABA的敏感度，來調控種子的休眠解除。

關鍵字

山櫻花 ； 休眠種子 ； 暖溫層積 ； 離層酸 ； 扣減抑制雜合反應

並列摘要

Seed dormancy is regulated by complex networks in order to optimize the timing of germination. Although over 5000 publications on seed dormancy have appeared in the past decades, the underlying molecular regulatory mechanisms are not fully understood. Fresh seeds of Formoan cherry (Prunus campanulata Maxim), a popular ornamental tree, are deeply dormant and require warm stratification followed by cold stratification for complete dormancy-breaking. This operation is special to Formoan cherry but not to Arabidopsis. Many studies have revealed the effect of cold stratification on dormancy-breaking, but the effect of warm stratification is unclear. In this study we used the subtractive suppression hybridization (SSH) to study the gene regulation of prunus seeds undergoing warm stratification. Formoan cherry seeds were harvested, cleaned and air-dried for 3 days to reduce the water content to 15% of fresh weight and followed by warm stratification. At the second and seventh days of warm treatment, seed RNAs were extracted for SSH experiments. After constructed the subtraction libraries of second and seventh days against zero day of warm stratification, we performed reverse northern assays to confirm the upregulated and down-regulated genes. Clones obtained from this study were sequenced after removing duplicated clone containing the same length of DNA fragments. Using the website to blast the fragment sequences, we obtained 59 genes were up-regulated and down-regulated that play functions in metabolism, transcription, translation, signal transduction, growth and development, nutrient storage and stress-related. Interestingly, two ABA negative regulators, RACK1 and PP2CA were highly expressed and one positive component, 1-phosphatidylinositol-4- phosphate 5-kinase was repressed. These three regulators could reduce the ABA sensitivity of prunus seeds in the beginning of warm stratification. Moreover, we found that the ABA content was significantly decreased at the seventh days of warm stratification. Taken together, warm stratification treatment alone can not promote the seed germination due to low GA content, but can reduce the ABA sensitivity by expressing ABA negative regulators and repressing positive components in the beginning when ABA content is still high and followed by reduced the ABA content in the seeds.

並列關鍵字

Formoan cherry ； seed dormancy ； warm stratification ； abscisic acid ； subtractive suppression hybridization

參考文獻

梁華軒。 (2005)。 利用抑制性扣減雜交及二維電泳尋找紅毛草在乾旱逆境下被誘導表現的基因和蛋白質。國立臺灣大學植物科學研究所碩士論文。1-89頁。

陳舜英。 (2008)。山櫻花和楊梅種子之休眠及解除機制。國立臺灣大學森林環境暨資源研究所博士論文。1-88頁。

Chien, C.T., Chen, S.Y., and Yang, J.C. (2002). Effect of stratification and drying on the germination and storage of Prunus campanulata seeds. Taiwan J. for Sci. 4: 413-420.

transcriptional activators in abscisic acid signaling. Plant Cell 15: 63–78.

Ahn, J.W., Atwell, B.J., and Roberts, T.H. (2009). Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis. BMC Plant Biol. 9: 52.