- 學位論文
山櫻花休眠胚軸受暖溫層積誘導之PcRACK1和PcPP2CA基因的選殖與分析
Cloning and characterization of PcRACK1 and PcPP2CA genes induced during warm stratification in dormant embryo axis of Prunus campanulata
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摘要
摘要 潤濕種子置於特定溫度下以促進發芽的操作稱為層積。層積是打破種子休眠的常用方法，而層積又分為暖溫層積和冷溫層積兩種，冷溫層積廣泛應用於打破深層的種子休眠，而暖溫層積則通常與冷溫層積合併使用以促進發芽。相關研究顯示，冷溫層積可以增加種子內GA生合成基因的表現，使種子內具生物活性GAs的含量提高，因而打破種子休眠。但目前對於暖溫層積的機制則尚不清楚。有研究報告指出山櫻花種子經過暖溫層積處理後，種子內的ABA含量會降低。 本實驗室先前的研究結果在暖溫層積後的山櫻花種胚中，找到兩個與阿拉伯芥中已知的ABA負調控基因AtRACK1B及AtPP2CA很相似的基因片段，本研究的目標是釣取命名為PcRACK1和PcPP2CA的這兩個基因的全長序列，並製作過度表現PcRACK1或PcPP2CA之阿拉伯芥轉殖株，以了解它們在ABA訊息傳導以及暖溫層積對山櫻花種子造成的效用中所扮演的角色。 研究結果顯示PcPP2CA OE line的種子在添加NaCl或ABA的環境下皆較野生型具有更高的發芽率，但PcRACK1 OE line的發芽率則低於野生型。在滲透壓逆境的測試中，PcRACK1 OE line和PcPP2CA OE line的存活率都明顯的高於野生型。在離體葉片失水速率實驗中，PcRACK1 OE line的失水率較野生型略低，而PcPP2CA OE line則較野生型高。紅外線攝影的結果顯示PcRACK1 OE line的葉部溫度與野生型接近，而PcPP2CA OE line則低於野生型。乾旱逆境實驗中，PcRACK1 OE line植株與野生型的乾旱耐受能力相近，而PcPP2CA OE line的乾旱忍受度則明顯低於野生型。因此推測PcPP2CA為ABA的負調控者。基於上述結果，我們推論暖溫層積的作用機制是誘導PcPP2CA大量表現，以降低山櫻花種子對ABA的敏感度。
並列摘要
Abstract Stratification is defined as imbibed seeds under specific temperature. There’s two kinds of stratification: cold stratification and warm stratification. Cold stratification is widely used to break seed dormancy, while warm stratification combining with cold stratification is also used to break deep seed dormancy. From literatures it is known that cold stratification induces genes involved in bioactive gibberellins synthesis and promote germination. The mechanism to break dormancy by warm stratification, however, is not clear yet. Previous report indicated that seeds of Prunus campanulata after treatment of warm stratification would decrease ABA concentration. Previous work in our lab found two gene fragments resembled AtRACK1B and AtPP2CA, which were known as negative regulators of ABA signaling in Arabidopsis thaliana. This study aims to obtain the full length of these two genes, named as PcRACK1 and PcPP2CA, and to express them in Arabidopsis thaliana to study their roles played in warm stratification of Prunus campanulata seeds. Seeds of PcPP2CA overexpression (OE) line had higher germination rate on medium containing NaCl or ABA than wild type plant, while the germination rate of PcRACK1 OE line was lower than WT. The survival rate of PcRACK1 OE line and PcPP2CA OE line on medium supplemented with salt were both higher than wild type plant. In experiments qunitified the rate of water loss(RWL) of excised leaves, the result indicated that PcRACK1 OE line had slightly lower RWL than WT, while PcPP2CA OE line had higher RWL than that of WT. Infrared image obtained by infrared thermometry showed that no marked difference in canopy temperatures between PcRACK1 OE line and wild type plant, whereas canopy temperature of PcPP2CA OE line were significantly lower than wild type. PcRACK1 OE line had slightly higher drought tolerance than wild type in the drought stress experiment, while PcPP2CA OE line had much lower drought tolerance. We suggest that PcPP2CA might be a negative regulator involved in ABA signaling transduction pathway. We inferred that the underlying mechanism of warm stratification of cherry seeds is to reduce the seed sensitivity to ABA.
參考文獻
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