Regulation of Sucrose Transporter Genes in Rice Embryos during Seed Germination

水稻種子發芽時期依賴的養分為胚乳澱粉所分解的醣類。由胚乳澱粉所分解而來的葡萄糖將在胚中之胚盤(Scutellum)中重新合成蔗糖，而蔗糖會進一步轉運至幼芽及根部做為幼苗生長所需之養分來源，顯示蔗糖於組織間的轉運效率為影響水稻發芽及幼苗生長之重要因子。為了解水稻發芽種子中醣類的運輸機制，本論文以carboxyfluorescein diacetate (CFDA)觀察醣分子利用原生質絲在各組織間運送之情形，另一方面，亦用real-time RT-PCR分析在種子發芽過程中，水稻蔗糖轉運蛋白基因家族(sucrose transporter gene family; OsSUTs)之表現，並利用各種醣類及其類似物處理水稻分離胚以探討OsSUT基因表現受醣類調控之情形及訊息傳導機制。種子由開始浸潤後直至四葉齡，胚乳、胚與莖部及根部之間可利用原生質絲進行醣類的運輸，並由螢光定量的結果推測胚乳醣份運送至幼芽及根部的比例會隨生長階段而有所不同。此外，在蔗糖轉運蛋白基因表現分析中，發現種子浸潤後，在胚中的 OsSUT1、2及4的表現量遠比OsSUT3及5高。其中，OsSUT2之表現明顯地隨種子浸潤後之天數而增加。外加各種醣類於培養分離胚(isolated embryo)的培養基中，發現100 mM葡萄糖、蔗糖皆會促使OsSUT1及2基因表現，但OsSUT1與2之表現對醣類反應的敏感度不同，而OsSUT4僅受100 mM葡萄糖之影響，並不受蔗糖調控。葡萄糖對OsSUT1的影響是藉由糖解作用中之中間產物進行訊息傳導;而葡萄糖調控OsSUT2及4則是藉由hexokinase-indepdent pathway。除了醣類分子，GA與光照亦為調控OsSUT基因表現之因子，GA會促使OsSUT4的表現上昇，而光照會促使OsSUT1及2的表現量增高。綜合實驗結果發現，雖然OsSUT1、2及4基因均在種子發芽時期明顯表現，但此三基因之表現調控機制卻有顯著地差異。Sugars degraded from endosperm starch are the major nutrient source for rice seed germination. Since glucose transported from endosperm to scutellum would be resynthesized to sucrose in scutellum and further transported to shoot and root for seedling growth, the efficiency of sucrose transport between different tissues could be an important factor to determine seedling establishment and growth. In order to clarify the mechanism of sucrose transport during rice seed germination, an effort was made to apply carboxyfluorescein diacetate (CFDA) into seeds for monitoring sugar transport from endosperm to embryo, shoot and root. In addition, the expressions of sucrose transporter family genes (OsSUTs) were analyzed by real-time RT-PCR during seed germination. To identify the sugar effect on OsSUT gene expressions and the signal transduction pathway involved in the sugar-mediated regulation, various sugar molecules and their analogs were supplied to isolated embryos cultures. The data presented in this thesis indicated that sugars can be transported from endosperm to embryo、stem and root mediated by plasmodemata before 4 leaf-old stage. The ratio of endosperm sugar transported to shoot and to root would be changed at different growth stages. During seed germination, the expression levels of OsSUT1、2 and 4 in embryos were higher than OsSUT3 and 5. OsSUT1 and 2 expressions could be enhanced by 100 mM glucose and sucrose, but their sensitivities in response to sugar were different. OsSUT4 expression was also up-regulated by glucose but not by sucrose. Even glucose was a common factor to control the expression of OsSUT1, 2 and 4, the sugar-mediated signal transduction pathways were different. The intermediate product of glycolysis was an essential signal for enhancing OsSUT1 expression. On the other hand, glucose-enhanced OsSUT2 and 4 expressions were controlled by hexokinase -independent pathway. In addition to sugar effect, GA and light effects on OsSUTs genes were also identified in this study. The results showed the expression of OsSUT4 could be enhanced by GA, and expressions of OsSUT1 and 2 could be stimulated by light.目錄-------------------------------------------------------------------------------------------------Ⅰ 圖表目錄-------------------------------------------------------------------------------------------Ⅲ 縮寫對照表----------------------------------------------------------------------------------------Ⅳ 中文摘要-------------------------------------------------------------------------------------------Ⅴ 英文摘要-------------------------------------------------------------------------------------------Ⅵ 壹、 前言------------------------------------------------------------------------------------------1 貳、 材料方法 Ⅰ、 植物材料-----------------------------------------------------------------------------10 Ⅱ、 水稻種子內澱粉分佈情形的觀測-----------------------------------------------10 Ⅲ、 水稻胚乳醣類經由原生質絲轉運之分析--------------------------------------10 Ⅳ、 水稻蔗糖轉運蛋白(Sucrose transporter; OsSUT )家族基因及Ubiquitin cDNA 片段的選殖----------------------------------------------------------------11 Ⅴ、 OsSUT家族基因的表現分析----------------------------------------------------15 Ⅵ、基因表現量的均值檢定-------------------------------------------------19 Ⅶ、 OsSUT家族基因啟動子的序列分析--------------------------------------------19 參、 結果 Ⅰ、 水稻種子內澱粉分佈之情形-----------------------------------------------------20 Ⅱ、 利用carboxyfluorescein diacetate (CFDA) 模擬醣類藉由原生質絲從胚乳轉運至幼芽及根部-----------------------------------------------------------------20 Ⅲ、 水稻種子浸潤後0至5天，胚部OsSUT家族基因表現-------------------21 Ⅳ、 不同醣類對分離胚中OsSUT1、2及4表現之影響---------------------------22 Ⅴ、 醣類調控OsSUT1、2及4基因表現之訊息傳導途徑------------------------23 Ⅵ、 不同濃度GA對分離胚中OsSUT1、2及4表現之影響-----------------------25 Ⅶ、 光對分離胚中OsSUT1、2及4表現之影響-------------------------------------25 Ⅷ、 OsSUT1、2及4啟動子之分析-------------------------------------------------26 肆、 圖表-----------------------------------------------------------------------------------------28 伍、 討論-----------------------------------------------------------------------------------------57 參考文獻------------------------------------------------------------------------------------------6