Molecular regulation of sink-source transition in rice leaf sheaths during the heading period

水稻上位葉葉鞘為醣類之暫存組織，其在抽穗前會大量累積澱粉，而在抽穗後其儲存之澱粉會快速分解，並合成蔗糖運送到充實中的穀粒，故在抽穗前後期，葉鞘將由儲存組織轉變成供源組織，此現象稱之葉鞘儲存-供源轉換。為了瞭解葉鞘儲存-供源轉換之分子調控機制，本論文以real-time RT-PCR分析在水稻抽穗期間，葉鞘中澱粉合成、澱粉分解與蔗糖轉運蛋白 (sucrose transporter ; OsSUT)等相關基因之表現變化，結果發現ADP-glucose pyrophosphorylase large subunit 2、granule-bound starch synthase II、soluble starch synthase I、starch branching enzyme (SBE) I、SBEIII及SBEIV等六個澱粉合成相關基因為影響葉鞘澱粉於抽穗前大量累積之主要基因，且其啟動子共同具有受荷爾蒙調控之序列。另一方面，澱粉分解基因α-amylase2A、β-amylase與蔗糖轉運蛋白基因OsSUT1、4在抽穗後表現量會增高，其很有可能分別參與葉鞘在抽穗後時期，澱粉分解及將蔗糖裝載到韌皮部之過程。進一步利用各種荷爾蒙處理水稻切離葉鞘以探討這些基因受荷爾蒙調控之情形，結果發現ABA會抑制AGPase及GBSS之酵素活性，但是會大量促進α-amylase與β-amylase的活性及OsSUT1及 4的基因表現量，顯示ABA可能為影響葉鞘中醣類代謝反應之因子之一。為了篩選參與水稻葉鞘儲存-供源轉換的調控因子，還藉由基因槍暫時性表現系統，分析OsSUT4基因之不同長度啟動子於抽穗前後的表現，鑑定出一段67 bp之啟動子片段，其可能包含控制OsSUT4基因於抽穗後大量表現之重要cis-acting elements。此外，以microarray技術分析抽穗前後之水稻基因組表現變化，發現sucrose synthase、β-D-glucan exohydrolase、sorbitol transporter、ammonium transporter及phosphate transporter等基因也於抽穗前後具有差異性表現，暗示不僅澱粉代謝與蔗糖轉運蛋白基因，還有其他醣類代謝途徑與胺基酸、無機磷之運送亦參與葉鞘儲存-供源之轉變過程。Upper leaf sheath of rice (Oryza sativa L.) serves as a temporary carbohydrate sink tissue before panicle heading. Starch pre-stored in upper leaf sheaths prior to heading would be remobilized to filling grains at post-heading stage. Thus, upper leaf sheaths could be converted to source tissues from sink tissue during heading period. The process of starch changes in leaf sheath is defined as the sink-source transition. The purpose of this project is to reveal the molecular mechanism of the sink-source transition in rice leaf sheaths during heading period. First, the expression profiles of genes involved in starch synthesis pathway were analyzed and compared to starch content in the second leaf sheath below the flag leaf. The results indicated the changes of ADP-glucose pyrophosphorylase large subunit 2 (AGP-L2), granule-bound starch synthase II (GBSSII), soluble starch synthase I (SSSI), starch branching enzyme (SBE) I, SBEIII, and SBEIV mRNA levels were highly correlated with starch content changes during the heading period in the leaf sheath, and these starch synthesis-related gene promoters shared several common hormone-responsive elements. In addition, the α-amylase2A and β-amylase were considered as major genes that regulated the starch degradation at the post-heading period. Of the five sucrose transporter (OsSUT) genes, OsSUT1 and OsSUT4 appeared to play an important role in sucrose loading into the phloem of source leaf sheaths. Besides, to reveal whether phytohormones were the fac-tors to control the starch metabolism-related enzyme activities and OsSUTs gene ex-pressions, the effects of GA、ABA and BAP on expressions of these enzymes and genes in detached leaf sheaths were examined. The results indicated that not only the expres-sions of OsSUT1 and 4 but the activities of α-amylase and β-amylase can be enhanced by ABA, suggested that ABA is one of the factors to regulate the carbohydrate metabolism in leaf sheaths. Since OsSUT4 gene was significantly up-regulated at post-heading stage, it was used as an indicator gene to identify the molecular mechanism of sink-source transition in leaf sheaths. In order to find the cis-acting elements on OsSUT4 promoter involved in controlling rice leaf sheaths sink-source transition during the heading period, we constructed nine various 5’-deletion OsSUT4 promoter fragments containing GUS reporter gene and analyzed the avtivities by particle bombardment assay. A 67-bp promoter fragment was identified, which might contain important regulatory elements involved in regulation of OsSUT4 gene up-expression in leaf sheath at post-heading stage. Moreover, microarray analysis was also applied to study the mechanism of sink-source transition in leaf sheaths during heading period. The data implied that the dominant processes associated with functional leaf sheath transition from sink to source were not only carbohydrate metabolism but also the translocation of the nitrogen sources and inorganic phosphate.目錄………………………………………………………………………….…………..1表與附錄目錄………………………………………………………………….……..3寫字對照表…………………………………………………………………….……..5文摘要…………………………………………………………………………….…..7文摘要…………………………………………………………………………….…..8言. 水稻葉鞘於抽穗前後期所扮演之角色……………………………..……..…..10. 葉片儲存-供源轉換之相關研究…………………………………………….….11. 水稻澱粉合成之相關基因……………………………………………………...12. 水稻澱粉分解之相關基因………………………………………………......….14. 澱粉代謝相關基因之影響因子…………………………………………..........15. 水稻蔗糖轉運蛋白基因之相關研究……………………………..………...…..16. 本論文之試驗架構及意義………………………………….………...….…..….18料與方法. 植物材料、種植與處理方法…………………………………………………….20. 醣類定性與定量分析………………………………………………………..….21. 基因表現分析………………………………………………………………..….23. Microarray分析……………………………………………………………...….24. 蛋白質萃取、定量與酵素活性分析…………………………………………….25. ABA萃取與含量分析………………………………………..…………………30. 啟動子釣取及序列分析…………………………………………………..….…32. 以暫時性表現系統進行啟動子活性分析…………...…………………………33. Electrophoresis Mobility Shift Assay (EM-SA) ……………..……..……………370. 以穩定性表現系統進行啟動子活性分析.……….……..……………………..411. 統計分析……………………………………………………………...……...…44果. 葉鞘中澱粉合成、澱粉分解與蔗糖轉運蛋白基因於抽穗期間之表現….……45.1. 水稻上位葉葉鞘在抽穗期間其澱粉與可溶性醣類含量變化……………45.2. 移除幼穗之水稻其葉鞘澱粉含量於抽穗期間之變化……………………45.3. 澱粉合成相關基因在葉鞘儲存-供源轉變期間之表現…………………...46.4. α-amylase, β-amylase及OsSUTs基因於葉鞘儲存-供源轉變期間之表現..47. 以Microarray分析儲存期及供源期葉鞘中之基因表現……………………....47. 探討荷爾蒙是否為調控葉鞘儲存-供源轉換之內在因子……………….….....49.1. 澱粉合成基因啟動子分析…………………………………………….…...49.2. 荷爾蒙傳導相關轉錄因子-OsDOF3與OsWRKY71在葉鞘中於抽穗期間之表現分析………………………………………………………..……..….50.3. 植物荷爾蒙對切離葉鞘中澱粉含量的影響…............................................50.4. 植物荷爾蒙ABA對切離葉鞘中澱粉代謝相關酵素活性及OsSUTs基因表現的影響……………………………………….............................................51.5. 抽穗期間水稻葉鞘中內生ABA含量與澱粉分解酵素活性變化………..52. 探討水稻葉鞘於儲存-供源轉換時，調控OsSUT基因表現之訊息因子……...52.1. 台農67水稻之OsSUT1、2、4基因之啟動子分子分析………………….52.2. OsSUT1、2及4基因啟動子於葉鞘中的暫時性表現分析………….....….53.3. OsSUT4基因不同長度啟動子片段之暫時性表現分析………………..….55.4. OsSUT4基因之DF(-550/-484)與DF(-643/-603)啟動子片段進行EMSA分析.....................................................................................................................56.5. OsSUT4基因不同長度啟動子之穩定性表現分析………………………...57論. 水稻TNG67之-2葉葉鞘為抽穗期間主要澱粉儲存組織…………….……...59. 稻穗的抽出及其碳源需求調控抽穗後時期葉鞘的供源能力…….………....59. 水稻葉鞘儲存-供源轉變之標識基因……………………………………..…..60. 影響葉鞘中澱粉代謝的內在因子…………………………………………….63. 影響葉鞘中蔗糖轉運蛋白表現的內在因子……………………………….....66. 調控葉鞘中OsSUT4在抽穗後大量表現的啟動子區域.……………………..68. 葉鞘組織暫時性表現系統之建立………………………………….…………70. 結語與未來展望……………………………………………………………….70考資料…………………………………………………..………………...……..…..72表與附錄目錄一、針對澱粉合成、澱粉分解相關基因及蔗糖轉運蛋白設計進行real-time RT-PCR之專一性引子………………………………………………………………...81二、水稻-2葉葉鞘於抽穗期間澱粉合成相關基因表現與澱粉含量變化之相關分析…………………………………………………………………………...…83三、針對Microarray所篩選出之基因設計進行Real-time RT-PCR或半定量RT-PCR之專一性引子……………………………………………………….84四、以real-time RT-PCR分析由Microarray所篩選出基因在抽穗期間之表現……………………………….………...........................................……...…86五、葉鞘澱粉合成主要基因之啟動子序列比對……….…….…………………….88六、設計合成OsSUTs與TRXh基因啟動子之專一性引子…………………...……89七、設計專一性引子，合成5’端deletion OsSUT4啟動子之片段與EMSA之DNA 探針………………………………………………………………………….…90八、OsSUT4基因不同長度啟動子於TNG67水稻之轉殖效率….……………...…91九、OsSUT4基因啟動子DF(-550/-484)片段上已知cis-acting ele-ments…………..92一、水稻葉片及葉環相對位置……………………………….……………………..93二、水稻上位葉葉鞘在抽穗期間的澱粉含量變化……….….…………………….94三、水稻抽穗期間-2葉葉鞘醣類含量變化……….…….……………………….…95四、正常抽穗與除去幼穗之水稻植株-2葉葉鞘澱粉變化趨勢…………….…..…96五、澱粉代謝相關基因於抽穗前之表現趨勢……………………………………...97六、葉鞘中澱粉分解相關基因於抽穗期間之表現…………………….………..…98七、葉鞘中OsSUTs基因於葉鞘於抽穗期間之表現…………………..……..…….99八、葉鞘於儲存及供源時期呈現差異性表現基因之分群與歸類….……………100九、葉鞘醣類代謝及分子轉運蛋白相關基因之表現………..…….……………..102十、葉鞘中荷爾蒙訊息傳導相關轉錄因子於抽穗期間之表現.............................103十一、植物荷爾蒙對水稻葉鞘生長及其澱粉含量之影響.....................................104十二、植物荷爾蒙ABA對水稻葉鞘澱粉合成與分解相關酵素活性之影響….105十三、植物荷爾蒙ABA對水稻葉鞘中OsSUTs基因表現之影響…..……………107十四、葉鞘中ABA含量與澱粉分解酵素活性在抽穗期間的變化………………108十五、OsSUT1 基因啟動子序列…………………….……….…...…….…………109十六、OsSUT2 基因啟動子序列……………………….....................………….…111十七、OsSUT4 基因啟動子序列……….....................………………………….…112十八、TRXh與Ubi啟動子之暫時性表現分析……………….……………………113十九、OsSUT promoter::GUS暫時性表現質體建構圖I…………………………..114二十、OsSUT promoter::GUS暫時性表現質體建構圖II……..………………….115二十一、OsSUT1、2及4啟動子於葉鞘中的暫時性表現分析…………….……...116二十二、OsSUT4 promoter::GUS表現質體建構圖……………...………………..117二十三、OsSUT4 啟動子5’-deletion片段活性分析………….………….…….…118二十四、EMSA所用DNA探針位置………………………………………………120二十五、OsSUT4啟動子片段與葉鞘核蛋白之結合分析……………………...…121二十六、OsSUT4啟動子片段與葉鞘核蛋白之競爭型結合分析………………...122二十七、OsSUT promoter::GUS穩定性表現質體建構圖………………...………123二十八、OsSUT promoter::GUS質體於轉殖水稻植株之表現………….…....…..124二十九、OsSUT promoter::GUS質體於轉殖水稻穎花之表現….…….……...…..125三十、OsSUT promoter::GUS質體於轉殖水稻之啟動子活性分析………….…..126錄一、木村氏水耕液配方……………………………………………………….…127錄二、水稻肥料施加表………………………………………………………….…128錄三、pAHC18 map……………………………………………………………..…129錄四、水稻基因轉殖各培養機配方………………………………………...….…13