劉麗飛臺灣大學：農藝學研究所劉秋萍Liu, Chiu-PingChiu-PingLiu2007-11-282018-07-112007-11-282018-07-112006http://ntur.lib.ntu.edu.tw//handle/246246/59165細胞週期為植物生長發育之重要過程，其中需要諸多重要基因之參與及調控，此些關鍵的調控者，除了參與DNA之複製及細胞分裂外，亦與生長發育過程中訊息的傳遞相關。為了瞭解植物細胞週期調控之機制及植物之生長分化，本試驗利用農桿菌轉殖法，並以CaMV35S啟動子將三個持續性表現的基因 (AtCDC25、AtR2、及OsR2) 轉殖至菸草中，探討此三個與細胞週期調控相關的基因對菸草生長發育及細胞分裂的影響。 試驗結果總計獲得45株轉殖株，包括27株單基因轉殖株，19株雙基因轉殖株，及2株三基因轉殖株；以RT-PCR分析T0代轉殖菸草，50%以上的轉殖株具有基因之表現。外表型方面，T0代轉殖株生長型態與非轉殖株差異不大，少數轉殖株葉緣有向上翻摺的情形，且轉殖株的開花時間有延遲之現象。T0代轉殖株葉片組織切片觀察發現，海綿細胞數目明顯增加，較非轉殖菸草增加約1.5倍以上，且部分轉殖株下表皮細胞數目亦有增加之趨勢；此外，將葉片細胞數對照轉殖株基因表現，可得知基因表現量高的轉殖株，葉片細胞數目增加情形略為明顯，且細胞堆疊排列的程度較為嚴重，尤以三基因組合的轉殖株效果最大；由此證明大量表現AtCDC25 ，AtR2，及OsR2基因可增加細胞的分裂，且使細胞形狀變小；此外，分析T1代轉殖株發芽能力，轉殖株發芽勢及發芽率均較非轉殖株為低。 綜合而言，大量表現AtCDC25 、AtR2、及OsR2三個與細胞週期調控相關的基因，會造成菸草開花時間延遲，細胞分裂的速度加快，細胞變小，並使後代發芽能力降低；而轉殖株外表型變異不大，則可能與擾動細胞週期後，基因本身之互補調控有關。Many essential genes are required to participate in and regulate the progress of cell cycle which is an important process for plant growth and development. These key regulators not only participate in DNA synthesis and cell division but also get involved in the transmission of signals during growth and development. In order to understand the molecular mechanisms in controlling plant cell cycle accompanied with plant growth and development, Agrobacterium - mediated gene transfer method was used to introduce three cell-cycle related genes (AtCDC25, AtR2, and OsR2) which are driven under CaMV35S promoter into tobacco (Nicotiana tobaccum). The effects of these genes on tobacco growth and development were then investigated. The results showed that totally 45 transgenic tobaccos were obtained, including 27 lines with single gene insertion, 19 lines with two genes insertion, and 2 lines with three genes inserted in tobacco genome. Analysis of the transgene expression in T0 transgenic tobaccos by RT-PCR indicated that there were more than 50% transgenic plants with foreign gene expressed. The phenotypes of T0 transgenic plants showed no obvious variation. However, a few of them displayed aberrant leaves and delayed flowering. Microscopic anatomy analysis of leaf morphology showed that there were 1.5 folds increase in the number of spongy cells in transgenic tobacco; also the cell number of the lower epidermis was increased. In addition, we noticed that transgenic tobacco with high levels of gene expression, especially lines with three genes inserted significantly correlated with much more cell numbers and serious mis-arranged cells in leaf. Our results demonstrated that overexpression of AtCDC25, AtR2, and OsR2 can enhance the cell division and reduce the size of cells. Finally, analysis of the germination ability of T1 transgenic plants suggested that both the germination tendency and germination rate were lower than those of wild type. Taken together, overexpression of AtCDC25, AtR2, and OsR2 leads to delay of flowering time, increase of cell division rate, reduction of the cell size, and decrease of the germination ability of transgenic tobaccos. The phenotypes of transgenic tobacco were similar to non-transgenic plants. The interpretation for this may be due to the compensatory mechanism in cell cycle after disruption of this process by overexpress of foreign genes.目錄………………………………………………………………………………. I, II 縮寫對照表………………………………………………………………………....III 中文摘要…………………………………………………………………………....IV 英文摘要…………………………………………………….……………….......V, VI 第一章、前言…………………………………………………………………1 第二章、文獻回顧…………………………………………………………………3 第一節、植物細胞週期………………………........……………….………….……3 第二節、細胞週期相關基因表現對植物生長發育的影響……...................………….…5 第三節、G1/S時期的OsR2基因……………………………...........…………….…6 第四節、持續性表現於細胞週期各時期之AtR2基因……………................…………7 第五節、G2/M時期的CDC25與WEE1基因………………………..............………7 第三章、材料與方法……………………………………………………………….... 8 第一節、試驗材料………………….......…………………………………….……8 第二節、基因之釣取…………………………….......……………………….……9 第三節、基因轉殖之質體製備……………………………………..........…………11 第四節、菸草基因轉殖…………………………………………........……………13 第五節、確認基因轉殖菸草插入之基因……………………………............………16 第六節、轉殖菸草基因表現分析…………………………………...........…………18 第七節、基因轉殖菸草之植物葉部形態切片分析…………………...........…………20 第八節、T1代轉殖菸草生長速率測試…………………………...............…………21 第四章、試驗結果…………………………………………………….……..……… 22 第一節、基因釣取……….......……………………………………………………22 第二節、構築質體………….......…………………………………………………23 第三節、轉殖效率……………….......……………………………………………23 第四節、轉殖菸草之確認………………………………........……………………24 第五節、轉殖菸草之生長發育…………………………………..........……………25 第六節、轉殖菸草之基因表現………………………………….........……………25 第七節、轉殖菸草葉片組織之變化……………………………...........……………26 第八節、T1代轉殖菸草種子發芽與幼苗生長特性………………..................………27 第五章、討論................................................................................................................29 參考文獻.................................................................................................................35 圖表..................................................................................................................42 附表..................................................................................................................66 附圖..................................................................................................................691449339 bytesapplication/pdfen-US細胞週期菸草:植物生長發育cell cycletobacco:plant growth and developmentthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59165/1/ntu-95-R93621102-1.pdf