王淑美臺灣大學：植物科學研究所林立傑Lin, Li-ChiehLi-ChiehLin2007-11-272018-07-062007-11-272018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/57962文心蘭在夏季高溫的環境下，可縮短花芽發育期，但花序之品質卻明顯降低，花軸分叉數和總花朵數都顯著減少，部分花朵亦有畸形的現象，且花軸長度也明顯短於在常溫下生長的花序。迄今我們對於影響文心蘭花序分叉數和花朵唇瓣發育的機制，以及參與調控之相關基因的種類及弁鄎o毫無所知。為期有效地改善夏季高溫影響花序及花苞發育的情形，本論文選殖在高溫下影響文心蘭唇瓣發育之相關基因，做為日後研究之基礎。實驗採用文心蘭（Oncidium）切花品種南西（Grower Ramsey）三年株齡之植物個體為材，分別培養在適溫（28/23℃）及高溫（35/30℃）之下，然後取其唇瓣，利用DDRT-PCR（differential display reverse transcription PCR）方法，挑出表現具有差異性之基因片段1323個，選殖獲得324個基因建構體，經定序比對，再從中挑選11個與已知的細胞壁合成、代謝及影響細胞正常生長之基因具有高度相似性者，設計引子以RT-PCR方式，研究上述基因在高溫影響下不同時期唇瓣之表現活性的變化模式。在適溫下發育的唇瓣，其OACS、OPHAN1、OPMEI、OV-ATPase、OKTN、OExp1和OAuxRFP等七個基因的表現活性均明顯高於高溫處理的唇瓣，顯示這些基因的表現可能對於唇瓣正常發育具正向的作用；而OASD、OCCR、OEGase和OGA2ox等四個基因，在高溫下的活性皆明顯上升，依這些基因被推論的弁遄A可能高溫刺激這些基因的活性而干擾了正常發育所必須的基因活性。又由GA噴灑試驗，推測GA是文心蘭唇瓣發育擴張的必要因子之一。本文為初步探討受高溫影響之文心蘭唇瓣發育的相關基因，及荷爾蒙可能參與的角色，詳細機制尚待進一步的研究Oncidium Grower Ramsey is one of major cut flower species in Taiwan, and the flower production is all over the year. However, high temperature of summer and autumn results in the poor quality of cut flowers, by that not only the branching and floral number of inflorescence drop severely, but the length of inflorescence is shorter than that developed in cool seasons. Besides, high temperature might cause the floral lips smaller and slightly deformed. I focused my study in the cloning of floral development-related genes that are interfered by high temperature. Pot plants with developing inflorescences were cultivated in growth chambers setting at regular (28/23℃) or high temperature (35/30℃), and floral lips at various developmental stages were collected for RNA extraction. By using DDRT-PCR technique, 1323 gene fragments shown differential expression were collected and 324 of them were cloned and sequenced. After Blast alignment, ten clones possessing high homology with known genes related to cell wall metabolism and cell expansion were selected and used as probes to examine the gene expression pattern during floral development. The expression of six genes（OACS, OPHAN1, OV-ATPase, OKTN, OExp1 and OAuxRFP）in lip tissues increased along with the normal development of floral bud; and high temperature hampered their gene activities. The expression of other four genes（OASD, OCCR, OEGase and OGA2ox）which were selected from high temperature-treated lips was confirmed to be enhanced by high temperature. OGA2ox was predicted to encode GA-2-oxidase which is a well-known enzyme to regulate the level of biologically active GAs in plant tissues. Although the GA content in floral lips were not determined, exogenous GA application to floral buds grown at high temperature was conducted to test the effect of GA on lip development. The results suggested that GA should be involved in the regulation of lip expansion.中文摘要…………………………………………………………………..i 英文摘要…………………………………………………………………..ii 前言……………………………………………………………………….1 材料與方法…………………………………………………...................9 結果…………………………………………………………..................25 一、 文心蘭花軸性狀與生長時期之溫度的關係.................................................25 二、 唇瓣發育與溫度的關係………………………………............................26 三、 mRNA Differential Display system之建立…………………….................26 四、 差異性cDNA片段定序與序列比對………………………………………...27 五、 以RT-PCR分析相關基因在不同時期唇瓣中的表現模式……………………….30 六、 文心蘭α-L-arabinofuranosidase之基因全長的選殖和分析…………………..33 七、 高溫下唇瓣的生長發育與GA作用…………………………………………35 討論…………………………………………………………..................37 參考文獻……………………………………………………..................44 圖表…………………………………………………………..................57 附圖…………………………………………………………..................901623357 bytesapplication/pdfen-US文心蘭荷爾蒙唇瓣高溫liphigh temperatureOncidiumGAotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57962/1/ntu-93-R90226021-1.pdf