臺灣博碩士論文加值系統

CONSTANS (CO) 為一於植物自生長期轉變為生殖期之光週期途徑中扮演重要角色的轉錄因子家族，其中包含了十七個COL基因，並在結構上分別於N端具有一或兩個與蛋白質交互作用相關的保守B-box 區域，以及C端含有帶入核訊息且和與HAP2具高度保守性且可進一步與HAP3/5進行下游基因調控之CCT區域。前人研究顯示，在植物花藥開裂的調控機制上，阿拉伯芥之COL9/10扮演著抑制者，而蘭科物種COL9/10則扮演著促進者的角色。為了探討COL9/10同源基因於不同物種中調控花藥開裂的功能，本研究進一步選殖並分析菸草（NbCOL9/10）、水稻（OsCOL3/11）及大根槽舌蘭（HolCOL9）中COL9/10同源基因之功能。NbCOL9/10、OsCOL3/11及HolCOL9之序列分別構築於結合抑制子之SRDX domain以及活化子的VP16 domain的載體，並將其轉殖入阿拉伯芥中以觀察及進行功能性分析。實驗結果發現大量表現NbCOL9/10及OsCOL3/11之轉殖株無法表現穩定的性狀，顯示NbCOL9/10及OsCOL3/11於阿拉伯芥中可能受到其他因素干擾。經由各物種中同源COL9/10的序列比對發現， NbCOL9/10及OsCOL3/11之CCT區域與AtCOL9/10及蘭科COL9/10具有差異，此差異可能影響NbCOL9/10及OsCOL3/11與阿拉伯芥中的HAP3及HAP5的交互作用，導致無法順利結合下游基因進行調控。HolCOL9與PaCOL9具有96 % 之胺基酸序列相同性，推測可能與PaCOL9具有相似的花藥發育調控功能。另一方面，本研究藉由顯微鏡切片觀察發現，具有自花授粉機制的香港綬草之花粉塊仍保有與阿拉伯芥相似的花藥室內壁次級細胞壁增厚機制，了解其COL9/10同源基因功能將有助於探討COL9/10是否參與花藥到花粉塊間的演化過程。

CONSTANS (CO) is a transcriptional factor that belongs to CONSTANS-Like (COL) family and plays an important role in regulating vegetative-to-reproductive transition. COL family contains 17 different CO/COL proteins share one or two B-box domains at the N-terminus involved in protein-protein interaction, and a CCT domain at the C-terminus contained nuclear localization signals and a region highly conserved with HAP2 to interact with HAP3/5 for DNA binding and downstream genes regulation. We have previously shown that orchid’s COL9/10 functioned as activators, whereas Arabidopsis COL9/10 played as repressors to regulate anther dehiscence. To further determine the role of COL9/10 homologs in controlling anther dehiscence in different plant species, COL9/10 homologs from Nicotiana benthamiana (NbCOL9/10), Oryza sativa (OsCOL3/11) and Holcoglossum amesianum (HolCOL9) were cloned and investigated in this study. The constructs contain these genes fused with either repressor domain SRDX (COL-SRDX) or activator domain VP16 (COL-VP16) were transformed into Arabidopsis for functional analysis. Our results showed that ectopic expression of NbCOL9/10 and OsCOL3/11 exhibit unstable phenotype in Arabidopsis. These observations indicated that some factors may disrupt the functions of NbCOL9/10 and OsCOL3/11 in Arabidopsis. In amino acid alignment, the CCT domain of NbCOL9/10 and OsCOL3/11 were distinct from AtCOL9/10 and orchid’s COL9/10. This may alter the abilities of COL9/10 to interact with HAP3/5, bind DNA and regulate downstream genes in transgenic plants. HolCOL9 share 96 % identity of amino acid sequence with PaCOL9. It could function similarly with PaCOL9 in regulating anther development. Furthermore, we discovered a self-pollinated orchid - Spiranthes hongkongensis, which maintains the similar mechanism of endothecial secondary cell wall thickening during the maturation of pullinia as seen in Arabidopsis. It would be of great interest to investigate if COL9/10 of Sprianthes hongkongensis participated in the mechanism in regulating the process from anther to pollinium during evolution

摘要 i
Abstract iii
前言 1
材料與方法 6
結果 14
一、各物種花藥室內壁結構觀察 14
二、菸草、水稻及大根槽舌蘭中CONSTANS-Like基因選殖及序列分析 14
三、CONSTANS-LIKE基因大量表現、強制抑制及強制活化重組載體之構築15
四、異位表現COL9/10於阿拉伯芥轉殖株所造成之花藥不開裂性狀不穩定 16
五、共軛焦顯微鏡觀察顯示轉殖株花藥不開裂性狀之成因並不一致 17
六、NbCOL9-2/9-5/10、OsCOL3/11序列差異造成於基因下游調控功能失活 17
七、綬草之花藥室內壁結構觀察 18
討論 19
參考文獻 23
圖表 27
表1、本研究中所使用之引子（primer）列表 27
表2、水稻OsCO/COL基因家族之基因位址及其同義名 29
圖1、不同類型花藥之外觀及其授粉機制 30
圖2、本氏菸草之生殖器官外觀及構造 31
圖3、大根槽舌蘭營養及生殖器官之外觀及構造 32
圖4、以共軛焦顯微鏡（confocal）觀察阿拉伯芥、菸草及大根槽舌蘭之花藥室內壁結構 33
圖5、COL9/10同源基因與各物種CO/COL基因家族成員之胺基酸序列演化
樹分析 35
圖6、本氏菸草NbCOL9-2 cDNA之序列及其相對應胺基酸 36
圖7、本氏菸草NbCOL9-5 cDNA之序列及其相對應胺基酸 37
圖8、本氏菸草NbCOL10 cDNA之序列及其相對應胺基酸 38
圖9、水稻OsCOL3 cDNA之序列及其相對應胺基酸 39
圖10、水稻OsCOL11 cDNA之序列及其相對應胺基酸 40
圖11、大根槽舌蘭HolCOL9 cDNA之序列及其相對應胺基酸 41
圖12、NbCOL9-2/9-5/10、OsCOL3/11及HolCOL9與CO/COL基因家族成員之胺基酸序列演化樹分析 42
圖13、各物種COL9/10同源基因之胺基酸序列比對 43
圖14、菸草NbCOL9-2基因之選殖及35S::NbCOL9-2/NbCOL9-2-SRDX/NbCOL9-2-VP16之構築 45
圖15、菸草NbCOL9-5基因之選殖及35S::NbCOL9-5/NbCOL9-5-SRDX/NbCOL9-5-VP16之構築 47
圖16、菸草NbCOL10基因之選殖及35S::NbCOL10/NbCOL10-SRDX/NbCOL10-VP16之構築 49
圖17、水稻OsCOL3基因之選殖及35S::OsCOL3/OsCOL3-SRDX/OsCOL3-VP16之構築 51
圖18、水稻OsCOL11基因之選殖及35S::OsCOL11/OsCOL11-SRDX/OsCOL11-VP16之構築 53
圖19、大根槽舌蘭HolCOL9基因之選殖及35S:: HolCOL9 / HolCOL9 -SRDX/ HolCOL9 -VP16之構築 55
圖20、異位表現35S::NbCOL9-2及NbCOL9-2-VP16於阿拉伯芥造成花朵雄蕊
及果莢性狀之分析 57
圖21、異位表現35S::NbCOL10、 NbCOL10-SRDX及NbCOL10-VP16於阿拉伯芥造成花朵雄蕊及果莢性狀之分析 58
圖22、異位表現35S::OsCOL11及OsCOL10-SRDX於阿拉伯芥造成花朵雄蕊及果莢性狀之分析 60
圖23、NbCOL10轉殖株之基因表現鑑定 61
圖24、OsCOL11轉殖株之基因表現鑑定 62
圖25、以共軛焦顯微鏡（confocal）觀察35S::NbCOL9-2及NbCOL9-2-VP16雄不稔轉殖株之花藥室內壁 63
圖26、以共軛焦顯微鏡（confocal）觀察35S::NbCOL10、NbCOL10-SRDX及NbCOL10-VP16雄不稔轉殖株之花藥室內壁 65
圖27、以共軛焦顯微鏡（confocal）觀察35S::OsCOL11及OsCOL11-VP16雄不稔轉殖株之花藥室內壁 67
圖28、CO/COL轉錄因子家族之下游基因結合調控機制 69
圖29、COL9/10同源基因之CCT區域序列比對 70
圖30、香港綬草營養及生殖器官之外觀及構造 71
圖31、以共軛焦顯微鏡（confocal）觀察阿拉伯芥、綬草及文心蘭之花藥室內壁結構 72
圖32、共軛焦顯微鏡（confocal）觀察綬草花藥室內壁結構之近觀 74
附錄1、花藥構造及發育期間之細胞分化途徑 75
附錄2、花藥發育各時期總覽 76
附錄3、阿拉伯芥中組織次級細胞壁生合成相關調控途徑 77
附錄4、NF-YA基因胺基酸序列及CO/COL基因之CCT domain胺基酸序列比較 78
附錄5、AtCOL9/10、OnCOL9/10、PaCOL9及CaCOL10於阿拉伯芥花藥發育功能性之假說 79
附錄6、水稻（Oryza sativa）之花藥結構橫切面圖 80
附錄7、阿拉伯芥花藥開裂過程中之花藥壁結構變化示意圖 81
附錄8、pGEM®-T Easy vector之載體圖譜（3018 bp） 82
附錄9、pEpyon-32K之載體圖譜（10624 bp） 83
附錄10、pEpyon-3aK之載體圖譜（10132 bp） 84
附錄11、pEpyon-3aK之載體圖譜（10080 bp） 85
附錄12、Gen-KB DNA ladder 86

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