華八仙(八仙花科)類花瓣構造與花部器官決定基因之研究

胡哲明臺灣大學：生態學與演化生物學研究所張天豪Chang, Tien-HaoTien-HaoChang2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/55097大而明顯的花瓣常是植物用來吸引傳粉者的構造，而一些植物會使用整個花序聚集形成一個如同一朵花的假單花(pseudanthium)構造來達到類似的功能。以八仙花科(Hydrangeaceae)植物為例，它的花序最外圍的花常為不孕性並且具有膨大而有顏色的花萼，使整個花序看起來像是一大朵顯眼的花，我們稱這樣膨大而可能扮演花瓣角色的構造為類花瓣(petaloid structure)。本研究採用華八仙(Hydrangea chinensis)作為實驗材料，希望能了解類花瓣的形成機制。本研究中從華八仙中釣取了花部器官決定的同源基因，包括一個A群基因HycFL、三個B群基因HycPI, HycAP3, HycTM6、一個C群基因HycAG以及一個E群基因HycMADS1，並藉由其保守的C端序列和譜系分析結果進行基因群的確認。利用RT-PCR檢視所釣取基因於花部各部位之表現情形發現，A群基因HycFL，一個B群基因HycTM6，和E群基因HycMADS1在所有的花部器官也均有表現；另外兩個B群基因中，HycAP3表現於花瓣、雄蕊、花柱以及與花萼合生的子房下半部，HycPI則除了在與花萼合生的子房下半部有微弱表現外，僅表現於花瓣以及雄蕊的部分；C群基因HycAG表現於花的內三輪器官但在雄蕊及心皮表現量較高。以掃描式電子顯微鏡觀察華八仙的花朵，其花的各部位細胞表面均有角質層的加厚，而僅有膨大花萼的近軸面細胞呈現圓錐狀形態。這種圓錐狀細胞通常存於植物的花瓣近軸面細胞，而在金魚草(Antirrhinum majus)的研究中已發現其中的MIXTA基因會調控花瓣近軸面表皮細胞形態，使其呈現圓錐狀突起。因此我們也在華八仙中選殖出了MIXTA的同源基因HycMYB1，RT-PCR的實驗結果顯示此基因會表現於膨大花萼，而在正常的花萼以及花瓣則僅有微弱的表現情形。依據我們的實驗結果，HycFL、HycTM6、HycMADS1和HycMYB1均在膨大花萼處有表現，顯示這四個基因可能均參與了華八仙中膨大花萼的形成過程。The inflorescence of Hydrangeaceae plants is often aggregated as a pseudanthium, which contains enlarged sepals on peripheral flowers of the inflorescence. The flowers with these petaloid sepals are generally sterile and may play important roles in attracting pollinators. In order to understand the molecular mechanism for the petaloidy formation, floral organ identity genes of Hydrangea chinensis was identified and examined the expression patterns among different organs. One A-class (HycFL), three B-class (HycPI, HycAP3, HycTM6), one C-class (HycAG) and one E-class (HycMADS1) floral homeotic genes were identified from H. chinensis, and all of the sequences show conserved C-terminal motifs for ABCE class genes, further confirmation for their identities were carried out by separate phylogenetic analyses. RT-PCR results show that the A class homolog HycFL, one B class homolog, HycTM6, and the E-class gene homolog HycMADS1 have ubiquitous expression in all floral parts. Another B-class homolog, HycAP3, is expressed in petals, stamens, styles, and lower part of carpels, which is fused to part of normal sepals. In contrast, HycPI is highly expressed in petals and stamens, and weakly in lower part of carpels. The C-class gene homolog, HycAG, is expressed in the inner three whorls of flowers but the expression is higher in stamens and carpels. Under Scanning Electromicroscope examination, the epidermal surfaces of cells on examined floral parts all have cuticular striation, while only adaxial epidermal cells of enlarged sepals show conical shape, a feature commonly found in the epidermal cells of a petal in other flowering plants. Such conical cell formation has been demonstrated to be controlled by a transcription factor, MIXTA, in Antirrhnicum majus. We have also successfully cloned HycMYB1, a homolog of MIXTA. The RT-PCR result shows that HycMYB1 is highly expressed in enlarged sepals and much less in normal sepals and petals. Since HycFL, HycTM6, HycMADS1 and HycMYB1 are all expressed in enlarged sepals, they are the candidate genes involved in the formation of petaloid sepals of Hydrangea chinensis.中文摘要..................................................1 英文摘要..................................................2 壹、實驗背景介紹..........................................4 一、花部形態與花被的演化..................................4 1. 花部形態...............................................4 2. 花被的演化.............................................4 3. 假單花.................................................5 4. 假單花於山茱萸目(Cornales)中的演化.....................5 二、花部器官發育的分子調控機制............................6 1. ABCDE model............................................6 2. MADS-box基因與Floral quartet model.....................7 3. 花瓣表皮細胞與MIXTA基因................................8 4. 類花瓣與ABCDE model....................................9 三、實驗假設與目的.......................................11 貳、材料與方法...........................................15 一、本實驗使用之八仙花科植物介紹.........................15 二、台灣產八仙花科植物樣本之收集.........................15 三、掃瞄式電子顯微鏡(SEM)樣本之製備與操作................18 四、台灣產八仙花科植物花部器官決定同源基因之選殖.........18 五、基因之譜系分析.......................................23 六、植物DNA的萃取........................................24 七、南方轉漬分析 (Southern blot analysis)................26 八、花部同源基因表現分析.................................28 參、結果.................................................44 一、華八仙花部形態觀察...................................44 二、華八仙花部同源基因以及MYB家族基因之選殖與南方轉漬分析法之檢測...................................................44 三、譜系分析.............................................47 四、以RT-PCR檢測華八仙花部同源基因與MYB家族基因於各花部器官之表現...................................................48 肆、討論.................................................76 一、華八仙花部表現細胞形態與傳粉機制.....................76 二、華八仙花部同源基因於不同花部之表現...................76 三、南方轉漬分析結果.....................................78 四、華八仙MYB家族同源基因之譜系分析......................78 五、膨大花萼之形成與候選基因.............................79 伍、結論.................................................82 陸、參考資料.............................................84 柒、附錄.................................................949067314 bytesapplication/pdfen-USMADS-box基因華八仙MIXTA類花瓣MADS-box genesHydrangeapetaloid sturcture華八仙(八仙花科)類花瓣構造與花部器官決定基因之研究Petaloidy and floral homeotic genes in Hydrangea chinensis (Hydrangeaceae)otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/55097/1/ntu-96-R94b44008-1.pdf