调控朵丽蝶兰及蝴蝶兰三唇瓣花朵发育相关CINCINNATA 基因之选殖.doc

調控朵麗蝶蘭及蝴蝶蘭三唇瓣花朵發育相關CINCINNATA 基因之選殖及表現分析 Cloning and analysis of CINCINNATA gene related to peloric flower development in Doritaenopsis and Phalaenopsis Orchids 研究生：陳怡汶 Yi-Wen Chen 指導教授：陳福旗 Fure-Chyi Chen 【摘要】 被子植物花朵大多為兩側對稱，而花朵對稱性主要受到TCP 基因家族的調控。本實驗探討自朵麗蝶蘭Doritaenopsis Sogo Vivien ‘F858’ 選殖CINCINNATA 基因DtpsSogo Vivien ‘F858’、Dtps Sogo Gotris ‘F1307’ 及 Brother Spring Dancer ‘KHM190’ 之野生型花朵為兩側對稱，當發生三唇瓣突變時，則轉變成輻射對稱，且在翼瓣基部會有突起及邊緣產生缺刻、嚴重時蕊柱花粉塊消失。利用掃瞄電子顯微鏡觀察翼瓣之橫切面，顯示突變株細胞層及細胞數皆較野生型多。經由南方氏雜合分析CIN 基因拷貝數，顯示這三種朵麗蝶蘭及蝴蝶蘭皆具多拷貝數。RT-RCR 分析CIN 基因在Dtps. Sogo Vivien ‘F858’、Phal. Brother Spring Dancer ‘KHM190’野生型花序、0.2-0.39 cm 花苞及翼瓣表現量較高，而Dtps. Sogo Gotris ‘F1307’ 則在突變株0.2-0.39 cm 花苞表現量較高。利用農桿菌轉殖方法將CIN 基因過量表現於菸草中，以GUS 活性組織化學染色分析，在葉片及花瓣中皆有表現。 關鍵字: 兩側對稱、輻射對稱、CINCINNATA基因、三唇瓣突變 【Abstract】 Flowers of most angiosperms are bilateral symmetry, The gene responsible for floral symmetry development belongs to the TCP gene family. This research is aimed at analyzing the role of CINCINNATA gene cloned from Doritaenopsis Sogo Vivien ‘F858’ in the floral symmetry. The deduced amino acid sequence of Doritaenopsis CIN gene contains a TCP domain and belongs to the TCP family. The wild type flowers of Dtps. Sogo Vivien ‘F858’, Dtps. Sogo Gotris ‘F1307’ and Phalaenopsis Brother Spring Dancer ‘KHM190’ are bilaterally symmetric, and their peloric mutant flowers changed to actinomorphic. Protuberance and marginal crinkle occurs in the petals of peloric mutants, and in severe case lacking pollinia. Cell numbers accrossing the protuberance area of a peloric petal are more than those in wild type. At least two copies of CIN gene occurs in the genomes of the Doritaenopsis and Phalaenopsis orchids. RT-PCR analysis showed that CIN gene was highly expressed in wild type inflorescence、0.2-0.39 cm buds and petals as compared to the peloric mutants, while in peloric Doritaenopsis Sogo Gotris ‘F1307’ highly expressed in 0.2-0.39 cm buds. The CaMV35S::CIN gene was transferred into the tobacco genome mediated by Agr