文心蘭中B和E功能性之MADS box開花基因之選殖與特性分析

Abstract

文心蘭(Oncidium Gower Ramsey)為一重要之蘭科植物。為了研究文心蘭花朵的發育，本研究經由分析cDNA微矩陣法，從文心蘭中選殖出其與花器發育形成相關之一個B群MADS box基因OMADS5，與兩個E群基因OMADS6和7。OMADS5由227個胺基酸所組成，其序列上與B群開花基因paleoAP3族群具高度同源性，在單子葉植物中，如百合之LMADS1基因與非洲菊之GDEF1基因皆屬於此一族群。此外，在OMADS6上亦比對出在B群基因中特有的高度保留區PI-衍生區及paleoAP3區域。進一步偵測基因在花器中mRNA層次上的表現，發現OMADS5除了在花器之唇瓣外皆有表現。OMADS6的序列與阿拉伯芥中E群開花基因之SEP3相似性較高，而OMADS7則與SEP1/2較相似。OMADS6及7基因在花器上各個器官皆有表現且相當類似。目前已建構出OMADS5和7之順式與去除MADS區域之構築體組合，透過農桿菌轉殖至野生種阿拉伯芥中，發現OMADS7會造成植株矮小、極度早開花，並且失去無限花序(inflorescence indeterminacy)的性狀。同時在阿拉伯芥中大量表現OMADS5也會使花器產生改變。這些結果對於文心蘭在B及E群開花基因的研究上將可提供相當有用的資訊。

cDNA for a B group MADS box gene OMADS5 and two E group MADS box genes OMADS6 and OMADS7 were isolated and characterized from Oncidium Gower Ramsey, an important species of orchid, by screening of cDNA microarray. OMADS5 encoding a 227 amino acid protein showed high sequence homology to paleoAP3 lineage of B group MADS box gene such as monocots AP3 homologue LMADS1 in lily and GDEF1 in Gerbera hybrida. In addition, consensus motifs such as PI-derived and paleoAP3 motifs identified in the C-terminal region of B group genes were also identified in OMADS5. OMADS5 mRNA was detected in all floral organs except in lips. OMADS6 showed high identity to E group MADS box gene SEP3 whereas OMADS7 showed high identity to E group MADS box gene SEP1/2 of Arabidopsis. OMADS6 and 7 showed a similar expression pattern by expressing their mRNA in all the floral organs tested. Constructs contained sense and truncated MADS-domain forms of OMADS5 and 7 were constructed and transformed into Arabidopsis for functional analysis. Ectopic expression of OMADS7 promotes flowering in transgenic Arabidopsis by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminacy. The flower organ alteration also observed in transgenic Arabidopsis ectopic expression of OMADS5. These results provide the usefully information regarding the function for B and E group MADS box genes in monocot orchid.