文心蘭中B功能性MADS Box基因之選殖與特性分析

Abstract

對植物來說，許多重要的生理發育過程是由MADS box基因所調控。而大部分影響花器形成的ABCDE基因皆屬於此類MADS box基因，其蛋白質結構中皆具有一段保守的DNA結合區域(MADS box domain)，後有一段和蛋白質相互作用相關的K domain。為了能更清楚了解蘭科植物花器形成機制，本研究從文心蘭（Oncidium Gower Ramsey）中選殖出OMADS8及OMADS9兩個基因進行功能性分析。OMADS8可轉譯出一個含210個胺基酸的蛋白質，其序列比對結果發現，和阿拉伯芥B class中之PI基因有高度相似性，屬於PI同源性基因。OMADS8在文心蘭葉、根及花皆有表現。進一步觀察花器內部表現，發現OMADS8在花萼及花瓣表現量較雄蕊、心皮來的高。將OMADS8基因大量表現於野生型阿拉伯芥中，發現會使植株矮小提早開花，並造成花萼轉變成似花瓣的構造。OMADS9可轉譯出一個含222個胺基酸的蛋白質，其屬於B class中之PaleoAP3族基因，例如鴿石斛蘭（Dendrobium）的DcOAP3B和文心蘭的OMADS5皆屬於此類。OMADS9C端部分具有兩個完整的paleoAP3演化分支特有的motif — PI-derived motif及paleoAP3 motif。OMADS9在文心蘭花器中表現量以花瓣及唇辦中最高。大量表現OMADS9於阿拉伯芥，觀察到早開之花朵白化但較晚開的花和野生型無異。此外，透過酵母菌雙雜交系統（yeast two hybrid），來分析所知的文心蘭B群基因的蛋白質間的相互關係，結果顯示，OMADS5及OMADS9有形成homodimer的能力，也會相互作用形成 heterodimer。OMADS8無法形成同質二聚體，也無法形成異質二聚體。

MADS box genes controlled many important developmental processes in plants. Most ABCDE class genes regulating flower formation are MADS genes that contained a DNA-binding domain (MADS box domain) and a protein interaction domain (K domain) in their encoded proteins. To investigate the flower formation in orchid, two B class MADS box genes, OMADS8 and OMADS9 from Oncidium Gower Ramsey, were isolated and characterized in this research. OMADS8 encoded a 210 amino acid protein showed high sequence homology to Arabidopsis B class gene PI. OMADS8 is expressed in all the organs tested including leaf, root and flower. In flowers, OMADS8 mRNA was especially detected higher in the sepal and petal than in stamen and carpel. Ectopic expression of OMADS8 significantly reduced the plant size and promoted flowering time in transgenic Arabidopsis. The conversion of sepal to petal-like structure was also observed in 35S::OMADS8 transgenic plants. OMADS9 encoded a 222 amino acid protein and showed sequence homology to PaleoAP3 lineage of B group MADS box gene such as DcOAP3B in Dendrobium and OMADS5 in Oncidium. In its C-terminal region, two completely consensus motifs such as PI-derived and paleoAP3 motifs which are unique to paleoAP3 lineage were identified. OMADS9 is highly expressed in petal and lip petal in flowers. The early developed flowers are whitened, but the late developed flowers in transgenic Arabidopsis ectopically expressing OMADS9 are indistinguishable from that of wild-type plants. Furthermore, to investigate the interaction among the Oncidium B class proteins such as OMADS8, OMADS9 and OMADS5, yeast two hybrid analysis was performed. The result indicated that OMADS5 and OMADS9 were able to form homodimer and heterodimer to each other. OMADS8 was not able to form either homodimer or heterodimer.