Characterization and Functional Analysis of B and C functional MADS Box Genes in Regulating Floral Organ Formation in Eustoma grandiflorum

Abstract

洋桔梗具有多樣的花色及花型而成為台灣切花市場廣受歡迎的切花植物。在阿拉伯芥調控花器發育的ABCDE模式中，A和E群基因調控花萼的發育，A、B和E群基因共同調控花瓣的發育，B、C和E群基因則調控雄蕊的發育，C和E群基因調控雌蕊的發育。大多的ABCDE 基因轉譯出MADS box蛋白，其所具有MADS domain與DNA的結合相關、Intervening domain 與二聚體的形成相關、Keratin-like domain與蛋白質間的交互作用相關及C-terminal domain具有多元的功能。為了進一步探討MADS box基因在洋桔梗中的功能，選殖出洋桔梗中B群的兩個同源基因EgAP3 和EgPI及C群的同源基因EgAG 進行功能性分析。本研究中，藉由轉入去除 MADS domain之基因包含EgAP3-∆M, EgPI-∆M, EgAG-∆M至阿拉伯芥及洋桔梗中，得到顯性抑制突變株(dominant negative mutants)。實驗結果顯示，在35S::EgAP3-∆M、35S::EgPI-∆M及 35S::EgAG-∆M阿拉伯芥轉殖株觀察到雄不稔之性狀，但並未造成花器的改變。而35S::EgPI-∆M洋桔梗轉殖株中，則可觀察到花瓣無法正常發育，其花瓣細胞轉變成花萼細胞的型態，此結果顯示EgPI確實參與了花瓣的調控。更進一步利用RNA干擾 (RNA interference)以靜默EgAP3, EgPI和EgAG等基因在洋桔梗的表現。本實驗基因轉殖植物的性狀觀察及分析仍持續進行中。期望在不久的將來可利用調控花器形成相關EgMADS基因，能藉由操控花型而增加洋桔梗的市場價值。

Eustoma grandiflorum is a popular cut flower in the international market because it contains many cultivars with diverse flower color and shape. In the ABCDE model of floral development in Arabidopsis thaliana, class A and E genes regulate sepal, class A, B and E genes regulate petal, class B, C and E genes regulate stamen, and class C, E genes regulate carpel. Most ABCDE genes encode typical MADS box proteins which contain a DNA-binding domain (MADS domain), a protein dimerization domain (Intervening domain), a protein interaction domain (Keratin-like domain), and a C-terminal domain with diverse function. To explore the function of EgMADS genes in regulating flower formation in Eustoma grandiflorum, two B functional genes EgAP3, EgPI and one C functional gene EgAG were isolated and characterized. In this study, dominant negative mutants for EgAP3, EgPI and EgAG were generated in Arabidopsis and E. grandiflorum by ectopic expression of the truncated form of these three genes in which the MADS box domain was deleted (35S::EgAP3-∆M, 35S::EgPI-∆M, 35S::EgAG-∆M). Male sterility was observed in the 35S::EgAP3-∆M, 35S::EgPI-∆M and 35S::EgAG-∆M transgenic Arabidopsis. However, no further alteration in flower organ formation was observed in these transgenic Arabidopsis plants. Interestingly, abnormal petal development was observed in the 35S::EgPI-∆M transgenic Eustoma. The petals were converted into sepal-like structures in the 35S::EgPI-ΔM transgenic E. grandiflorum flowers. This result indicated that the function of EgPI is involved in petal development. In addition, down-regulation of EgAP3, EgPI and EgAG function by using RNA interference (RNAi) and phenotypic analysis of all the RNAi transgenic plants has also been undertaken to elucidate the function of these three genes in transgenic E. grandiflorum. In the future, the manipulation of these EgMADS genes in controlling floral shape for E. grandiflorum would increase its market value.