蘭科植物為開花植物中數量最龐大的一科，目前約有25,000種以上。蘭科植物具有相當獨特的花部形態，其生殖構造是由雄蕊及雌蕊癒合形成單一個合蕊柱(gynostemium or column)，此特徵可作為辨識蘭科植物的重要指標。
　　本研究利用野生型國蘭及叢瓣化（multitepal mutant）突變植株為材料，探討C群基因在蘭花和合蕊柱發育中扮演之角色。我們選殖到國蘭之兩個C群基因之全長cDNA序列分別可轉譯出233及234個胺基酸，並命名為CeMADS1及 CeMADS2。親緣演化分析結果顯示，此二序列為蘭花之C群基因。透過比對CeMADS1 及CeMADS2在野生型及叢瓣型突變株序列分析，我們發現在叢瓣型突變株中CeMADS1 及CeMADS2的核苷酸皆出現數個點突變的情形，且會造成叢瓣型突變株type B胺基酸轉譯時的改變。空間表現模式分析結果顯示，CeMADS1只在合蕊柱中表現，在其他花部器及營養組織中皆不表現。而CeMADS2則主要表現於合蕊柱中，亦微量表現於其他花器，然而卻不會表現於營養組織。在花苞發育過程中，CeMADS1在初期的stage 1有較大量的表現，而CeMADS2則在stage 2以及後來的stage中有較明顯的表現。比較CeMADS1 及CeMADS2在野生型及叢瓣型突變株花苞中表現結果顯示，CeMADS1表現於野生型國蘭花苞中，但不表現於叢瓣型突變株中，而CeMADS2 則一致的表現於野生型及叢瓣型突變株花苞。另外，透過南方墨點法（Southern blot analysis）分析此二基因在國蘭基因體之存在情形，發現此二基因皆為single copy gene，且在突變株中並未改變其基因結構。我們更進一步利用酵母菌雙雜交系統(Yeast two-hybrid analysis)，探討C- 及 E群基因的蛋白質交互作用。結果顯示，CeMADS1、CeMADS2和PeMADS8會形成同型或異型二聚體 (homo- or heterodimer)，且CeMADS1同型二聚體及其和PeMADS8異型二聚體之交互作用能力皆比CeMADS2同型二聚體及其和PeMADS8異型二聚體強。最後，功能性分析部份，透過農桿菌轉殖法在阿拉伯芥中大量表現CeMADS1會導致花朵之花瓣和雄蕊萎縮以及雙雌蕊之外表型改變，而大量表現CeMADS2則並沒有改變野生型阿拉伯芥花形。
綜合以上結果，我們推測CeMADS1在國蘭合蕊柱發育中扮演關鍵性的initiation之決定基因，而CeMADS2可能是存在的一個duplicate且在合蕊柱發育可能扮演maintenance的角色。

The Orchidaceae is one of the most species-rich plant families and are renowned for their spectacular floral diversity. A gynostemium, comprising stamen filaments adnate to a syncarpous style, is one of the unique features of orchid reproductive organ. To understand MADS-box genes involved in gynostemium development of Cymbidium ensifolium, we isolated the full-length cDNA of two C-class genes with 233 and 234 amino acids, and named as CeMADS1 and CeMADS2, respectively. Phylogenetic analysis results showed that these two genes were clustered with other orchid C-class genes and classified into the C-lineage of the AG subfamily. The spatial expressions of CeMADS1 and CeMADS2 in various floral organs were analyzed by using northern blot, quantitative real-time RT-PCR and in situ hybridization. Results showed that both CeMADS1 and CeMADS2 expressed exclusively in the reproductive organs. In addition, CeMADS1 only expressed in the column, while CeMADS2 expressed in all floral organs. Both CeMADS1 and CeMADS2 were detected in all the developmental stages of flower buds. CeMADS1 was expressed more strongly in stage 1; while CeMADS2 was expressed more noticeably at subsequent stage than CeMADS1. We also characterized these genes from multitepal mutant of C. ensifolium. The flower of multitepal mutant of C. ensifolium has lost its gynostemium. Instead, it is replaced by a new emerged flower, and this ecotopic flower continues to produce sepals and petals centripetally. Comparing the nucleotide and amino acid sequences of CeMADS1 and CeMADS2 from the multipetal mutant with those of wild type, we found that point mutations occurred at the nucleotide level that lead to transition of amino acid sequences from Cysteine (C) to Tyrosine (Y) in CeMADS1, and from Lysine (K) to Asparagine (N) in CeMADS2 of multitepal mutant type B. Meanwhile, the expression of CeMADS1 was reduced to 1% and 11% in the flower buds of two multitepal mutants as compared to that in the wild-type plant. In contrast, the expression of CeMADS2 remained as 90% and 66% in two multitepal mutant flowers as compared to that of the wild-type plant. in situ hybridization results showed that both CeMADS1 and CeMADS2 transcripts were detected in the very early stage of floral primordium. At the later stages, they were more concentrated in the adaxial face of developing column. Southern blot analysis showed that both CeMADS1 and CeMADS2 were single-copy genes in the Cymbidium genome. In addition, there were no genomic structure alterations detected in the multitepal mutant. Moreover, homodimer as well as heterodimer formations were detected for CeMADS1, CeMADS2 and PeMADS8 proteins by using yeast two-hybrid analysis. Quantification of the interaction strength revealed that the interaction strength of CeMADS1 homodimer was similar to that of CeMADS1-CeMADS2 heterodimer but stronger than that of CeMADS2 homodimer. In addition, the interaction strength of CeMADS1-PeMADS8 heterodimer was also stronger than that of CeMADS2-PeMADS8 heterodimer. Functional analysis of these two genes were carried out by ectopic expression of these genes in transgenic Arabidopsis. Results showed that ectopical overexpression of CeMADS1 in transgenic plants caused the flowers with reduced petals and stamens. In contrast, no alterations in flower morphology were observed when ectopically overexpressing CeMADS2.
In conclusion, the CeMADS1 is the column identity gene in Cymbidium orchids, while the CeMADS2 could be a duplicate of CeMADS1 but did not play an essential role in orchid column development. It is possible that CeMADS1 may play a role in the initiation step for column morphogenesis, while CeMADS2 may play a role in the maintenance step.

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