洋桔梗中E功能之MADS box開花基因之選殖與特性分析

Abstract

ABCDE model 說明了在植物中花器的形成是由model中的五群基因互相作用調控的，由於大部分的ABCDE功能性基因為包含了MADS box domain和K domain的MADS box 基因，因此MADS box 基因在開花的過程中必是扮演重要的角色。MADS box domain是靠近N端且保留性高的DNA-binding domain，而位在蛋白質中間的K domain則是putative protein dimerization domain。為了了解洋桔梗的開花發育的過程，本研究選殖出兩個和阿拉伯芥中E function的MADS box基因(SEPALLATA1/2/3)具有高度相似性的基因，分別命名為EgMADS5( Eustoma grandiflorum MADS box Gene5)和EgMADS6。EgMADS5基因可以轉譯出一個含244個胺基酸的蛋白質，其序列和阿拉伯芥中的SEP3基因具有68.5%的相同性。EgMADS6基因則可轉譯出一個含246個胺基酸的蛋白質並且和阿拉伯芥中的SEP1/2基因具有63%的相同性。EgMADS5和EgMADS6在不同花苞時期都有mRNA的表現。而在各個花器方面，EgMADS6基因不管是在花萼、花瓣、雄蕊、雌蕊等各部位均有表現，但EgMADS5則只有在花瓣、雄蕊及雌蕊表現，在花萼部位則不表現。這些結果顯示洋桔梗的EgMADS5和EgMADS6基因應是有調控花器形成的功能的。在轉基因植物方面，已成功將EgMADS5基因轉至阿拉伯芥中，結果發現轉殖基因的植物植株較小，開花時間明顯提早許多，並且出現終結花的現象。

ABCDE model predicts the formation of any flower organs by the interaction of five classes of homeotic genes in plants. Most "ABCDE" functional genes are MADS box proteins which contained a conserved DNA-binding domain (the MADS box domain) at the N-terminus and a putative protein dimerization domain (K domain) at the center, indicating the central role for MADS box genes in flower development. To investigate the flower development of lisianthus (Eustoma grandiflorum), two cDNAs showed high homology to the E functional MADS box genes of Arabidopsis SEPALLATA1/2/3 were isolated and characterized. EgMADS5, encodes a 244 amino acid protein that showed 68.5% identity to SEP3 of Arabidopsis. EgMADS6, encodes a 246 amino acid protein that showed 63% identity to SEP1/2 of Arabidopsis. EgMADS5 and EgMADS6 mRNA was detected in floral buds of different developmental stages. In flower, similar to SEP1/2, EgMADS6 was expressed in all four flower organs, sepal, petal, stamen and carpel. By contrast, similar to SEP3, EgMADS5 was expressed in petal, stamen and carpel and absent in sepal. These results indicate that EgMADS5 and EgMADS6 are the putative SEP3 and SEP1/2 homologues of lisianthus and functions in regulating the flower organ formation. Transgenic Arabidopsis, which ectopically expresses EgMADS5, showed novel phenotypes by significantly reducing plant size, flowering extremely early, and loss of floral determinacy.