臺灣博碩士論文加值系統

花器發育是由一群基因如APETALA1 (AP1)，APETALA2 (AP2)，APETALA3 (AP3)及AGAMOUS (AG) 所調控。這其中的AP1，AP3，AG都含有一段保留度很高的DNA結合區域(MADS box domain)，顯示MADS box基因在花朵發育中佔了相當重要的角色。為了研究MADS box基因在不同植物裡的功能與將來更進一步的應用，本實驗以選殖重要的經濟花卉洋桔梗的MADS box基因為目標。以花苞的RNA為模版及用RT-PCR及5’-與3’-RACE的方式，三個MADS box基因的cDNA全長被選殖出，分別名為EgMADS1、2與3 （E. grandiflorum MADS box gene 1,2 and 3）。EgMADS1，可轉譯出一個由223氨基酸構成的蛋白，與矮牽牛裡控制胚珠發育Floral Binding Protein gene 7 (FBP7)有80 %的一致性及91 %的相似性。在MADS box的58個氨基酸序列中，EgMADS1與FBP7便有53個氨基酸序列完全相同。在K box，也就是負責兩個蛋白質結合的區域，這兩個基因之間更有88 %的一致性及96 %的相似性。南方墨點法分析中發現，EgMADS1在洋桔梗中是以單一拷貝（single copy）的方式存在。而北方墨點法分析結果指出，EgMADS1專一表現在胚珠，並且可能與FBP7在矮牽牛中的扮演相似的功能。EgMADS2，可轉譯出一個由225氨基酸構成的蛋白，與擬南芥中的AP3，一個控制花瓣與雄蕊形成的基因，有51 %的一致性及67 %的相似性，而在MADS box區域58個氨基酸序列中，44個氨基酸序列完全相同。EgMADS3，可轉譯出一個由258氨基酸構成的蛋白質，與擬南芥裡的AGL6，一個可能與花朵發生（floral initiation）有關的基因有50 %的一致性及62 %的相似性。這兩個基因在MADS box區域58個氨基酸序列中有44個完全相同。北方墨點法分析指出EgMADS2與EgMADS3均在花器中有專一表現。將來更進一步基因功能的分析，可利用在轉基因擬南芥中異位表現這三個基因的方式進行。這些結果不僅能使我們對MADS box基因有更深入的瞭解，而且也可以提供在未來有效運用這些基因的策略。

Floral organs are specified by a set of flower organ identity genes including APETALA1 (AP1), APETALA2 (AP2), APETALA3 (AP3) and AGAMOUS (AG). Since AP1, AP3, and AG all contain the MADS box, a conserved DNA-binding domain, indicating the central role for MADS box genes in flower development. To investigate the function and further application of MADS box genes in different plant species, a PCR-based strategy was used to clone MADS box genes from Eustoma grandiflorum, an economically important flowering plant in the world. Full-length cDNA sequence for three MADS box genes, EgMADS1, 2, and 3 (E. grandiflorum MADS box gene 1, 2 and 3), were isolated using RT-PCR and 5''-, 3''-RACE. EgMADS1, encoding a putative 223 amino acid protein, showed 80% identity and 91% similarity to petunia Floral Binding Protein gene 7 (FBP7) which controlling the ovule development. Among the 58 amino acid in MADS box domain (DNA binding domain), 53 are identical between EgMADS1 and FBP7. K domain, a putative protein dimerization domain, showed 88% identity and 96% similarity between these two genes. Southern analysis indicates that EgMADS1 is present in E. grandiflorum genome in a single copy manner. Northern blot analysis indicated that EgMADS1 was specifically expressed in ovules and may have functional equivalency to FBP7 in petunia. EgMADS2, encoding a 225 amino acid protein, showed 51% identity and 67% similarity to Arabidopsis AP3 which controlling the petal and stamen formation. Among the 58 amino acid in MADS box domain, 44 are identical in EgMADS2 and AP3. EgMADS3, encoding a 258 amino acid protein, showed 50% identity and 62% similarity to Arabidopsis AGL6 which may involve in the floral initiation. 44 out of 58 amino acid in MADS box domain are identical in EgMADS3 and AGL6. Northern blot analysis indicated that EgMADS2 and 3 were specifically expressed in floral organs. Further functional analysis by ectopic expression of these three genes in transgenic Arabidopsis plants is necessary. The result should not only lead to a deeper understanding of the MADS box genes but also provide useful strategy to manipulate these genes in the future.

中文摘要-------------------------------------------------- 1
英文摘要-------------------------------------------------- 3
壹、前言-------------------------------------------------- 5
貳、前人研究---------------------------------------------- 7
參、材料與方法-------------------------------------------- 13
肆、結果-------------------------------------------------- 39
伍、討論、------------------------------------------------ 48
陸、參考文獻---------------------------------------------- 53
柒、圖---------------------------------------------------- 62
捌、表---------------------------------------------------- 81
玖、附圖-------------------------------------------------- 82

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