- 學位論文
台灣肖楠MADS-box基因之選殖、特性分析及其蛋白質交互作用之分析
Molecular cloning and characterization of MADS-box gene from Calocedrus formosana and protein interaction analysis between BCFMADS and CCFMADS
摘要
在開花植物中，花器的形成是受到A、B、C三種不同功能型之MADS-box基因控制，更也包含了其他功能型之基因參與其中，本研究從台灣特有種台灣肖楠（Calocedrus formosana）中選殖到兩個MADS-box基因：BCFMADS以及CCFMADS。透過親緣關係圖以及胺基酸序列C端區塊之相似性分析，可將BCFMADS以及CCFMADS兩基因分別歸類於B和C功能型之家族（lineages）。更進一步透過序列分析結果得知，發現BCFMADS基因與柳杉中的CjMADS1和CjMADS2基因最相近。雖然許多的研究認為柏科與杉科植物為並源之演化關係，但是由此BCFMADS的基因分析發現，此基因在台灣肖楠與日本柳杉之間有數量之差異。CCFMADS基因則在序列上面和C功能型與D功能型之基因C-terminal之區塊相似，並與其他裸子植物無太大的差異性。本研究透過轉殖阿拉伯芥來觀察其CCFMADS基因的結果進而瞭解其功能性。結果發現，CCFMADS之轉殖和DAL2之轉殖結果相似，在SHP2之轉殖結果中也有部分相似。都會影響其營養生長之狀況，如葉小，或是葉片捲曲等表現性狀。MADS-box基因中，基因並不會單獨的表現，而是透過形成同質二元體（homodimer）、異質二元體（heterodimer）或是蛋白質複合體（complex）來表現其基因的功能性，因此本研究為欲證實BCFMADS以及CCFMADS基因間可否形成同質二元體或是異質二元體，乃利用E. coli表現此二基因之蛋白質，進行胞外之pull down分析。結果顯示，由於蛋白質不能夠順利且大量的表現在可溶性蛋白中，因此本論文之結果並不能完全的證實BCFMADS與CCFMADS基因可否形成同質二元體或是異質二元體。
並列摘要
In flowering plants, the formations of floral organs are controlled by different functional types of MADS-box genes, including types A, B, and C as well as other functional type genes. In this research, two MADS-box genes, BCFMADS and CCFMADS genes, were cloned from Calocedrus formosana. Through phylogenetic analyses and the similarities at specific C-terminal motifs, BCFMADS and CCFMADS genes were categorized into B-class and C-class lineages. BCFMADS gene was also confirmed identical to both CjMADS1 and CjMADS2 from Cryptomeria japonica based on sequence analysis. Although Calocedrus and Cryptomeria have evolutionally shared paraphyletic relationship, the copy numbers of BCFMADS in Calocedrus formosana are different from those of CjMADS1 and CjMADS2 in Cryptomeria japonica. However, CCFMADS specific C-terminal motif has high similarity in C and D functional type of genes, and does not show much divergence with other gymnosperms. In addition, transgenic Arabidopsis thaliana was used to observe and further determine the function of CCFMADS gene. We found that the transgene of CCFMADS is similar to transgene DAL2 and partially the same as transgene SHP2. All sense transgenic lines would affect vegetative growth, such as the occurrence of smaller and curling leaves. During the last decade, many studies have hypothesized that the MADS-box proteins could form specific dimers, which would be further assembled into tetrameric complexes to have more functions. In order to prove whether BCFMADS and CCFMADS genes can interact with each together to form either homodimer or heterodimer, we used an in vitro approach (pull down assay) to analyze protein expressions for both genes. However, since BCFMADS and CCFMADS fusion proteins cannot express in soluble from of E. coli, we cannot prove that BCFMADS and CCFMADS genes can produce either homodimer or heterodimer in this study.