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標題: Functional Analysis and the Agricultural Application of Mads Box Genes Involved in Regulating the Perianth Developmental Process in Oncidium Gower Ramsey
文心蘭中參與花萼花瓣及唇瓣發育調控之MADS box基因之功能性分析及應用
作者: 楊長賢
關鍵字: 農藝;商品化;Oncidium Gower Ramsey;MADS box genes;lips;flower shape regulation
Oncidium Gower Ramsey is a popular orchid with delicate flower shapes, and withhigh economic values in the international cut flower markets. The exploration of themechanism controlling flower development and the increasing of the diversity of theflower shapes for O. Gower Ramsey through biotechnology are important. Comparedwith other orchids, the large and well-expanded lips make O. Gower Ramsey a specialcase for the study of the diverse functions of MADS box genes during the flowerdevelopment. Up to date, four B class MADS box genes, including three paleoAP3homologs (OMADS3/OMADS5/OMADS9) and one PI homolog (OMADS8), inregulating perianth formation in O. Gower Ramsey have been cloned andcharacterized in our laboratory. The OMADS8 and OMADS3 mRNAs were expressedin all four floral organs. The OMADS9 mRNA was only detected in petals and lips.The mRNA for OMADS5 was only detected in sepals and petals. This result revealed apossible negative role for OMADS5 in regulating lip formation. The determination ofthe final organ identity for the sepal/petal/lip likely depended on the presence orabsence of OMADS5. The presence of OMADS5 caused short sepal/petal formation.When OMADS5 was absent, cells may proliferate, resulting in the possible formationof large lips. The prime objective of this proposal is to generate transgenic O. GowerRamsey with various modified flower shapes by ectopic expressing sense or RNAi ofOMADS5. For example, the knock down of the OMADS5 expression by RNAi strategy(pOMADS5::OMADS5-RNAi) specifically in the sepals and peals will potentiallyconvert these two organs into lips, and then produced flowers with multiple (6) lips.By contrast, ectopic expression of OMADS5 (pOMADS9::OMADS5) in lips willconvert lip into sepal/petal and resulted in a flower with 6 sepal/petals. For theobjective to identify the sepal/petal/lip-specific interacting protein complexes forOMADS3/5/9/8 in O. Gower Ramsey, in vitro co-immunoprecipitation assay (co-IP),mass spectrometry (MS) and fluorescence resonance energy transfer (FRET) will beperformed in this study. To further gain insight into the effects of C-terminal sequenceon the dimerization and function of paleoAP3 homologs (OMADS3 and OMADS5)and PI homolog (OMADS8), yeast two-hybrid analysis, electrophoretic mobility shiftassay (EMSA) and complementation analysis in ap3 or pi mutants by usingOMADS3/5/8 with serial deletions in the C-terminal region will be performed. Theseresults will help to know how these Oncidium B function MADS box proteins formcooperative complexes during sepal/petal/lip development and will lead to a betterunderstanding of the “orchid code” for Oncidium floral organ identity.

文心蘭歷年出口值穩定成長,是少數能以單一品系及花色達到如此高經濟效應之蘭花,若能對文心蘭花形調控加以研究並進一步透過生物技術加以改造,相信對文心蘭之產業將更有助益。文心蘭的花朵構造中,唇瓣是觀賞價值中極為重要的部分,本實驗室已自文心蘭選殖四個與花萼/花瓣/唇瓣發育相關之B 功能MADSbox 基因: OMADS3/OMADS5/OMADS9 屬於paleoAP3 分支,OMADS8 屬於PI 分支。研究發現OMADS3 與OMADS8 是花萼/花瓣/唇瓣發育必須存在的成員,同時加入OMADS5 與OMADS9 之表現時會促成花瓣發育,只加入OMADS5 之表現時促成花萼發育,只加入OMADS9 之表現則促成唇瓣發育。由此基因表現模式可知OMADS5 在唇瓣發育上扮演的是負調控的角色,在花萼/花瓣/唇瓣中只要OMADS5不表現則會使花器分化為唇瓣。基於此,本計劃首要目的在改造文心蘭花形,預計建構多種促進或抑制OMADS5 表現之載體,並進行基因轉殖,預期轉殖pOMADS5::OMADS5-RNAi 載體之文心蘭花萼/花瓣會變成唇瓣化的現象,進而可產生多唇瓣花形之轉殖文心蘭。另一方面以表現於唇瓣的OMADS9 起動子,建構pOMADS9::OMADS5 載體,使OMADS5 表現於唇瓣中,則轉殖文心蘭之唇瓣將轉化成花萼/花瓣,產生花形變小之文心蘭。此外依據ABCDE model 的理論,調控文心蘭花萼/花瓣/唇瓣的發育除了需要B 功能基因群所組成的複合物以外,亦須A/E 功能基因群及其他非MADS box 基因的參與,本計劃將以免疫共沉澱法(co-immunoprecipitation, co-IP) , 分別以OMADS3、OMADS8、OMADS5 及OMADS9 專一性抗體分離花萼、花瓣或唇瓣中MADS box 蛋白質複合體,再以質譜分析(mass spectrometry, MS) 專一性抗體所沉澱下來的蛋白質複合體成份,以分析調控花萼、花瓣或唇瓣之蛋白質複合體組成為何。另外我們也將利用螢光共振能量轉移系統(Fluorescence resonance energy transfer, FRET),觀察植物活體細胞中OMADS3、OMADS8、OMADS5 及OMADS9 兩兩形成雙聚體進入細胞核的能力。本計劃亦將進一步分析B 功能paleoAP3 同源蛋白質OMADS3/OMADS5 及PI 同源蛋白質OMADS8 C 端胺基酸序列影響兩者形成雙聚體及執行其功能的能力。將對OMADS3、OMADS5 及OMADS8 的C 端序列作漸進式截切,以酵母菌雙雜合系統及EMSA (electrophoretic mobility shift assay)分析其形成雙聚體之能力,並將其進行在阿拉伯芥ap3 或pi 突變株中之互補試驗, 以了解OMADS3/OMADS5/OMADS8 C 端胺基酸序列與其調控花萼/花瓣/唇瓣功能之關係。
其他識別: NSC100-2313-B005-004-MY3
Appears in Collections:生物科技學研究所

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