文心蘭之MADSBox基因及其下游調控基因之功能性分析

Abstract

文心蘭是重要的單子葉觀賞花卉，是台灣第三大之外銷切花，主銷售市場為日本，台灣主要栽培品種為「南茜」 (Oncidium Gower Ramsey)，栽培幾乎遍布全省，在切花市場上極具經濟價值，但文心蘭花朵發育相關的研究卻不多，本實驗室許多學長姐多年的研究，主要在選殖及分析文心蘭花朵發育相關之MADS box基因，本研究的目的在接續前人自文心蘭中所選殖到的MADS box基因並對這些基因的功能與特性做進一步的分析。 文心蘭OMADS1基因和阿拉伯芥之 AGL6具有高度相似性，將OMADS1轉入阿拉伯芥後出現植株矮小、提早開花與花序異常的性狀。為進一步討論OMADS1 在阿拉伯芥中調控的下游基因，構築35S::OMADS1-GR 轉基因植物以DEX來誘導，再配合cDNA microarray的方式得到一些受OMADS1 促進與抑制的基因；進一步探討ㄧ個受到OMADS1促進的基因ChaCL (ChaC-like family protein)。為了解ChaCL在阿拉伯芥中表現情形與功能，分別進行promoter分析、在阿拉伯芥中大量表現ChaCL與以RNAi抑制ChaCL表現。發現ChaCL的表現位置在根部髓心、保衛細胞、葉脈、葉毛、葉毛基座細胞、莖頂分生組織、成熟花粉等部位有大量表現。在阿拉伯芥中大量表現ChaCL，35S::ChaCL轉基因植物會出現提早開花的性狀，與野生型相比提早約1~2週，其他外型性狀無顯著差異。35S::ChaCL-RNAi 阿拉伯芥轉基因植株，會出現開花時間延遲的性狀，約延遲3~5天，且營養葉數目較多。以real-time PCR相對定量分析檢測35S::ChaCL轉基因植物中GI, CO, FT, SOC1, LFY, AP3之mRNA表現量發現並無顯著差異，顯示ChaCL促進提早開花的機制可能不是光週期調控路徑。藉由分析OMADS1所調控下游基因的功能與特性，將更了解開花調控機制。(第一章) 文心蘭之OMADS5屬於B class中之PaleoAP3族基因，可轉譯出一個含227個胺基酸的蛋白質。其功能可能是在負調控唇瓣的形成。預期若在文心蘭中大量表現OMADS5，可能會抑制唇瓣的形成，使其轉型為花瓣或花萼，相反的若在文心蘭中抑制OMADS5的表現，則可能會促進唇瓣的形成，使文心蘭的花萼花瓣轉型為唇瓣。本研究已完成35S::OMADS5及35S::OMADS-RNAi的構築及文心蘭之基因轉殖，待轉殖文心蘭植株抽花序後將觀察性狀進一步印證推論。本研究另將全長及去除MADS domain之OMADS5轉殖入阿拉伯芥中大量表現，初步觀察性狀發現轉基因植株與野生型相比並無發現顯著差異。(第二章)

Orchids are among the most important plants in the flower market in Taiwan, however little research on MADS box genes has bene reported. Therefore, the isolation of MADS box genes and further study of their roles on orchid (Oncidium Gower Ramsey) flower development is the goal for this study. Oncidium OMADS1 showed high homology to Arabidopsis MADS box gene, AGL6. Trangenic Arabidopsis ectopically expressed OMADS1 showed novel phenotypes by significantly reducing plant size, flowering extremely early, and losing inflorescence indeterminancy. To analyze the downstream genes of OMADS1 in Arabidopsis, construct contaning 35S::OMADS1 fused with glucocorticoid receptor (GR) was transformed into Arabidopsis. Genes up- or down-regulated by OMADS1 were identified by using cDNA microarray for DEX treated transgenic plants. One gene ChaCL showed up-regulated by OMADS1 were further characterized. To explore the ChacL expression in Arabidopsis, the promoter region of ChacL was fused to the reporter gene encoding β-glucuronidase (GUS) and introduced into Arabidopsis plant by Agrobacteria-mediated gene transfer. GUS activity was detected in root pith, guard cells, venation, trichome, shoot apical meristem and mature pollen of ChacL::GUS plants. Ectopic expression of ChacL caused the early flowering phenotype. By contrast, late flowering phenotype was observed in 35S::ChaCL-RNAi transgenic Arabidopsis. The expression of flowering time genes such as GI, CO, FT, SOC1 and LFY was however not affected in the 35S::ChacL transgenic plant. This data suggested that ChacL promotes flowering through a pathway independent from photoperiod flowering pathway.(Chapter 1) To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, one B function paleoAP3 gene OMADS5 was characterized. The OMADS5 encodes a 227 amino acid protein. The mRNA for OMADS5 was strongly detected only in sepals and petals and was absent in the lips. This result revealed a possible negative role for OMADS5 in regulating lip formation. Ectopic expression of OMADS5 may cause the conversion of lip into sepal/petal whereas ectopic expression of OMADS5 RNAi may cause the conversion of sepal/petal into lips in Oncidium. In this study, 35S::OMADS5 and 35S::OMADS5-RNAi constructs were constructed and transformed into Oncidium for functional analysis. Further phenotypic analysis for sepal/petal/lip formation in transgenic plants should reveal the function for OMADS5 in regulating flower development in Oncidium. Furthermore, ectopic expression of either full length or truncated MADS box form of OMADS5 caused no phenotypic change in transgenic Arabidopsis.(Chapter 2)