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Download 題目: Regulatory mechanism of floral coloration in Oncidium cultivars
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題目: Regulatory mechanism of floral coloration in Oncidium cultivars 作者:邱崇益 Chung-Yi Chiou 指導老師:葉開溫 D03 Abstract Oncidium are among the most important plants in the flower market in Taiwan, however little research on pigmentation biosynthetic genes has been reported. Therefore, the isolation of pigmentation biosynthesis related genes and further study color patterning in floral tissue of Oncidium Gower Ramsey and carotenoid-related genes determines diversified carotenoid coloration in Oncidium Gower Ramsey “Sunkist” and Oncidium Gower Ramsey “White Jade” In this study, chromatography analysis revealed that the red coloration in floral tissues was composed of malvidin-3-O-galactoside, peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside compounds. By contrary, these pigments were not detected in yellow lip tissue. Four key genes involved in anthocyanin biosynthetic pathway, i.e. chalcone synthase (OgCHS), chalcone isomerase (OgCHI), dihydroflavonol 4-reductase (OgDFR) and anthocyanidin synthase (OgANS) were isolated and their expression patterns were characterized. Northern blot analysis confirmed that although they are active during floral development, OgCHI and OgDFR genes are specifically down-regulated in yellow lip tissue. Bombardment with OgCHI and OgDFR genes into lip tissue driven by a flower-specific promoter, Pchrc (chromoplast-specific carotenoid-associated gene), demonstrated that transient expression of these two genes resulted in anthocyanin production in yellow lip. Further analysis of a R2R3 MYB transcription factor, OgMYB1, revealed that although it is actively expressed during floral development, it is not expressed in yellow lip tissue. Transient expression of OgMYB1 in lip tissues by bombardment can also induce formation of red pigments through the activation of OgCHI and OgDFR transcription. These results demonstrate that differential expression of OgMYB1 is critical to determine the color pattern of floral organ in Oncidium Gower Ramsey. The differential expression of OgMYB1 is caused by DNA methylation. Three cultivars of Oncidium orchid with varied coloration, such as Oncidium Gower Ramsey (yellow), Sunkist (orange) and White Jade (white), were analyzed for carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The HPLC analysis revealed that yellow Gower Ramsey accumulates violaxathin, 9-cisviolaxathin and neoxanthin, orange Sunkist accumulates an additional β-carotene, and White Jade is devoid of carotenoid compounds. Molecular characterization indicated that the three Oncidium cultivars exhibited varied expression pattern and level in carotenoid-biosynthetic pathway. Among them, high expression level of β-hydroxylase (OgHyb) and zeaxathin epoxidase (OgZep) was displayed in yellow Gower Ramsey, relative to the down-regulation of OgHyb and OgZep exhibited in orange Sunkist, which results in the accumulation of β-carotene and orange coloration in floral tissues. However, White Jade is caused by the up-regulation of CCD1 (Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites. Methylation assay of CCD1 promoter in White Jade and Gower Ramsey revealed that a high level of DNA methylation was present in CCD1 promoter region of Gower Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by bombardment confirmed its function of disintegrating carotenoid compounds. Our results suggest an evolutionary significance that genetic variation of carotenoid-related genes in Oncidium generates the complexity of floral pigmentation, and consequently provides the profound varieties in Oncidium population. Tissue-specific promoters are required for plant molecular breeding to drive a target gene in the appropriate location in plants. In this study, a chromoplast specific carotenoid-associated gene (OgCHRC) and its promoter (Pchrc) were isolated from Oncidium orchid and characterized. Northern blot analysis revealed that OgCHRC is specifically expressed in flowers, not in roots and leaves. Transient expression assay of Pchrc by bombardment transformation confirmed its differential expression pattern in floral tissues of different horticulture plants and cell-type location in conical papillate cells of adaxial epidermis of flower. These results suggest that Pchrc could serve as a useful tool in ornamental plant biotechnology to modify flower color.