Anthocyanin Biosynthesis And Flower Coloration Controlled By Cineraria PCFH Gene

Cineraria (Senecio cruentus Masson ex L'Herit) is a groundsel which is popularly potted. Cineraria is plentiful in varieties of colors and has typical blue variety which is rare in Compositae. By studying the mechanism of cineraria's color formation and gene regulation, we will be able to do better in color modification and in molecular breeding of novel color, especially the breeding of blue Chrysanthemum (Chrysanthemum×morifolium Ramat.). It will also provide a reference to the key genes of capitate plants which lead to a blue variety.In this study, Senecio cruentus'Jester'Serises including pure white, pure light yellow, pure pink, pure carmine and pure blue in color are chosed as materials. PCFH gene was isolated from the Cineraria and PCFH sequence is homologous with F3'5'H. We also take tobacco (Nicotiana tabacum) as the acceptor to analyze the effects of heterogenous expression of PCFH gene and discuss its function in plants in further. Results obtained as follows.By using visual color measurement, RHSCC colorimetry and colorimeter to analyse the variety of phenotype of different color's cineraria during different period flower development. And by using HPLC-PAD/DAD mcthod to analyze the changes of flavonoids compound's content, while different color phenotypes are defined and distinguished. We find anthoxanthins are main pigments in pure white and pure light yellow cineraria, while anthocyanins are main pigments in pure pink, pure carmine and pure blue cineraria. That is to say the color phenotypes of cineraria are directly relative to the variety and the content of flavonoids compounds.The composations of anthocyanins are analyzed by using HPLC-ESI-MS method. We find that the main anthocyanidins of pure pink, pure carmine and pure blue cineraria are pelargonidin, cyanidin, and delphinidin, respectively. This result elucidates the types of anthocyanidin are the main factor on forming different colors.By RT-PCR method examining the expression of the key gene PCFH during the synthese of anthocyanin in five colors, seven stages of cinerarialingulate florets, its transcripts were only detected in early stages of inflorescence development. This result indicates that PCFH is the key gene in the synthese of delphinidin and its derivative and in the formation of blue variety ulteriorly.By using Agrobacterium-mediated leaf discs transformation we transform the heterogenous gene PCFH to tobacco'NC89'.The fourteen transgenic To generation plants get the phenotype of luminance receded and colors deepen, becoming redder and bluer. A kind of new delphinin are found in corolla and the content of cyanidin also rose. All forenamed results indicate that cineraria F3'5'H coding by PCFH has the function of 3',5'-hydroxylation to form delphinium and 3'-hydroxylation to increase the content of cyanidin. The T1 generation has similar characterizations with the parent, comparing with wild plants, there are remarkable changes in both color phenotypes and anthocyanin contents. It indicates that through sexual propagation, the heterogenous gene PCFH can inherit steadily and is still effective.As a result, PCFH plays a key role in the anthocyanin biosynthetic pathway and the flower color formation of cineraria. In this study, we first study the heterogenous expression of F3'5'H gene, which belongs to CYP75B subfamily, in tobacco, and we prove that cineraria F3'5'H coded by PCFH, the key gene to form blue cineraria, has the function of 3',5'-hydroxylation and 3'-hydroxylation in plant. This study clarify the effect of PCFH during variety of color's formation, and it also provide a reference to the function of Compositae-specific F3'5'H in plant, which belongs to CYP75B subfamily.