Influence Of Transformation Of Key Structural Genes Involved In Anthocyanin Synthesis On Flower Coloration Of Chrysanthemum (Chrysanthemum×Morifolium Ramat.)

Chrysanthemum is a traditional famous flower in China, and is also an important cutted and potted flower, with the capacity to earn foreign exchange through exports. All color serials except blue have been bred through traditional breeding. Because of the limitation of gene resources, the novel blue serial can't be bred through traditional breeding. The gene transformation technique can break the species restrictions, and it is likely to attain blue flower color varieties through induction new gene(s) into the gene pool of chrysanthemum. However, the mechanism of chrysanthemum flower coloration, chemical analysis of pigment and molecular breeding are very preliminary. Variety specificity and low genetic transformation efficiency have not been effectively resolved in chrysanthemum transgenic event. In this paper, we analysed the genes expression involved in anthocyanin synthesis, and established high efficiency shoots regeneration and gene transformation system. Then we analyzed the influence of key structural genes which affected the formation of blue flower chrysanthemum on flower color and gene expression of chrysanthemum.In this paper, we established adventitious buds regeneration system of chrysanthemum internode transverse thin cell layers (tTCLs). Adventitious buds regeneration did not demonstrate variety specificity using tTCLs as explants. Buds regeneration efficiency was influenced by explants'position, cultural temperature and concentration of agar. The latter two could influence the efficiency of buds vitrification. Evaluation of different explants in buds regeneration and transformation indicate that petiole tTCLs are the best explants for chrysanthemum transformation, with the best medium, MS+6-BA3.0+NAA0.5mg/L, for buds regeneration and MS+6-BA1.0+2,4-D0.1+Kan15+Carb400mg/L for resistant cell regeneration after Agrobacterium infection.The results of anthocyanin accumulation and RT-PCR analysis of key structural gens involved in anthocyanin synthesis of ray florets indicated that ray florets only accumulate cyanidin with the expression of F3'H in 9 different developmental capitulums of'Riqietaohong'('DF-3'). The pattern of anthocyanin accumulation was similar to that of structural genes expression, but the latter was slightly earlier than the former. CmDFR and CmANS only expressed in ray florets. Besides, In addition, inactivation of transcriptional factors would suppress the expression of several genes involved in anthocyanin synthesis, and thus can't accumulate anthocyanin in ray florets.After that, we optimized the concentration of selection pressure for buds regeneration and rooting. Then, we induced F3'5'H homologous gene, SCFH into chrysanthemum by Agrobacterium mediated. 865 explants were carried out, and 8 individual resistant lines were attained, with the efficiency of 0.92%.6 of 8 were SCFH positive lines after PCR amplification. SCFH transcript was detected in 5 of 6 lines. The color of transgenic lines was darker than control. The content of anthocyanin significantly increased in transgenic lines, but new type of anthocyanin was not detected by HPLC. To verify the activity of DFR to reduce DHM, full length cDNA of DFR homologus CmDFR was isolated from ray florets of'DF-3'through RT-PCR and RACE. Overexpression and RNAi of CmDFR in chrysanthemum indicated that CmDFR directly related to coloration of chrysanthemum flower. Overexpression could accumulate more anthocyanin, while suppression expression by RNAi could down regulate other key structural genes involved in anthocyanin synthesis.36 resistant lines were attained through 1250 chrysanthemum petiole tTCLs Agrobacterium mediated cotransformation of SCFH and F3'H RNAi vector, with the efficiency of 2.48%.10 lines were F3'5'H overexpression lines,5 lines were F3'H RNAi lines, and 16 lines were cotransgenic lines, with the ratio of 51.6%. The expression of F3'Hi lines was down regulated, but the extend of flower color alteration was limited. The flower color of F3'5'H heterologous expression and F3'H RNAi chrysanthemums became redder and bluer.In this paper, we established the chrysanthemum tTCLs transgenic system, and resolved the problems of variety specificity and low transformation efficiency. CHS, CHI, F3H, F3'H, DFR and ANS are key structural genes involved in anthocyanin synthesis. Down regulation or suppression the expression of these genes would influence the synthesis of anthocyanin. F3'5'H did not involve in the synthesis of anthocyanin. DFR, F3'5'H and F3'H were key structural genes which affected the formation of blue color chrysanthemum. Heterologous expression of F3'5'H could enhance the content of anthocyanin, and make the flower color redder and bluer. Overexpression of DFR in chrysanthemum could enhance the content of anthocyanin, and make the flower color redder. However, interference the expression of DFR in chrysanthemum through RNAi would decrease the content of anthocyanin, and make the flower color paler. Interference the DFR expression in chrysanthemum would down regulate the expression of other key structural genes involved in anthocyanin synthesis, which indicated that DFR involved in coloration of chrysanthemum directly, and the role of DFR was very important. The influence of expression suppression of F3'H on chrysanthemum coloration was limited, which indicate that may exist other F3'H copy(ies) in chrysanthemum genome. Heterologous expression and suppression the expression of F3'H in chrysanthemum could make the flower color redder and bluer.