Cloning And Transformation Of Color Gene F3＇5＇H And DFR In Petunia Hydrida

Object: There are various colors in the nature, but some important flowers lack blue color, therefore, research of color breeding has been the work of the major targets. In the biosynthesis of anthocyanins, F3'5'H (Flavonoid-3',5'-hydroxylase) is the key enzyme in the formation of blue flowers, which can divert the production of delphinidin pigments. DFR (Dihydro flavonol-4-reductase) play a decisive role in the final formation of anthocyanin, it can specifically catalyse dihydro flavonol reduced to colorless proanthocyanidins, which will further generate blue-violet anthocyanin. F3'5'H gene as an important component for the blue flowers, identified the expression of F3'5'H gene will build the foundation for the research of improved variety by genetic engineering technology.Methods: The total RNA was isolated from the purple flower of Petunia hybrida. A specific F3'5'H fragment and DFR fragment of the expected size were amplified by RT-PCR and sequenced .The F3'5'H and DFR fragments were respectively constructed in a sense-orientation driven by CaMV 35S promoter, and then we successfully obtained plant expression vector pBI-F3'5'H and pBI-DFR. Subsequently, we transformed pBI-F3'5'H into pink Petunia hybrida by using Agrobacterium tumefacien mediated method.Results: The sequence analysis indicated that,the cloned 1.7kb cDNA of F3'5'H fragment including an open reading frame of 1521bp by which encodes a predicted poly peptide of 506 amino acids. Compared with the F3'5'H gene (D14588) in GeneBank ,the homology of nucleotide sequence could reach 99. 34%, and 99.6% at the amino acid sequence,only two amino acid differences. And the cloned 1.2kb cDNA of DFR fragment including an open reading frame of 1143bp by which encodes a predicted poly peptide of 380 amino acids. Compared with the DFR gene(X15537) in GeneBank, the homology of nucleotide sequence could reach 99.39%, and 100% at the amino acid sequence.A construct of F3'5'H in a sense-orientation driven by CaMV 35S promoter was constructed and transformed into pink flower of Petunia hybrida by using Agrobacterium tumefacien mediated method. The transgenic plants were confimed that F3'5'H gene was interated into genome of pink Petunia hybrida by identification of PCR and RT-PCR. we have already obtained transformed plants by transgenic technology, and we will continue studing color variation under the influence of F3'5'H gene, in order to get color changed plants.Conclusion: We have successfully cloned F3'5'H and DFR gene of Petunia hybrida which coding the two key enzymes in the biosynthesis pathway of anthocyanins. Furthermore, a plant expression vector pBI-F3'5'H was constructed and the regeneration system of pink Petunia hybrida was established. At present, we have primaryly obtained transformed petunia plants by transgenic technology already, so as to build the foundation for the further research of flower color improving by genetic engineering technology.