Flavonoid 3 ', 5' Hydroxylase Gene Cloning And Transformation Of Petunia Study

Flavonoid-3',5'-hydroxylase (F3',5'H) corresponding to the genetic loci Hfl and Hf2,a member of the cytochrome P450 family,is the key enzyme in the anthocyanin biosynthesis,which is generally required for blue or purple flowers. To isolate Hfl -,Hf2 gene encoding flavonoid-3',5'-hydroxylase (F3',5'H) from petunia hybrida of the purple flower,we used a polymerase chain reaction (PCR) amplification using the genomic DNA as a template. The result indicated that the Hfl DNA was 2797bp in total length and encoded 506 amino-acid,containing three extron and two intron;Hf2 DNA was 2223bp in total length and encoded 509 amino-acid,containing three extron and two intron. Comparison of the sequence with Genbank databases revealed that Hfl DNA and Hf2 DNA shared significant nucleotides identity (98.97% and 99.04% respectively) and amino-acid identity (99.41%and 99.41%respectively) .We excised the PGN fragment,which contains a CaMV 35S promoter a GUS gene and a Nos terminator from the plant expression vector pBI121 with EcoR I and Hindlll and inserted it between the EcoR I and Hindlll sites of the plant expression vector pCAMBIA3301,yielding pC3301-PGN. The Hf2 fragment was excised from pG-HO with Xba I and Sac I and then cloned between the Xba I and Sac I sites of the plant expression vector pC3301-PGN to produce pC3301-Hf2. In this construct,Hf2 DNA should be expressed under the control of the CaMV 35S promoter. The construct was transformed to petunia hybrida of the light pink via Agrobacterium tumefaciens EH A105 using the leaf disc method. In the end,three transgenetic plants were obtained by screening with the phosphinothricin resistance and PCR amplification.The achievement of transgenetic plants would provide a scientific basis and lay firm foundation for the transformation of Hfl and Hf2 gene into other ornamental flowers.