Abstract：Flavanone 3-hydroxylase (F3H) is a key enzyme in the metabolic pathways of flavonoid compounds in plants. The open reading frame(ORF) of F3H gene, which included 1 107 basic group and encoded a 368 amino acids protein, was cloned from tea (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 41.46 kD and its theoretical isoelectric point was 5.61. The results of Real-time fluorescent quantitative PCR showed that CsF3H had high expression levels in the leaves, and was influenced by light condition. The gene was cloned into the expression vector PRSF for expression in prokaryotic cells. The SDS-PAGE results showed that the F3H peotein was expressed in Escherichia coli BL21. The optimal inducing conditions including temperature, isopropyl-β-d-thiogalactoside(IPTG) concentration and time were studied , and were 28 ℃, 1.0 mmol/L, 5 h, respectively. The activity of the recombinant protein in vitro was tested by high performance liquid chromatography(HPLC) method. Experiment results showed that purified protein had the F3H enzyme activity and transfered naringenin(N) and eriodictyol(E) respectively to dihydrokaempfero(DHK) and dihydroquercetin(DHQ), while the recombinant protein had a much more preference for eriodictyol(E). The research provides basic data for the kinetic study and organ specificity of CsF3H protein.
