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CsF3H is a coding sequence that codes for flavanone 3-hydroxylase, or called Flavonol synthase, in Camellia sinensis. It is dioxygenase involved in the flavonoid biosynthesis which has ligand with iron, metal-binding and Vitamin C. In nature, the enzyme expressed in young seedlings (at protein level). Expressed in roots, emerging leaves, shoot-root transition zone, trichomes, flowers and siliques. In cotyledons, expressed mostly on the adaxial side and only in guard cells on the abaxial side. The flavanone 3-hydroxylase catalyzes the formation of flavonols from dihydroflavonols. It can act on dihydrokaempferol to produce kaempferol, on dihydroquercetin to produce quercitin and on dihydromyricetin to produce myricetin. In vitro catalyzes the oxidation of both enantiomers of naringenin to give both cis- and trans-dihydrokaempferol. In term of its catalytic activity, 2-oxoglutarate + a (2R,3R)-dihydroflavonol + O2 = a flavonol(naringenin) + CO2 + H2O + succinate In our project, the sequence will express flavanone 3-hydroxylase to convert naringenin into dihydroflavonol.

Figure 1: [1] The schematic of catalytic activity

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CsF3H in Level1 plasmid assembly

Figure 2: [2] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CsF3H

The construction schematic of CsF3H sequence demonstrated in Fig.2. The initiation of the CsF3H sequence is done by promoter pGAL7, with termination done by tENO2. The sequence ConL1 and ConR2 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are imposed to enact selection through colonies PCR, results shown in Fig.9 B The band length 2755bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

CsF3H in Level2 plasmid assembly

Figure 3: [3] A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

CsF3H is successfully inserted into the vector type9 C-mTurquoise-Ura.