Part:BBa_K4012011

AaDFR( Arabidopsis thaliana dihydroflavonol 4-reductase)

AaDFR is a coding sequence that is responsible for coding dihydroflavonol 4-reductase,also called Tetraketide alpha-pyrone reductase 1, in Anthurium andraeanum. In nature, the enzyme in Anthurium andraeanum is Involved in the biosynthesis of hydroxylated tetraketide compounds that serve as sporopollenin precursors (the main constituents of exine), which is essential for pollen wall development. To achieve this, the enzyme acts on tetraketide alpha-pyrones and reduces the carbonyl function on the tetraketide alkyl chain to a secondary alcohol function. (a (2R,3S,4S)-leucoanthocyanidin + NADP+ = a (2R,3R)-dihydroflavonol + H+ + NADPH) In our project, we recombine it into to plasmid to convert dihydroflavonol into leucoanthocyanidin

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 758
  

AaDFR in Level1 plasmid assembly

Figure 2: [1] A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence AaDFR

The construction schematic of AaDFR sequence demonstrated in Fig.11. The initiation of the AaDFR sequence is done by promoter pTDH3, with termination done by tENO1. The sequence ConL3 and ConR4 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.2B The band length 2377bp match with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

AaDFR in Level2 plasmid assembly

Figure 3: [2] 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

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