Part:BBa_K3458003

35S Promoter + HQT

This part is the L. japonica. hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) coding region, with a composite 35s promoter element.

Usage and Biology

Because this is a composite of promoter and the coding region for the HQT sequence, it can be expressed by its two components--please see BBa_K3458001 and BBa_K414002.

The flower buds of Lonicera japonica are widely used in Chinese medicine for their anti-inflammatory properties. The reason why L. japonica has potent and significant effects is that it contains various active components, especially chlorogenic acid (CGA). This is a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encoding a protein of 439 amino acids from L. japonica..

Fig. 1 A simplified diagram of the three alternative routes for chlorogenic acid biosynthesis. The product names appear underneath the structures. Enzymes involved in this pathway are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-hydroxycinnamoyl CoA ligase; HCT, hydroxycinnamoyl CoA shikimate/quinate hydroxycinnamoyl transferase; C3′H, p-coumaroyl 3′-hydroxylase; HQT, hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase; UGCT, UDP glucose: cinnamate glucosyl transferase; HCGQT, hydroxycinnamoyl D-glucose: quinate hydroxycinnamoyl transferase.

The biosynthetic route of synthesizing CGA from caffeoyl-CoA and quinate using hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) has been reported as the most important way for plants to synthesize CGA. And the result of recent study also showed that tissue distribution of HQT was in accordance with the pattern of CGA content.

35S is a plant specific promoter, obtained from Cauliflower Mosaic Virus, and the part is intended for use as a constitutive promoter for gene expression experiments. In order to express HQT in Oryza sativa L. protoplast we choose the plant specific constitutive promoter 35S.

Characterization

* Western Blot

We used the composite part 35S Promoter + HQT (BBa_K3458003) for expression. Western Blot was performed to detect protein expression after transfection of Oryza sativa L. protoplasts.

Fig. 2The result of the Western Blot

As shown, the HQT gene is expressed successfully in protoplasts. Then we measured the protein expression in the protoplast every 3 hours and measured the gray value of each band. These results provide a reference for the subsequent retransfection of protoplasts into HPLC.

* HPLC

Fig. 3 The result of the HPLC

We use HPLC to compared the wile type of protoplast, chlorogenic acid standard samples and our product sample obtained from the transfected protoplast. The peak value of the product measured in our product sample was basically consistent with the peak value of chlorogenic acid standard samples. However, no overlapping peak with the protoplast standard sample was found in the wild-type liquid chromatography, which proved that wild protoplasts do not produce chlorogenic acid，and we can determine that the transfected protoplast can produce chlorogenic acid.

References

Peng X, Li W, Wang W, et al. Cloning and characterization of a cDNA coding a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase involved in chlorogenic acid biosynthesis in Lonicera japonica[J]. Planta Med., 2010,76(16):1921-1926.

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 BsaI site found at 191