Composite

Part:BBa_K3458004

Designed by: Jiahua Zou   Group: iGEM20_GDSYZX   (2020-08-16)
Revision as of 03:33, 17 August 2020 by Kawah (Talk | contribs) (→‎Characterization)


GluD-1 Promoter + HQT

This part is the L. japonica. hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) coding region, with a composite GluD-1 promoter element.


Usage and Biology

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.

Oryza sativa L. glutelin (GluD-1) is a member of the glutelin family. The expression of GluD-1 gene has unique temporal and spatial characteristics. GluD-1 was predominantly expressed in the inner starchy endosperm beginning about 5 DAF and steadily increased until maturity at 30 DAF. Its spatial specificity is caused by the characteristics of the GluD-1 promoter . Studies have shown that Only about 0.2 kb of the GluD-1 promoter was suffificient to confer inner starchy endosperm-specifific expression. Since our goal is to increase the content of chlorogenic acid in rice seeds specifically, we chose the 1.2kb GluD-1 promoter.

Characterization

The GluD-1 promoter cannot express the gene in rice protoplasts due to its specific expression in Oryza sativa L. endosperm. To verify this, we designed two composite parts and used western blot to compare GluD-1 promoter and 35S constitutive promoter).（more information on BBa_K3458003 and BBa_K3458004)

Fig. 2 File:The result of the Western Blot .png

The results of western blot showed that the 35s promoter can initiate the expression of HQT gene in Oryza sativa L. protoplasts while the GluD-1 promoter cannot.This result can prove the specificity of GluD-1 promoter expression.

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.rc site found at 225


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Parameters
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