Difference between revisions of "Part:BBa K3458001"

 
 
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Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encoding a protein of 439 amino acids from ''L. japonica.''.（After codon optimized according to the codon preference of ''Oryza sativa L.''.）
  
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===Usage and Biology===
 
===Usage and Biology===
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'''Because this is just the coding region for the HQT sequence, it is not meant to be expressed on its own. For an expressible HQT biobrick part, please see [https://parts.igem.org/Part:BBa_K3458003 BBa_K3458003] and  [https://parts.igem.org/Part:BBa_K3458004 BBa_K3458004].'''
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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.''.
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[[File:HQT_Pathway.png|none|thumb|650px|'''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.]]
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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.
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Our team aims to increase the content of chlorogenic acid in rice to make rice health product effect. In order to increase the expression level of chlorogenic acid in ''Oryza sativa L'', our team optimized the codon of the HQT gene of ''Lonicera japonica'' and tested the expression level in the rice expression system. Our work is of significance for further studies concerned with strengthening the efficacy of ''Oryza sativa L.'' by means of improved CGA content. (You can find more related information on our wiki)
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===Characterization===
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'''* Western Blot'''
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We used the composite part 35S Promoter + HQT ([https://parts.igem.org/Part:BBa_K3458003 BBa_K3458003])  for expression. Western Blot was performed to detect protein expression after transfection of ''Oryza sativa L.'' protoplasts.
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[[File:WB_of_35S--HQT.png|none|thumb|650px|]]
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[[File:WB_of_35S--HQT-2.png|none|thumb|650px|'''Fig. 2'''The result of the Western Blot]]
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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.
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'''* HPLC'''
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[[File:Result_of_HPLC.png|none|thumb|650px|'''Fig. 3''' The result of the HPLC]]
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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.
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===References===
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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.
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Latest revision as of 03:13, 17 August 2020

HQT(Hydroxycinnamoyl-CoA Quinate Hydroxycinnamoyl Transferase)

Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encoding a protein of 439 amino acids from L. japonica..（After codon optimized according to the codon preference of Oryza sativa L..）


Usage and Biology

Because this is just the coding region for the HQT sequence, it is not meant to be expressed on its own. For an expressible HQT biobrick part, please see BBa_K3458003 and BBa_K3458004.

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.

Our team aims to increase the content of chlorogenic acid in rice to make rice health product effect. In order to increase the expression level of chlorogenic acid in Oryza sativa L, our team optimized the codon of the HQT gene of Lonicera japonica and tested the expression level in the rice expression system. Our work is of significance for further studies concerned with strengthening the efficacy of Oryza sativa L. by means of improved CGA content. (You can find more related information on our wiki)

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.

WB of 35S--HQT.png
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
    COMPATIBLE WITH RFC[1000]