Difference between revisions of "Part:BBa K3458004"

(→‎References)
(→‎Characterization)
 
(2 intermediate revisions by the same user not shown)
Line 18: Line 18:
  
 
===Characterization===
 
===Characterization===
−
'''* Western Blot'''
+
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 [https://parts.igem.org/Part:BBa_K3458003 BBa_K3458003] and  [https://parts.igem.org/Part:BBa_K3458004 BBa_K3458004])
 +
[[File:WB_of_35s--HQT_and_GluD-1--HQT.png|none|thumb|650px|'''Fig. 2''' The result of the Western Blot]]
 +
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.
  
−
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.
+
===References===
−
[[File:WB_of_35S--HQT.png|none|thumb|650px|]]
+
[1]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.
−
[[File:WB_of_35S--HQT-2.png|none|thumb|650px|'''Fig. 2'''The 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'''
+
[2]Kawakatsu T, Yamamoto M P, Hirose S, et al. Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm[J]. J. Exp. Bot., 2008,59(15):4233-4245.
  
−
[[File:Result_of_HPLC.png|none|thumb|650px|'''Fig. 3''' The result of the HPLC]]
+
[3]Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305.
−
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===
+
[4]Paine JA, Shipton CA, Chaggar S, et al. 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnolgy 23, 482–487.
−
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.
+
  
 +
[5]Qu LQ, Takaiwa F. 2004. Evaluation of tissue specifificity andexpression strength of rice seed component gene promoters intransgenic rice. Plant Biotechnology Journal 2, 113–125.
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:45, 17 August 2020


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 The result of the Western Blot

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

[1]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.

[2]Kawakatsu T, Yamamoto M P, Hirose S, et al. Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm[J]. J. Exp. Bot., 2008,59(15):4233-4245.

[3]Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I. 2000. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305.

[4]Paine JA, Shipton CA, Chaggar S, et al. 2005. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnolgy 23, 482–487.

[5]Qu LQ, Takaiwa F. 2004. Evaluation of tissue specifificity andexpression strength of rice seed component gene promoters intransgenic rice. Plant Biotechnology Journal 2, 113–125. 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