Team:GDSYZX/Parts
PARTS
| Name | Type | Description | Length |
|---|---|---|---|
| BBa_K3458001 | Coding | HQT(Hydroxycinnamoyl-CoA Quinate Hydroxycinnamoyl Transferase) | 1320bp |
| BBa_K3458002 | Promoter | GluD-1 Promoter | 1228bp |
| BBa_K3458003 | Composite | 35S Promoter + HQT | 2355bp |
| BBa_K3458004 | Composite | GluD-1 Promoter + HQT | 2554bp |
| BBa_K3458005 | Coding | Apis Mellifera Silk Fibroin 3 With Codon Optimization | 951bp |
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HQT
http://parts.igem.org/BBa_K3458001
Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encodes a protein of 439 amino acids from L. japonica.. (After codon optimized according to the codon preference of Oryza sativa L..)
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 by codon preference in rice. In order to meet the assembly requirements, we also removed the illegal restriction site.
For the purpose of expressing HQT in Oryza sativa L. and conducting further tests, we also designed two composite originals, GluD-1::HQT.
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GluD-1 Promoter
http://parts.igem.org/BBa_K3458002
The promoter of the Oryza sativa L. glutelin gene (GluD-1), due to the deviation in the GCN4 motif, this pomoter has the characteristics of being specifically expressed in the endosperm of Oryza sativa L..
GluD-1 is only a specific promoter, which can not be expressed alone. The expression of starch (daf-30d) in endosperm of rice increased steadily, and the expression of glu-5d protein began to increase steadily. The endosperm specificity of GluD-1 promoter (1228 bp) is responsible for its spatial specificity. Compared with other grain SSP gene promoters (such as GlB-1 and GluB-1), GluD-1 has endosperm specific expression, which makes it more suitable for the expression of recombinant protein in starch endosperm. GluD-1 specific promoter is the key to endosperm expression in rice, which provides a great help for the project of increasing chlorogenic acid content in rice seed by inserting HQT gene, which is also a gap of expression.
Studies have shown that 0.2kb GluD-1 promoter is enough to endoplasmic endosperm specific expression, and 1.2kb GluD-1 promoter has the best endosperm specific expression. Therefore, we chose 1.2kb GluD-1 promoter as the experimental promoter. Since we chose protoplasts from rice stems and leaves as experimental objects, the GluD-1 promoter could not express specificity. We conducted a control experiment with 35S promoter.
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GluD-1Promoter+HQT
http://parts.igem.org/BBa_K3458003
This part is the L. japonica. hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) coding region, with a composite GluD-1 promoter element.
The flower buds of Lonicera japonica are widely used in Chinese medicine for their anti-inflammatory properties and the chlorogenic acid (CGA) is one of useful active components. This is a hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) gene encoding a protein of 439 amino acids from L. japonica.
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. Its spatial specificity is caused by the characteristics of the GluD-1 promoter . Since our goal is to increase the content of chlorogenic acid in rice seeds specifically, we chose the 1.2kb GluD-1 promoter.
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.
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35S Promoter + HQT
http://parts.igem.org/BBa_K3458004
This part is a composite part containing the L. japonica. hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) coding region, with a composite 35s promoter element.
Because the GluD-1 promoter cannot express the gene in rice protoplasts due to its specific expression in Oryza sativa L. endosperm, we choose the plant specific constitutive promoter 35S to express HQT in Oryza sativa L. protoplast. 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.
Western Blot was performed to detect protein expression after transfection of Oryza sativa L. protoplasts and the HQT gene is expressed successfully in protoplasts.
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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Apis Mellifera Silk Fibroin 3 With Codon Optimization
http://parts.igem.org/BBa_K3458005
According to the codon preference of Arabidopsis thaliana, our team optimized the codon of BBa_K1763000 to make it more suitable for expression in plants, providing a basis for subsequent research on this part in plants.