Team:GDSYZX/Engineering
ENGINEERING
Project Background Investigation
For more information and references, please see our PROJECT DESCRIPTION.
↓ The application of traditional Chinese medicine in the prevention and treatment of COVID-19.
↓ Commonness of traditional Chinese medicine prescriptions for COVID-19-Honeysuckle.
↓ The main active ingredient in honeysuckle-chlorogenic acid.
↓ Chlorogenic acid has clinical antiviral effects.
↓ Honeysuckle is not cheap and the production area is scarce.
↓ The price of rice is relatively cheap and the acceptance is high.
↓ Rice is a good choice for food tonic as a Chassis creature.[1]
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STEP 1 : Expression of Key Enzymes for Chlorogenic Acid Synthesis In Rice
Research → Imagine → Design → Build → Test → Learn → Improve → Research...
Research
HQT is a key enzyme for the synthesis of chlorogenic acid in honeysuckle, and rice contains precursor substances required for the synthesis of chlorogenic acid.[2]
Imagine
The key enzyme HQT for chlorogenic acid synthesis can be expressed heterologously in rice.
Design
Use 35s as a promoter, pUC19-HA as a vector to express HQT heterologously in rice protoplasts.
https://2020.igem.org/Team:GDSYZX/DesignBuild
HQT (BBa_K3458001);
35S Promoter + HQT (BBa_K3458003)
https://2020.igem.org/Team:GDSYZX/PartsTest
Use Western Blot to detect the expression level of HQT in rice protoplasts.
Learn
Western Blot results showed that the target band of HQT could be detected in rice. HQT can be expressed heterologously in rice.
https://2020.igem.org/Team:GDSYZX/ResultsImprove
Confirm whether the expression of HQT can actually increase the content of chlorogenic acid.
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STEP 2 : HQT Expression Increases The Content of Chlorogenic Acid
→ Imagine → Test → Learn → Improve → Research...
Imagine
The expression of HQT gene can actually increase the content of chlorogenic acid in rice protoplasts.
Test
Use the HPLC to detect the content of chlorogenic acid in p35S::HQT transfected rice protoplasts, and compared with the chlorogenic acid standard and wild-type.
Learn
The p35S::HQT transfected rice protoplasts can detect the same peak value as the standard product, but the wild type does not. The expression of HQT can actually increase the content of chlorogenic acid in rice.
https://2020.igem.org/Team:GDSYZX/ResultsImprove
The HQT gene is specifically expressed in rice seeds.
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STEP 3 : Specific Expression of HQT In Rice Seeds
Research → Imagine → Design → Build → Test → Learn → Improve → Research...
Research
The GluD-1 promoter can promote gene specific expression in rice endosperm.[3]
Imagine
GluD-1 promoter can promote the specific expression of HQT gene in rice endosperm.
Design
Use GluD-1 as a promoter, pUC19-HA as a vector to transfected rice protoplasts.
Build
GluD-1 Promoter(BBa_K3458002)；
HQT (BBa_K3458001)；
GluD-1 Promoter + HQT (BBa_K3458004)
https://2020.igem.org/Team:GDSYZX/PartsTest
Use Western Blot to detect the expression level of HQT in rice protoplasts and use the HPLC to detect the content of chlorogenic acid in pGluD-1::HQT transfected rice protoplasts, compare with the p35S::HQT transfected rice protoplasts and wild-type.
Learn
Western Blot results showed that the HQT couldn't be detected in pGluD-1::HQT transfected rice protoplasts. HPLC results showed that the pGluD-1::HQT transfected rice protoplasts cannot detect the same peak value as the standard product. Due to the endosperm specificity of the GluD-1 promoter, in rice protoplasts, pGluD-1::HQT transfection has the same characteristics as wild-type. The GluD-1 promoter is specific.
https://2020.igem.org/Team:GDSYZX/ResultsImprove
Do further testing in rice seeds.
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Conclusion
We successfully expressed the HQT gene heterologously in rice protoplasts, and verified that the expression of the HQT gene can indeed increase the content of chlorogenic acid in rice. In order to express HQT specifically in rice seeds, we introduced the endosperm-specific promoter GluD-1 and confirmed the specificity of GluD-1 promoter.
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Further Research Directions
Due to the COVID-19, the laboratory has temporarily closed. Also, considering the safety of GMO crops, our experimental plan stops at the stage of protoplasts. Although it is a pity that we have not been able to further verify our ideas in rice seeds and conduct clinical tests, we have some ideas for future research directions, which may be helpful.
· Use pGluD-1::HQT transfected protoplasts to obtain transformation seedlings;
· Detect the expression in other stages and parts of the transformed seedlings to verify the specificity of expression;
· Cultivate transformed seedlings to obtain rice seeds;
· Test the HQT expression and chlorogenic acid content in rice seeds;
· Testing medicinal value;
· Edible safrty test;
· …………
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References
[1]Engineering plants with increased levels of the antioxidant chlorogenic acid Ricarda Niggeweg1,3,Anthony J Michael2 & Cathie Martin
[2]Cloning and Characterization of a cDNACoding a Hydroxycinnamoyl-CoAQuinate Hydroxycinnamoyl Transferase Involved in Chlorogenic Acid Biosynthesis in Lonicera japonica Xiaoxiao Peng, Weidong Li, Wenquan Wang, Genben Ba
[3]Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm Taiji Kawakatsu1, Masayuki P. Yamamoto1,*, Sakiko Hirose1, Masahiro Yano2 and Fumio Takaiwa1,