Honeysuckle is an important Chinese medicine, which has been recorded in ancient books such as Lei Gong's Medicinal Herbs Solution, Materia Medica Fengyuan and Materia Medica Huiyan. The main medicinal component of honeysuckle is chlorogenic acid. Honeysuckle and chlorogenic acid have been widely used in the fields of food, medicine and chemical industry. In order to produce adequate chlorogenic acid, our team hopes to improve the work done by “2020 GDSYZX” to achieve high expression of chlorogenic acid in model plant called Arabidopsis Thaliana.
We optimized their HQT gene sequence according to the codon preference of Arabidopsis Thaliana and named it AtHQT gene (BBa_K3880011). The optimized AtHQT gene does not contain the prohibited restriction sites of iGEM's bio-brick, and then the optimized sequence was synthesized in Tianyi Huiyuan Company. In order to express HQT and AtHQT protein in Arabidopsis Thaliana protoplast, we linked the 35S promoter and AtHQT gene to pUC19, and marked the end of the gene with an HA tag to construct pUC19:: HQT vector. After that, we extract plasmids, transfer the plasmids into the protoplasts of Arabidopsis Thaliana and culture the protoplasts for 12 hours to run protein gel electrophoresis and Western blot to test the expression quantity of HQT and AtHQT. We hope that by overexpression of HQT in Arabidopsis Thaliana protoplast, we can increase the production of chlorogenic acid.
Results and discussion
1. Clone of AtHQT gene
The Fig.1A is the DNA gel electrophoresis of the AtHQT gene after PCR. From it we can see that there is clearly a bright band between 1000bp and 2000bp, which is basically the same size as our target gene (the length of the AtHQT gene is 1320bp), so we speculate that the band amplified by PCR is the HQT gene. Later, we linked AtHQT gene to the pUC19-HA vector via recombination method and then transformed this recombinant vector into E.coli DH5α.
2. The colony PCR of AtHQT gene
The Fig.1b shows the colony PCR of recombinant E.coli DH5α. The gene we designed to amplify is HQT. The length of the AtHQT. gene is 1320bp. The electropherogram shows that the 1-5 wells have bands around 1000bp. We selected these colonies for further sequencing verification.
3. Western Blot detects the expression of the target protein
We respectively transformed pUC19:: AtHQT and pUC19:: OsHQT (35s::HQT, BBa_K3458003) recombinant vector into protoplasts of Arabidopsis Thaliana. The results of western blot showed that both AtHQT and OsHQT could be detected in protoplasts. However, the expression level of AtHQT was higher than OsHQT which was in line with our expectations.
Outlook and perspective
Chlorogenic acid is one of the most important medicine and have been widely used in the fields of food, medicine and chemical industry. However, the content of self-synthesizing chlorogenic acid in Honeysuckle and other plants is low. The amount of chlorogenic acid on the market cannot meet people's needs. In order to produce adequate chlorogenic acid, team “2020 GDSYZX” developed a project to achieve high expression of chlorogenic acid in rice. As rice has a long growth cycle, our team improved their part by overpression AtHQT in model plant called Arabidopsis Thaliana. We successfully expressed AtHQT in protoplasts of Arabidopsis Thaliana. However, we could not do more work as the limitation of time. But, our project is not end. We will do more work about our project in the future.