Team:GDSYZX/Basic
Basic
This year GDSYZX contributed 3 basic parts, including 4HPAAS, UGT33 and d35s.
4HPAAS & UGT33
4HPAAS(referring to 4-hydroxyphenylacetaldehyde synthase) protein coding region. It catalyzes L-tyrosine to produce 4-hydroxy-phenylacetaldehyde. It is the first enzyme in the biosynthesis of salidroside. →4HPAAS's page UDP-glucose-4-O-glucosyltransferase(T8GT) protein coding region.UGT33 is the most active gene among R. rosea Tyrosol-Modifying UGTs. The protein it catalyzes reduces tyrosol to produce salidroside. UGT33 is used as the last gene to complete salidroside biosynthesis from tyrosine. →UGT33's page
Experiment
The pattern plant Arabidopsis and E. coli DH5α were used in this experiment . we use Arabidopsis protoplast to express our target genes and use HPLC y（high performance liquid chromatograph） to detect the targeted product. In the first place, the genes 4HPAAS and UGT33 were optimized and synthesized according to the preference codon of Arabidopsis, We construct the expression vectors of 4HPAAS and UGT33: firstly, we conducted the enzyme digestion of vector pUC19-HA by BamHI and StuI. Then the two genes were cloned and recoveryed by DNA gel afterward. Secondly, we conducted Gibson Assembly to combine the target segments and the vectors. After that, the vector was transformed into E. coli DH5α and were selected by running PCR and sequence verification. After the expression vectors containing the target genes 4HPAAS and UGT33 were obtained, we extracted plenty plasmid and transformed them into protoplast of Arabidopsis . We first conducted western blot experiment to test the expression of protein and found both of them could expressed in protoplast of Arabidopsis.As the expression level of protein is corelated with the production of salidroside ,we then conducted western blot experiment to test the expression of protein at different times to acquire the best expression time of protein. After that, the target product was detected by HPLC. The results showed salidroside expressed in our protoplast.
The result proves that our experimental scheme is feasible and we can reasonably to imagine a future in which the wide-used component presented in Chinese medicine, salidroside, is much less prohibitive than it is today.
And in order to find a suitable detection time for salidroside, we explored the rule that the expression of 4HPAAS and UGT33 in Arabidopsis protoplasts in respond to the change of time. It was found that the expression of 4HPAAS and UGT33 reached the highest level 10 hours after transfection, so we speculated that this time should be the time with the highest synthesis rate of salidroside, so we chose 12 hours after transfection as the best time to detect salidroside.
A: Western blot was used to detect the expression of 4HPAAS and UGT33 in respond to the change of time; B: We used “i mage J” to analyze the relative expression of 4HPAAS in respond to the change time (set the band gray value of 4hours as 1); C: i mage J software and UGT33s with time (set the band gray value of 4 hours as 1).
d35s
d35S refers to Double GoldenGate compatible 35S Promoter. 35S is a plant specific promoter obtained from the Cauliflower Mosaic Virus. →d35s's page We add two 35s promotor together to get a d35s promotor, then we merge GFP gene with d35s promotor to make a binary expression vector. After that, we extract plasmids, transfer the plasmids into the protoplasts of Arabidopsis thaliana and culture the protoplasts for 12 hours to view fluorescence intensity run protein gel electrophoresis and Western blot to test the GFP expression rate.
