Team:GDSYZX/Design

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Design

The strain we used in this experiment was E. coli dh5a, and the pattern plant was Arabidopsis. we use the Arabidopsis protoplast system to express our target genes and use liquid chromatography to detect the target products.

In the first place, we optimized the genes 4HPAAS and UGT33 according to the codon preference of Arabidopsis thaliana, and synthesized them in the company. That’s how we construct the vectors of 4HPAAS and UGT33: first, we conducted the enzyme digestion of vector PUC19-HA, then we clone the segment of the two genes that are mentioned; we did the DNA gel recovery afterward. Second, we did the enzyme digestion and connection of the target segments. At the begining, we planned to ligate two target genes which are UGT33 and 4HPAAS and their vectors through T4 ligase. After several experiments, we found that this way was inefficient and could not make positive clone. Due to time constraints, we decided to use the method of Gibson recombination to carry out the recombination connection, and this method successfully connected these two target genes to their vectors and this turned out that the latter method succeeded.

After that, the vector recombination by transforming E.coli was proceeded; positive clones were selected by running PCR and sequence verification.



After the expression vector containing the target genes 4HPAAS and UGT33 was obtained, we extracted plenty of protoplast and then the target gene was transformed into Arabidopsis thaliana cells by protoplast transfer technique. Before we test them with chromatography, we conduct the Western blot experiment to test the expression of protein. After the cells were cultured at room temperature for 12 hours, we collected the cells and extracted them with organic solvents to obtain the extract containing salidroside. After that, the target product was detected by prepared high performance liquid chromatography (HPLC). The results show that the amount of salidroside expressed in our protoplast is similar to the counterpart in wild R. rosea, which is the main producer of salidroside. 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.