Team:GDSYZX/Experiment

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Experiment

1.Selection of research object

Salidroside is the main active ingredient of traditional Chinese medicine Rhodiola rosea. Previous studies have shown that Salidroside has high medical value However, there are fewer and fewer Rhodiola rosea in nature. Therefore, other methods are urgently needed to solve the current problem. The synthetic pathway of salidroside in Rhodiola rosea has been found. 4HPAAS and UGT33 are the key enzymes in the synthetic pathway of salidroside. As we all know, Arabidopsis thaliana is a model plant and has many advantages than other plants, such as short life cycle. Protoplast of Arabidopsis has used in study of many biological phenomena. We propose that transferred 4HPAAS and UGT33 into the protoplast of Arabidopsis thaliana, can we obtain salidroside in the protoplast ?

2.Synthesis of UGT33 and 4HPAAS

3.Vector construction

We digest PUC19-FLAG and PUC19-HA, cloned both genes, recycled the DNA segments and then link vectors and the target segments by Gibson Assembly. Next, we transferred the combined vector to E.coli. We cloned the bacteria and select the positive clones. At last we sent the extracted plasmid to sequencing company for sequencing.

4.Massive plasmid extraction

We use a kit to extract the plasmid.

5.Preparation of protoplast of Arabidopsis thaliana

6.Transfection of the protoplast

7.Western Blot

We use Western blot to verify the successful transfection of both genes to the protoplast.

8.Extraction and detection of purpose products

We use ethanol for extraction and detect salidroside by high performance liquid chromatography.

Developments of Project

1.Decide the topic of research project and read scientific essays

2.Select the target sequence (UGT33 & 4HPAAS) and send it to the biological company for synthesis to transfer into the plasmids (PUC19-HA & PUC19-FLAG)

3.Transfect the protoplast of Arabidopsis Thaliana with the plasmids.

4.Extract and detect salidroside in the protoplast.

Protocols