Team:GDSYZX/Results

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Result

1. Gene cloning

We first carried out codon optimization for our two target genes 4HPAAS and UGT33 according to the preference of Arabidopsis codon. After obtaining the target gene, we obtained a large number of target gene fragments by PCR gene cloning in vitro.

2. Carrier construction

As shown in the figures below, the carrier puc-ha and the carrier puc-flag were digested and glued. From the results of running gel, the enzyme digestion products of both vectors were 4000bp. Therefore, we judge we got purpose fragment.

3. The colony PCR

After the gene was transferred to the vector, we transferred the plasmid to e. coli overnight. The next day, PCR was performed to test whether the gene transfer was successful. The figure shows that the size of both UGT33 gene and 4HPAAS gene is around 500dp, and the result is positive. After the PCR result of bacterial liquid was positive, we made a small plasmid and sent it to test, and obtained the transferred escherichia coli successfully.

4. Test the expression products

We transfected two genes into the protoplast simultaneously. For the transformed protoplast, we first performed western blot using protoplast. Western blotting was performed at 4 hours, 8 hours, 10 hours, 12 hours and 14 hours, respectively, and the gray values were compared. We found that the highest expression efficiency of UGT33 and 4HPAAS was 12 hours after they were transferred into the protoplast. This proves that the protein can be expressed successfully and provides reference time for liquid chromatography.

Below is the result of a liquid chromatograph. The first row is the wild type, the second row is the protoplast samples of Arabidopsis Thalianaafter transfection, and the third row is the standard sample of salidroside. The peak value of product measured in the second sample was basically consistent with the peak value of salidroside standard sample, while there was no corresponding peak in the detection results of wild type. Therefore, we can determine that the protoplast of the untransfected Arabidopsis Thaliana does not contain salidroside, while the protoplast of the transfected Arabidopsis Thaliana can produce salidroside.

5. To summarize

Based on the above results, 4HPAAS and UGT33 are the key genes for producing salidroside. In the case that the salidroside anabolism pathway in Arabidopsis has not been explored, we successfully predicted two key genes and obtained the expression of salidroside after transferring into Arabidopsis.