Glycosyl transferase PpUGT73E5 and application thereof in synthesis of polyphyllin
A technology of glycosyltransferase and papaya saponin, which is applied in the direction of transferase, application, genetic engineering, etc., and can solve problems such as difficult analysis
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Embodiment 1
[0047] Example 1. Discovery, Cloning and Expression of Glycosyltransferase Gene PpUGT73E5
[0048] 1. Gene Discovery
[0049] Using Dianzhonglou as the research material, we calculated the correlation coefficient between all genes in the transcriptome of Chonglou through weighted gene co-expression network analysis, and grouped genes with similar expression patterns into the same module, and found out that it is highly synergistic with the distribution and accumulation of Chonglou saponins. module, in which a glycosyltransferase gene was found and named PpUGT73E5. The open reading frame (ORF) of the PpUGT73E5 gene contains 1398 bases, its nucleotide sequence is shown in SEQ ID NO:2, and the encoded amino acid sequence is shown in SEQ ID NO:1. In order to verify the function of PpUGT73E5 gene in the synthesis of steroidal saponins, we cloned and expressed the gene.
[0051] 2.1 Extraction of total RNA from leaves of C. chinensis
[0052] The RNAprep...
Embodiment 2
[0124] Example 2. Functional identification of glycosyltransferase PpUGT73E5
[0125] Step 1: Enzyme activity detection
[0126] Enzymatic activity experiment of PpUGT73E5 protein: Accurately weigh 400 μg steroidal sapogenin (diosgenin / pianogenin), 570 μg UDP-glucose, 50 μL purified PpUGT73E5 protein solution (1 mg / mL), dissolve in PBS phosphate buffer (0.01 M, pH 8.0) to a final volume of 300 μL. After being placed at 37°C to react for 2 hours, the same volume of methanol as the reaction system was added to stop the enzymatic activity, the product was spin-dried under reduced pressure, and then dissolved in 500 μL of chromatographic methanol for testing.
[0127] Set the empty vector control: use the prokaryotic expression product of the pGEX-6p-1 empty vector (that is, the protein obtained from the Rosetta (DE3) cells containing the pGEX-6p-1 empty vector after protein-induced expression and protein purification) instead of PpUGT73E5 protein. The enzymatic activity experim...
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