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

Pending Publication Date: 2022-07-29
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The genome of Patricia genus is huge and there are many kinds of saponins, so it is very difficul

Method used

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  • Glycosyl transferase PpUGT73E5 and application thereof in synthesis of polyphyllin
  • Glycosyl transferase PpUGT73E5 and application thereof in synthesis of polyphyllin
  • Glycosyl transferase PpUGT73E5 and application thereof in synthesis of polyphyllin

Examples

Experimental program
Comparison scheme
Effect test

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.

[0050] 2. Gene Cloning

[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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Abstract

The invention relates to a saponin metabolic pathway, in particular to glycosyl transferase PpUGT73E5 and application of the glycosyl transferase PpUGT73E5 in synthesis of polyphyllin. The glycosyl transferase is a protein as shown in a1 or a2: a1, a protein with an amino acid sequence as shown in SEQ ID NO: 1; a2, a protein with glycosyl transferase activity, which is formed by substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence as shown in SEQ ID NO: 1. The glycosyltransferase can catalyze glycosylation of steroid sapogenin, and a gene resource is provided for subsequent obtaining of multiple polyphyllin.

Description

technical field [0001] The present invention relates to a saponin metabolic pathway, in particular to a glycosyltransferase PpUGT73E5 and its application in the synthesis of C. chinensis. Background technique [0002] Chonglou is a general term for plants of the genus Chonglou in the Liliaceae family. It can be used as the main raw material of Chinese patent medicines such as Yunnan Baiyao, Gongxuening and Reduqing, and has extremely high medicinal and economic value. Steroidal saponins are the main chemical constituents of Polyphyllin plants, and more than 160 species have been isolated and identified, mainly including polyphyllins I, II, III, V (diosgenin) and VI, VII (polyphyllins). ), with a wide range of pharmacological activities. Anti-tumor is the main effect of salicylic acid, salicylic acid saponin I induces autophagy and cell cycle arrest by inhibiting PDK1 / Akt / mTOR signaling pathway and down-regulating cyclin B1 in human gastric cancer HGC-27 cells (He et al. 201...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P33/20
CPCC12N9/1048C12P33/20
Inventor 薛哲勇宋伟华欣袁强
Owner NORTHEAST FORESTRY UNIVERSITY
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