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Glycosyltransferase genes related to glycyrrhizic acid biosynthesis and coding products and application thereof

A glycyrrhizic acid, coding technology, applied in the direction of glycosyltransferase, transferase, plant gene improvement, etc., can solve the problem of UGT gene not being successfully cloned, etc.

Inactive Publication Date: 2017-08-04
刘春生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The biosynthesis of glycyrrhizic acid in plants requires a series of catalytic reactions by related enzymes in the biosynthetic pathway. At present, the genes of most enzymes in the glycyrrhizic acid biosynthetic pathway have been successfully cloned and can be artificially synthesized by biotechnology methods glycyrrhetinic acid, however, the UGT gene that catalyzes the last step in glycyrrhetinic acid synthesis has not yet been successfully cloned

Method used

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  • Glycosyltransferase genes related to glycyrrhizic acid biosynthesis and coding products and application thereof
  • Glycosyltransferase genes related to glycyrrhizic acid biosynthesis and coding products and application thereof
  • Glycosyltransferase genes related to glycyrrhizic acid biosynthesis and coding products and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Example 1: Cloning of UGT in Glycyrrhiza uralensis

[0073] We extracted total RNA from licorice (Glycyrrhiza uralensis Fisch) roots with an RNA extraction kit (TAKARA, Japan), and obtained cDNA with a reverse transcription kit (TAKARA, Japan). The primers of the enzyme cutting site are used for PCR amplification with high-fidelity HS taq enzyme (TAKARA company in Japan), and the PCR products are sequenced and spliced ​​to obtain the full-length nucleosides of the UGT gene related to the biosynthesis of glycyrrhizic acid involved in the present invention The acid sequence is represented by SEQ ID No.1, the length of its full-length open reading frame (ORF) is 1458bp, encoding 485 amino acids, and the detailed amino acid sequence is shown in SEQ ID No.2 in the sequence listing.

Embodiment 2

[0074] Embodiment 2: Construction of recombinant expression vector

[0075] The nucleotide fragment (SEQ ID No.1) of glycyrrhizic acid UGT obtained in Example 1 and the expression vector pET-32a (+) were both digested with restriction endonucleases (Japanese TAKARA company) simultaneously, and the enzyme The cleaved product was purified and recovered using the method of an agarose gel recovery kit (TAKARA, Japan), and the recovered product was ligated overnight in a metal bath at 16° C. with DNA ligase to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli DH5α competent cells and plated on a solid medium containing ampicillin for screening. The positive clones were sent for sequencing verification again, and the correct clones verified by sequencing could be recovered by the method of the plasmid purification kit (TAKARA Company, Japan). A recombinant expression vector with the nucleotide sequence of SEQ ID No.1 is obtained.

Embodiment 3

[0076] Embodiment 3: the construction of host bacterium

[0077] We transformed the Escherichia coli BL21 (DE3) competent cells with the recombinant expression vector obtained in Example 2 according to the competent cell transformation manual (TAKARA company in Japan) and plated them on solid medium containing ampicillin for screening, positive clones Sent again for sequencing verification, and the correct clone of the sequencing verification is used as the host bacterium, which contains the recombinant expression vector with the nucleotide sequence of SEQ ID No.1.

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Abstract

The invention discloses glycosyltransferase (UGT) genes participating in glycyrrhizic acid biosynthesis and coding products and application thereof, and belongs to the field of medicinal plant genetic engineering. The genes are cloned from radix glycyrrhizae for the first time, are key enzyme genes for glycyrrhizic acid synthesis metabolic pathways and can directly catalyze production of glycyrrhizic acid. A nucleotide sequence provided by the invention is shown as SEQ ID No. 1, and a protein sequence coded by the nucleotide sequence is shown as SEQ ID No. 2. The glycosyltransferase genes, provided by the invention, are closely related to the glycyrrhizic acid biosynthesis, and have significance on regulating contents of the glycyrrhizic acid in plants and artificial production of the glycyrrhizic acid.

Description

technical field [0001] The invention relates to a glycosyltransferase (UGT) gene related to the biosynthesis of glycyrrhizic acid, and uses an in vitro enzymatic reaction method to analyze the functional activity of the protein encoded by the gene, and relates to the UGT gene and its encoded product in the biosynthesis pathway of glycyrrhizic acid The invention and application belong to the field of genetic engineering of medicinal plants. Background technique [0002] Glycyrrhizic acid is a triterpenoid saponin natural product derived from Glycyrrhiz, which has a wide range of biological activities, including anti-inflammatory, anti-allergic and anti-viral. At present, glycyrrhizic acid has been developed into an injection for clinical treatment of chronic hepatitis B and acute liver injury. According to reports, the annual sales of Tianqingganmei injection, which is made with glycyrrhizic acid as the main ingredient, can reach 1.6 billion yuan. At the same time, because ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12P33/20C12N15/82A01H5/06
CPCC12N9/1051C12N15/8242C12P33/20C12Y204/01017
Inventor 徐国杰刘春生
Owner 刘春生
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