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CYP450 gene catalyzing oxidation of C-28 site of amyrin and coding product and application of CYP450 gene

A technology for catalyzing incense and resinol, applied in the fields of application, genetic engineering, oxidoreductase, etc., to achieve the effect of short nucleotide sequences

Active Publication Date: 2020-02-11
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although there are a large number of CYP450s related to the oxidative modification of the triterpene backbone that have been identified so far, there is no report that discloses the protein encoded by the natural short-sequence cytochrome P450 enzyme gene that can catalyze the oxidation of balsamic resin at the C-28 position

Method used

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  • CYP450 gene catalyzing oxidation of C-28 site of amyrin and coding product and application of CYP450 gene
  • CYP450 gene catalyzing oxidation of C-28 site of amyrin and coding product and application of CYP450 gene

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Cloning of embodiment 1 Gangmei IaAO2 gene

[0062] Use the plant RNA extraction kit (Magen) to extract the total RNA of the young leaves of Gangmei, and use the reverse transcription kit (full gold) to reverse transcribe to obtain cDNA, design specific primers according to the transcriptome data, and use the cDNA as a template for PCR Amplification, agarose gel electrophoresis showed that a specific fragment appeared at about 1200bp, the target fragment was recovered with an agarose gel recovery kit (Magen), cloned into pEASY vector (full gold), and positive clones were identified and verified by sequencing , for the construction of recombinant expression vectors.

[0063] 1. Construction of yeast expression vector

[0064] By designing primers with BamH I and Xho I restriction sites, the cloned IaAO2 gene was inserted between the BamH I and Xho I restriction sites of the yeast expression vector pESC-TRP (Stratagene) by PCR , obtained the recombinant plasmid pESC-T...

Embodiment 2

[0069] Example 2 Homologous gene IaAO1 and 5'-terminal truncated new gene tIaAO1

[0070] In the early stage, our laboratory cloned and identified another balsamol C-28 oxidase gene IaAO1 from the medicinal plant Prunus mellifera, and constructed a Saccharomyces cerevisiae expression vector pESC-TRP-IaAO1. Comparing the sequence of IaAO2 with the homologous gene IaAO1, the latter has an extra nucleotide fragment at the 5'-end than the former. According to the overlapping sequence of IaAO2, IaAO1, design specific primers, carry out PCR amplification with pESC-TRP-IaAO1 as the template, agarose gel electrophoresis shows that specific fragments appear at about 1200bp place, use agarose gel recovery kit ( Magen) recovered target fragments, cloned into pEASY vector (full gold), identified positive clones and performed sequencing verification for the construction of recombinant expression vectors.

[0071] 1. DNA sequence alignment

[0072] Comparing the sequences of IaAO2 and t...

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Abstract

The invention discloses a CYP450 gene IaAO2 catalyzing the oxidation of the C-28 site of amyrin. A protein which catalyzes the oxidation of the C-28 site of amyrin is encoded, and the CYP450 gene IaAO2 can be induced and expressed in engineered strains to catalyze alpha-amyrin and beta-amyrin to be oxidized into ursolic acid and oleanolic acid separately. The nucleotide sequence of the CYP450 geneIaAO2 is as shown in SEQ ID No.1, and the amino acid sequence of the CYP450 gene IaAO2 is as shown in SEQ ID No.2. The invention further discloses an expression vector and host bacterium of the CYP450 gene IaAO2 and application of the CYP450 gene IaAO2 to preparation of triterpenoid compounds, compounds catalyzing the oxidation of the C-28 site of pentacyclic triterpenes and compounds catalyzingthe oxidation of alpha-amyrin and beta-amyrin of pentacyclic triterpenes. The invention further discloses a method for performing homologous gene modification based on the sequence characteristics ofthe gene IaAO2 and a novel gene tIaAO1 obtained by the method. The IaAO2 gene has a shorter nucleotide sequence than a homologous gene, a genetically modified organism can be constructed by using theIaAO2 gene to synthesize a pentacyclic triterpenoid compound with important economic value, and good application prospects are achieved.

Description

technical field [0001] The invention relates to a gene that catalyzes the C-28 oxidation of balsamic resin and its coded product and application, and belongs to the field of genetic engineering of medicinal plants, in particular to a CYP450 gene that catalyzes the C-28 oxidation of balsamic resin and its coded product and application. Background technique [0002] Cytochrome P450 monooxygenases (CYP450) are a family of heme proteases that widely exist in nature and have oxidation functions, and participate in various metabolic reactions in microorganisms, animals, and plants. In plants, CYP450 plays important physiological functions in the biosynthesis of terpenes, flavonoids, alkaloids, plant hormones and other substances. In the downstream of the biosynthetic pathway of triterpene saponins, CYP450 plays a role in modifying the triterpenoid skeleton, enriching the structural diversity, and is one of the factors that contain a wide variety of triterpenoids in plants. Theref...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/80C12N1/19C12N15/11C12P33/00C12R1/865
CPCC12N9/0071C12Y114/14C12N15/80C12P33/00
Inventor 徐晖
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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