Lupeol C28 oxidase gene and preparation method and application thereof

A lupeol and oxidase technology, applied in biochemical equipment and methods, applications, genetic engineering, etc., can solve problems such as destruction of plant resources, high production costs, and unreported P450 genes, and achieve great application prospects

Inactive Publication Date: 2012-05-02
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there have been many reports on the cloning of 2,3-epoxysqualene cyclase gene, but the downstream P450 gene has not been reported at home and abroad
[0003] The compound betulinic acid is mainly distributed in Betulaceae plants, and is synthesized in the bark. At present, the method of obtaining this anticancer drug mainly depends on plant resources; directly extracting and purifying from plants, the production cost is high, which seriously leads to the loss of plant resources. Destroyed and Endangered

Method used

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  • Lupeol C28 oxidase gene and preparation method and application thereof
  • Lupeol C28 oxidase gene and preparation method and application thereof
  • Lupeol C28 oxidase gene and preparation method and application thereof

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Embodiment Construction

[0035] Below in conjunction with accompanying drawing and embodiment describe in detail:

[0036] 1. Acquisition of the full-length cDNA sequence of the lupeol C28 oxidase gene

[0037] According to GenBank ( www.ncbi.nlm.nih.gov ) provided in the 3 sequence expression tags (the registration numbers of the sequence tags in GenBank are FD660831, FD661068 and FD661229) information, use RACE-PCR technology to obtain the full-length cDNA sequence information of this gene, and then use the following general PCR The reaction system can be obtained.

[0038] 25 μl PCR reaction system: 2 μl cDNA template, 1 μl each of primers P1 and P2, 5 μl 5×Buffer, 2 μl dNTP Mixture (2.5 mmol / L each), 0.2 μl Phusion enzyme (NEB Biotechnology Co., Ltd.), ddH 2 O 13.8 μl;

[0039] The reaction conditions were as follows: pre-denaturation at 98°C for 3 minutes; then 35 cycles at 98°C for 10s, 62°C for 30s, and 72°C for 45s; finally, extension at 72°C for 5 minutes.

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Abstract

The invention discloses a lupeol C28 oxidase gene and a preparation method and application thereof and relates to a technology of biosynthesis of pentacyclic triterpene compounds. The lupeol C28 oxidase gene is derived from periwinkle leaves and is named as CYP716AL1. Gene sequences are a gene sequence (1) or a gene sequence (2): the nucleotide sequence of the gene sequence (1) is DNA (deoxyribonucleic acid) molecules shown in SEQIDNO: 1; and the gene sequence (2) is hybridized with a limited DNA sequence under strict conditions and codes proteins or DNA molecules with more than 90 percent ofhomologous proteins. The lupeol C28 oxidase gene is introduced into yeast cells, and transgene yeast can be subjected to stepwise oxidation reactions at a C28 position of lupeol by taking the lupeol as a substrate and finally forms betulinic acid serving as an anti-cancer compound.

Description

technical field [0001] The invention relates to the biosynthesis technology of pentacyclic triterpene compounds, in particular to a lupeol C28 oxidase gene and its obtaining method and application. Background technique [0002] Pentacyclic triterpene compounds are widely distributed in the plant kingdom, and there are mainly three types: ursane type, oleanane type and lupine type; the representative compounds are ursolic acid, oleanone acid (oleanolic acid) and betulinic acid (betulinic acid). Among them, betulinic acid has significant anti-HIV (AIDS virus) and anti-tumor activities, and is an excellent lead for searching for anti-HIV and anti-tumor drugs. At present, through the structural modification of betulinic acid, many derivatives with excellent biological activity and new mechanism of action have been obtained. The biosynthesis of betulinic acid is mainly through the cyclization of 2,3-epoxy squalene to form lupeol, which is then generated by the downstream cytoch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/10C12N1/19C12P33/00G01N30/02
Inventor 章焰生黄莉莉李长福黎佳
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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