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Alisma squalene epoxidase and its application

A technology of ene epoxidase and Alisma shark, which is applied in the field of molecular biology, can solve the problems of narrow distribution of resources, low content, and inability to meet utilization needs, and achieve the effect of improving synthesis efficiency

Active Publication Date: 2022-07-01
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Alisma orientale (Alisma orientale) is a plant belonging to the family Alismaceae. Its main medicinal ingredients are proterane-type tetracyclic triterpenoids, which have obvious anti-hyperlipidemia, hypotension, and anti-HIV1 effects, especially It has remarkable anticancer activity and broad prospects for its development and application. However, its resource distribution is narrow and its content is low. It only exists in a few plant groups such as Alisma genus, which cannot meet the needs of utilization. Bioengineering is one of the effective ways to increase the yield of medicinal ingredients

Method used

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  • Alisma squalene epoxidase and its application
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  • Alisma squalene epoxidase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example specifically illustrates the full-length cloning method of the Alisma squalene epoxidase of the present invention.

[0039] Alisma is harvested from the Jianou planting base in Fujian Province, and identified as the Alisma orientale (Sam.) Juzep by Professor Gu Wei of Nanjing University of Traditional Chinese Medicine. After collection, they were transplanted to the flower room of Nanjing University of Traditional Chinese Medicine. After the plants grew stably, the fresh leaves were taken and aliquoted into cryopreservation tubes, and stored in liquid nitrogen for future use.

[0040] 1.1 Extraction and detection of total RNA from Alisma

[0041] The extraction of total RNA was carried out according to the operation steps of Tiangen total RNA extraction kit (batch number: DP419):

[0042] ①Take 100mg of plant tissue, transfer it to a pre-cooled mortar, and grind it into powder in liquid nitrogen;

[0043] ②Add 1 mL of lysis solution to the powder, and aft...

Embodiment 2

[0067] This example illustrates the prokaryotic expression method of the gene of the present invention.

[0068] 2.1 Vector construction: The primers (SF5, SR5; SF6, SR6) shown in Table 2 were designed according to the sequences of Alisma orientalis AoSE1 and AoSE2, and SE was cloned into the pGHn vector. The reaction system and procedure are as follows:

[0069] Table 2 Primer sequence list in the text

[0070]

[0071] The reaction system is:

[0072]

[0073]

[0074] Reaction program: 94°C for 5min; 94°C for 30sec, 54°C for 30sec, 72°C for 1min, 30cycles; 72°C for 1min.

[0075] The PCR product was mixed with Loading Buffer and then loaded on a 1% agarose gel, and the target band was detected in a gel imaging system. The recovery, ligation, PCR identification and clone sequencing of the target fragment. The gene AoSE ORF was cloned into the pCzn1 expression vector between NdeI and XhoI, and the target gene was fused with 6X his tag protein to obtain the pCzn1-A...

Embodiment 3

[0097] This example illustrates the functional validation of AoSE1 and AoSE2 enzymes.

[0098] In order to test the catalytic function of AoSE1 and AoSE2, an in vitro enzymatic reaction system was established.

[0099] The reaction volume is 500 μl: 0.1 mM DTT, 25 mM MgCl 2, 490μM squalene, 0.14mM NADPH, 0.5% Triton X-100, 1mM FAD, 10μL target protein, add Tris-HCl (PH 7.4) to 500μL reaction system, incubate the reaction at 25°C for 20h, add 15 μL to each test tube The experimental reaction was terminated by %KOH / MeOH and extracted twice with equal volume of n-hexane for content determination. Negative controls were performed under the same conditions, but using E. coli extracted with an empty vector.

[0100] Metabolites were qualitatively analyzed by HPLC, chromatographic conditions: YMC C 18 Chromatography column (5 μm, 4.6×250 mm). Mobile phase: (phase A: water, phase B: acetonitrile); elution program: 0–20min, 25%–65%B; 20–40min, 65%–85%B; 40–50min, 85%B; 50 -60min, ...

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Abstract

The invention discloses an Alisma squalene epoxidase and an application thereof. The Alisma squalene epoxidase has the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4. The invention clones the full length of the Alisma squalene epoxidase gene for the first time, provides an Alisma squalene epoxidase, a prokaryotic expression method of the enzyme gene, and prepares the polyclonal antibody of the enzyme protein , the ELISA detection method and the Western Blot detection method of the protein are provided, and the invention can be used to improve the biosynthesis efficiency of Alisma proterane type triterpenes and monitor the dynamic accumulation process of medicinal components.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to Alisma squalene epoxidase and its application. Background technique [0002] Alisma orientale is a plant belonging to the genus Alisma, and its main medicinal components are orthoterpane-type tetracyclic triterpenoids. Its anticancer activity is remarkable, and its development and application prospects are broad, but its resource distribution is narrow and its content is low, and it only exists in a few plant groups such as Alisma, which cannot meet the needs of utilization. Bioengineering is one of the effective ways to improve the yield of medicinal components. . Squalene epoxidase (SE) is a key enzyme in the biosynthesis of orthoterane-type triterpenes, which can be used to improve the biosynthesis efficiency of orthoterane-type triterpenes to meet the demand. SUMMARY OF THE INVENTION [0003] The purpose of the present invention is to provide a squal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C07K16/40C07K16/06G01N33/573
Inventor 谷巍吴启南巢建国张阿琴徐飞李思蒙田方
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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