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Swertia bimaculata ceraniol-10 hydroxylase gene SmG10H and uses thereof

A technology of geraniol and hydroxylase, applied in application, genetic engineering, oxidoreductase, etc., can solve the problems of unexplored biosynthesis steps and achieve the effect of improving accumulation

Inactive Publication Date: 2009-08-19
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Swertiamarin is an iridoid compound. Starting from the synthesis of GPP (geranyl pyrophosphate), the framework of its metabolic pathway is basically clear, but its complete biosynthetic steps have not yet been ascertained.

Method used

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  • Swertia bimaculata ceraniol-10 hydroxylase gene SmG10H and uses thereof
  • Swertia bimaculata ceraniol-10 hydroxylase gene SmG10H and uses thereof
  • Swertia bimaculata ceraniol-10 hydroxylase gene SmG10H and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Cloning of embodiment 1, SmG10H

[0020] 1.1 Extraction of total RNA from Western Sichuan Swertia

[0021] (1) Put the Swertia japonica material in western Sichuan into a pre-cooled mortar, add liquid nitrogen and quickly grind it into a uniform powder. Pay attention to ensure that the material is immersed in liquid nitrogen during the grinding process.

[0022] (2) After the liquid nitrogen volatilizes, quickly transfer the material to a pre-cooled centrifuge tube, add 1ml TRIZOL solution for every 50-100mg of tissue material, mix well, place at room temperature for 5min, centrifuge at 12000r / min, 2-8℃ for 10min To precipitate.

[0023] (3) Add 0.2ml of chloroform to every 1ml of TRIZOL solution, oscillate for 15sec to mix well, leave at room temperature for 5min, centrifuge at 12000r / min, 2-8°C for 15min.

[0024] (4) Take the supernatant, add 0.5ml of isopropanol to every 1ml of TRIZOL, mix well, and place at room temperature for 10-20min.

[0025] (5) Centrifuge...

Embodiment 2

[0109] Example 2, Functional Verification of Western Sichuan Swertia geraniol-10 Hydroxylase Gene SmG10H Expressed in Yeast

[0110] 2.1 Construction of yeast expression vector

[0111] 2.1.1 Obtaining the target gene

[0112] (1) Primer design: Design a pair of PCR amplified bands with EcoRI and KpnI restriction sites, both of which are SmG10H genes.

[0113] P2: 5'- GAATTC ATGGATTTTGATTTCCTCACC-3'

[0114] EcoRI

[0115] P3: 5'- GGTACC TCACAGAGGAGTCGCAACA-3′

[0116] KpnI

[0117] (2) PCR reaction system:

[0118] 10×PCR buffer 2μl

[0119] MgCl 2 (25mmol / L) 1.5μl

[0120] dNTP (each 250μmol / L) 0.2μl

[0121] P2 (4μmol / L) 1μl

[0122] P3 (4μmol / L) 1μl

[0123] Recombinant plasmid pMD18-T-SmG10H 1μl

[0124] Ex Taq (TaKaRa) 0.2μl

[0125] Sterile water 13.1μl

[0126] (3) PCR reaction conditions are

[0127] 94°C for 3min; 94°C for 30sec, 55°C for 30sec, 72°C for 1.5min, 35 cycles; finally 72°C for 10min. The PCR products were recovered...

Embodiment 3

[0225] Example 3. Functional verification of geraniol-10 hydroxylase gene SmG10H in Swertia japonica plants in western Sichuan

[0226] 3.1 Functional verification of Swertia geraniol-10 hydroxylase gene SmG10H in western Sichuan

[0227] 3.1.1 Protein extraction of Swertia geraniol-10 hydroxylase SmG10H from Western Sichuan Swertia

[0228] (1) Take Swertia japonica plants in western Sichuan, put them in a mortar, add liquid nitrogen and grind for 10 minutes.

[0229] (2) The ground cell fragments were quickly added to the 0°C pre-cooled extract [50mM dipotassium hydrogen phosphate buffer (pH7.6) containing 0.5M sucrose, 1mM EDTA, 1mM DTT and 10μM leupeptin; 2mL / g fresh weight], ice bath for 5 minutes.

[0230] (3) After the above cell mixture was filtered through a nylon membrane with a pore size of 48 mm, it was centrifuged at 200×g for 20 minutes at 4°C.

[0231] (4) Remove the supernatant, collect the precipitate, and centrifuge at 20,000×g for 60 minutes at 4°C.

[0...

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Abstract

The invention discloses a swretia mussotii franch geraniol-10 hydroxylase gene SmG10H, a yeast expression vector and a plant expression vector of the gene SmG10H. The invention also discloses application of catalyzing geraniol to generate 10-hydroxy geraniol by the gene SmG10H in pichia and swretia mussotii franch plants. Tests prove that the the swretia mussotii franch geraniol-10 hydroxylase gene SmG10H, the yeast expression vector and the plant expression vector of the gene SmG10H provide theory basis and practical foundation for improving content of the target product 10-hydroxy geraniol in the pichia and swretia mussotii franch plants, and the transgenic plant is greatly improved on accumulating performance of swertiamarin over a non-transgenic plant.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a gene related to medicinal components of Swertia japonica in western Sichuan—geraniol-10 hydroxylase gene SmG10H and its application. Background technique [0002] Swertia mussotii Franch, an alpine Tibetan medicinal herb, belongs to the family Gentianaceae and belongs to the genus Swertia. It grows on the hillsides, shrubs, grasslands, forest margins, and river beaches at an altitude of 3600-3800 meters on the Qinghai-Tibet Plateau, and is most concentrated on the banks of the Tongtian River. It contains flavonoid glycoside (flavonoidglycoside), triterpenoid coumarin trace alkaloids, flavin, mangiferin, oleanolic acid, bitter gentioside, and eight kinds of xanone ingredients. It is cool, rough, and bitter, and has the functions of clearing the liver and gallbladder, and reducing all heat. For jaundice hepatitis, viral hepatitis and blood disease, st...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/81C12N15/82C12N9/02C12R1/84
Inventor 向凤宁王俊峰
Owner SHANDONG UNIV
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