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Gentiana straminea beta-amyrin synthetase gene GsAS1 and use thereof

A technology of synthetase and prickly ash, applied in the direction of steroids, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problem of unreported research on the relationship with oleanolic acid, etc.

Inactive Publication Date: 2009-07-08
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although the β-amyrin synthetase gene has been cloned in many species, the gene has not been cloned, functionally verified and applied in plant transformation for the Gentianaceae plant Gentianae There is no report on the relationship between aranolic acid

Method used

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  • Gentiana straminea beta-amyrin synthetase gene GsAS1 and use thereof
  • Gentiana straminea beta-amyrin synthetase gene GsAS1 and use thereof
  • Gentiana straminea beta-amyrin synthetase gene GsAS1 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Cloning of embodiment 1, GsAS1

[0025] 1.1 Extraction of total RNA from Gentiana japonica

[0026] (1) Put the genita material 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.

[0027] (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.

[0028] (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.

[0029] (4) Take the supernatant, add 0.5ml of isopropanol to every 1ml of TRIZOL, mix well, and place at room temperature for 10-20min.

[0030] (5) Centrifuge at 12000r / min for 10min at 2-8°C, d...

Embodiment 2

[0114] Example 2, Functional Verification of Gentiana β-amyrin Synthetase Gene GsAS1

[0115] 2.1 Construction of yeast expression vector

[0116] 2.1.1 Obtaining the target gene

[0117] (1) Primer design: Design a pair of PCR amplification bands with EcoRI and XhoI restriction sites, both of which are GsAS1 genes.

[0118] P01: 5'- GAATTC ATGTGGAGGCTAAAAATCGG-3'

[0119] EcoRI

[0120] P12: 5'- CTCGAG CGTCTCCTACGGCACTTGCTTGCG-3'

[0121] wxya

[0122] (2) PCR reaction system:

[0123] 10×PCR buffer 2μl

[0124] MgCl 2 (25mmol / L) 1.5μl

[0125] dNTP (each 250μmol / L) 0.2μl

[0126] P01 (4μmol / L) 1μl

[0127] P02 (4μmol / L) 1μl

[0128] Recombinant plasmid pMD18T-AS1 1μl

[0129] rTaq (TaKaRa) 0.2μl

[0130] Sterile water 13.1μl

[0131] (3) PCR reaction conditions are

[0132] 94°C for 5min; 94°C for 30sec, 60°C for 30sec, 72°C for 2min, 35 cycles; finally 72°C for 7min. The PCR products were recovered by electrophoresis and used in subsequent reactions.

...

Embodiment 3

[0202] Embodiment 3, the construction of plant expression vector

[0203] 3.1 Obtaining the target gene

[0204] Primer design and PCR reaction system and conditions:

[0205] (1) Design the following pair of PCR amplification primers, and introduce the two enzyme cutting sites SacI and XbalI contained in the plant expression vector pROK2 (purchased from Bao Biological Engineering Co., Ltd.).

[0206] P11: 5'-CGAGCTATGTGGAGGCTAAAAATCGG-3'

[0207] SacI

[0208] P12: 5'-GCTCTAGACGTCTCCTACGGCACTTGCTTGCG-3'

[0209] wxya

[0210] (2) PCR reaction system:

[0211] 10×PCR buffer 2μl

[0212] MgCl 2 (25mmol / L) 1.5μl

[0213] dNTP (each 250μmol / L) 0.2μl

[0214] P11 (4μmol / L) 1μl

[0215] P12 (4μmol / L) 1μl

[0216] Recombinant plasmid pMD18T-AS1 1μl

[0217] rTaq (TaKaRa) 0.2μl

[0218] Sterile water 13.1μl

[0219] (3) PCR reaction conditions are

[0220] 94°C for 5min; 94°C for 30sec, 60°C for 30sec, 72°C for 2min, 35 cycles; finally 72°C for 7min. The PCR products ...

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Abstract

The invention discloses a twisted Gentiana beta-amyrin synthetase gene GsAS1 and yeast and plant expression vector containing the gene GsAS1, also discloses a twisted Gentiana beta-amyrin synthetase gene GsAS1 and the applications of the plant expression vector containing the gene GsAS1 in the preparation of the oleanolic acid. The invention provides a theoretical basis and practical basis for improving the oleanolic acid content in the target products.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to an oleanolic acid-related gene—beta-amyrin synthetase gene GsAS1 and its function verification. Background technique [0002] Plant secondary metabolism refers to the process in which some organisms use certain primary metabolites as "raw materials" to form some special chemical substances under the catalysis of a series of enzymes. These special chemical substances are secondary metabolites, such as Alkaloids, flavonoids, terpenoids, organic acids, lignin, etc., are a large class of small molecule organic compounds that are not necessary for plant growth, but play an irreplaceable role in the survival and development of plants in complex environments. role. [0003] Plant secondary metabolites have important economic value. Many clinically applied drugs are secondary metabolites derived from plants. About 25% of western clinical drugs are secondary ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/81C12N15/82C07J63/00
Inventor 向凤宁刘艳玲
Owner SHANDONG UNIV
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