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Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene

A technology of tanshinone content and hairy root, applied in the field of genetic engineering, can solve problems such as poor stability, complex tanshinone structure, and reduced content of active ingredients, and achieve reliable effects, increase total tanshinone content, and low cost

Inactive Publication Date: 2013-05-29
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to long-term cultivation quality degradation of Salvia miltiorrhiza; reduced content of active ingredients; and long growth cycle of Salvia miltiorrhiza, the yield of tanshinone is low, which cannot meet the needs of human medical treatment for tanshinone
The structure of tanshinone is complex, the chemical synthesis process is cumbersome, and the cost of artificially synthesizing tanshinone is high; and the production process is easy to cause environmental pollution; the accumulation of cell active components obtained by the cell culture method under in vitro conditions is low and the stability is poor

Method used

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  • Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene
  • Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene
  • Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] (1) Extraction of total RNA from Salvia miltiorrhiza and synthesis of the first strand of cDNA:

[0073] Using the RNA prep pure plant kit provided by TIANGEN, follow the steps specified in the instructions in the kit to extract total RNA from the transgenic hairy roots of Salvia miltiorrhiza. The fresh weight of the transgenic hairy roots of Salvia miltiorrhiza used to extract total RNA is 0.1g, and the DNA in the sample during the extraction process is removed with DNase working solution. Measure the absorbance of the extracted RNA on a spectrophotometer to calculate the purity and concentration of the extracted RNA. After calculating the concentration of different RNA samples, 0.5μg RNA was used as the initial amount to synthesize the first-strand cDNA with reverse transcriptase XL (AMV), and the operation steps were in accordance with the instructions provided by Promega.

[0074] (2) SmGGPPS Design of coding sequence-specific primers and obtain target gene fragments:

...

Embodiment 2

[0077] With SmGGPPS Construction of plant expression vector of gene.

[0078] (1) Intermediate vector pCAMBIAl304 + The build:

[0079] Use pBIl21 and pCAMBIAl304 as materials to construct plant expression vector pCAMBIAl304 + . Specifically, HindIII / EcoRI double enzyme digestion of pBI121 and pCAMBIAl304; recovery of the pBI121-GUS expression cassette and pCAMBIAl304 large fragment; ligation transformation, picking of monoclonal colonies to extract plasmids for verification. The results showed that the plant expression vector pCAMBIAl304 + The build was successful.

[0080] (2) Plant expression vector pCAMBIAl304 + - SmGGPPS The build:

[0081] PCAMBIAl304 successfully constructed in the above + Based on the use of cloned from Salvia SmGGPPS Gene replacement above GUS gene. The specific method is BamHI / SacI double enzyme digestion pMD18T- SmGGPPS And pCAMBIAl304 + ; Recycling SmGGPPS Gene and pCAMBIAl304 + Large fragments; ligation transformation, picking of monoclonal colonies f...

Embodiment 3

[0084] Agrobacterium rhizogenes mediated SmGGPPS Genetically transformed Salvia miltiorrhiza to obtain transgenic hairy roots.

[0085] (1) Containing plant expression vector pCAMBIAl304 + - SmGGPPS Obtaining of Agrobacterium rhizogenes engineering bacteria:

[0086] Include in Example 2 SmGGPPS Gene expression vector pCAMBIAl304 + - SmGGPPS Transform into Agrobacterium rhizogenes C58C1, pick a single colony for PCR verification. The results show that containing SmGGPPS The plant expression vector of the gene has been successfully constructed into Agrobacterium rhizogenes strain C58C1.

[0087] (2) Mediated by Agrobacterium rhizogenes SmGGPPS Genetic transformation of Danshen.

[0088] (3) Pre-culture of explants:

[0089] Cut the robust aseptic seedling leaves of Salvia miltiorrhiza 0.5 cm 2 , Inoculate on the pre-culture medium MS, cultivate in the dark at 25 ℃ for 2 days.

[0090] (4) Co-cultivation of Agrobacterium and explants:

[0091] Soak the above-mentioned pre-cultured Salvia m...

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Abstract

The invention belongs to the technology for gene engineering, and discloses a method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene. In the prior art, the growth cycle of the salvia miltiorrhiza is long; the tanshinone content is low, and the requirement of the human medical treatment for the tanshinone content cannot be met. The method comprisesthe following steps of: cloning a coding sequence of the SmGGPPS gene from salvia miltiorrhiza, and constructing a plant high-efficient expression vector; genetically transforming salvia miltiorrhizaleaves to obtain the salvia miltiorrhiza hairy roots with transferred SmGGPPS gene; analyzing the expression condition of the SmGGPPS gene in the transgenic salvia miltiorrhiza hairy roots by a semi-quantitative RT-PCR method; measuring the total tanshinone content including cryptotanshinone, tanshinone I and tanshinone II A of the transgenic salvia miltiorrhiza hairy roots by high performance liquid chromatography; and measuring the antioxidant activity of cryptotanshinone of the transgenic salvia miltiorrhiza hairy roots by a diphenyl picryl-hydrazyl (DPPH) method. The method has the advantages of obviously increased total tanshinone content of the salvia miltiorrhiza hairy roots, reliable effect, low cost of tanshinone, and no environmental pollution during manufacturing.

Description

Technical field [0001] The present invention belongs to genetic engineering technology, specifically a kind of transformation SmGGPPS A method for genetically increasing the tanshinone content of Salvia miltiorrhiza hairy roots. Background technique [0002] Salvia ( Salvia miltiorrhiza Bunge ), also known as purple salvia, red root, red ginseng, and blood stimulating ginseng, a perennial herbaceous plant belonging to the sage of the family Lamiaceae. Danshen is an important traditional Chinese medicine in the medicine of the motherland, and it is clinically used for the treatment of cardiovascular diseases. The medicinal ingredients of Danshen include: tanshinone fat-soluble diterpene quinone compounds and water-soluble phenolic acid compounds. Modern technology shows that the tanshinone substances contained in Danshen, such as cryptotanshinone, tanshinone I, and tanshinone IIA, have multiple pharmacological activities such as anti-oxidation, anti-atherosclerosis, anti-tumor, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/82C12Q1/68G01N30/89G01N21/31A01H5/00A01H5/06
Inventor 开国银许辉廖攀周伟张林
Owner SHANGHAI NORMAL UNIVERSITY
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