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

A technology for tanshinone content and hairy roots, applied in the field of genetic engineering, can solve the problems of complex tanshinone structure, poor stability, and reduced active ingredient content, and achieve the effects of increasing total tanshinone content, reliable effect and low cost

Inactive Publication Date: 2011-02-02
SHANGHAI NORMAL UNIVERSITY
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  • 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

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

[0074] (2) SmGGPPS Design of coding sequence-specific primers and acquisition of targe...

Embodiment 2

[0077] contain SmGGPPS Construction of plant expression vectors for genes.

[0078] (1) Intermediate vector pCAMBIA1304 + The build:

[0079] Using pBI121 and pCAMBIA1304 as materials, construct the plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII / EcoRI; the pBI121-GUS expression cassette and the large pCAMBIA1304 fragment were recovered; ligation transformation was carried out, and single-clonal colonies were picked to extract plasmids for digestion verification. The results showed that the plant expression vector pCAMBIA1304 + The build was successful.

[0080] (2) Plant expression vector pCAMBIA1304 + - SmGGPPS The build:

[0081] The successful pCAMBIA1304 constructed above + Based on, using cloned from Salvia miltiorrhiza SmGGPPS gene replacement above GUS Gene. The specific method is BamHI / SacI double digestion of pMD18T- SmGGPPS and pCAMBIA1304 + ;Recycle SmGGPPS gene and pCAMBIA1304 + Large fragmen...

Embodiment 3

[0084] Agrobacterium rhizogenes mediated SmGGPPS Genetic transformation of Salvia miltiorrhiza to obtain transgenic hairy roots.

[0085] (1) Containing plant expression vector pCAMBIA1304 + - SmGGPPS Obtaining Agrobacterium rhizogenes engineering bacteria:

[0086] Contain in embodiment 2 SmGGPPS Plant expression vector pCAMBIA1304 + - SmGGPPS Transformed into Agrobacterium rhizogenes C58C1, picked a single clone colony for PCR verification. The results showed that containing SmGGPPS The plant expression vector of the gene has been successfully constructed in Agrobacterium rhizogenes strain C58C1.

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

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

[0089] Cut 0.5 cm leaves of healthy aseptic seedlings of Salvia miltiorrhiza 2 , inoculated onto the pre-culture medium MS, and cultured in the dark at 25°C for 2 days.

[0090] (4) Co-cultivation of Agrobacterium and explan...

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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 invention belongs to genetic engineering technology, specifically a kind of transgenic SmGGPPS Genetic method for increasing the content of tanshinone in the hairy root of Salvia miltiorrhiza. Background technique [0002] Salvia ( Salvia miltiorrhiza Bunge ), also known as purple salvia, red root, red ginseng, blood ginseng, Labiatae Salvia genus, perennial herb. Salvia miltiorrhiza is an important traditional Chinese medicine in motherland medicine, and it is clinically used for the treatment of cardiovascular diseases. The medicinal components of Danshen include: tanshinone lipid-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 various pharmacological activities such as anti-oxidation, anti-atherosclerosis, anti-tumor, anti-bacterial and anti-inflammatory, and have a huge ...

Claims

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Application Information

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