Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation

A technology for tanshinone content and hairy roots, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the problems of low accumulation of active ingredients, high cost, poor stability, etc., and achieve the goal of increasing the total tanshinone content, Alleviate the shortage of drug sources and have reliable effects

Inactive Publication Date: 2012-12-19
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, the complex structure of tanshinone leads to its complex chemical synthesis process, high cost, and the synthesis process is easy to cause environmental pollution; the cells obtained by the cell culture method under in vitro conditions have very low accumulation of active ingredients and poor stability.

Method used

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  • Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation
  • Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation
  • Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation

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Experimental program
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Effect test

Embodiment 1

[0073] (1) Extraction of total RNA from Salvia miltiorrhiza and synthesis of the first strand of cDNA:

[0074] The RNA prep pure plant kit provided by TIANGEN was used to extract total RNA from the transgenic hairy roots of Salvia miltiorrhiza, and the extraction steps followed the instructions in the kit. The fresh weight of Salvia miltiorrhiza transgenic hairy roots used to extract total RNA is about 0.1 g, and the DNA in the sample has been removed with DNase working solution during the extraction process. Measure the relative absorbance value of the extracted RNA on a spectrophotometer, and calculate the purity and concentration of the extracted RNA. After calculation based on the concentration of different RNA samples, 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.

[0075] (2) wxya and SmGGPPS Design of codi...

Embodiment 2

[0078] Contains Danshen wxya and SmGGPPS Construction of plant expression vectors for genes.

[0079] (1) Intermediate vector pCAMBIA1304 + The build:

[0080] 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 fragment of pCAMBIA1304 were recovered; ligation transformation was carried out, and single-clonal colonies were picked to extract plasmids for digestion and verification. The results showed that the plant expression vector pCAMBIA1304 + The build was successful.

[0081] (2) Plant expression vector pCAMBIA1304 + - SmGGPPS The build:

[0082] The successful pCAMBIA1304 constructed above + Based on the cloned from Salvia miltiorrhiza SmGGPPS gene replacement GUS Gene. Specifically, BamHI / SacI double enzyme cut pMD18T- SmGGPPS and pCAMBIA1304 + ;Recycle...

Embodiment 3

[0087] Agrobacterium rhizogenes mediated wxya and SmGGPPS Genetic transformation of Salvia miltiorrhiza to obtain transgenic hairy roots.

[0088] (1) Containing plant expression vector pCAMBIA1304 + - wxya - SmGGPPS Obtaining Agrobacterium rhizogenes engineering bacteria;

[0089] Contain in embodiment 2 wxya and SmGGPPS Gene plant bivalent expression vector pCAMBIA1304 + - wxya - SmGGPPS Transformed into Agrobacterium rhizogenes C58C1, picked a single clone colony for PCR verification. The results showed that containing wxya and SmGGPPS The plant expression vector of the gene has been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0090] (2) Mediated by Agrobacterium rhizogenes wxya , SmGGPPS Genetic transformation of salvia miltiorrhiza:

[0091] ① Pre-cultivation of explants: cut leaves of healthy and sterile seedlings of Salvia miltiorrhiza (0.5 cm 2 ), inoculated onto the pre-culture medium (M...

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Abstract

The invention discloses a method for enhancing the content of tanshinone in salvia miltiorrhiza hairy root by double genetic transformation, belonging to methods for enhancing the content of tanshinone in medicinal material salvia miltrorrhiza. Tanshinone substance has a plurality of pharmacological activities like antitumor, bacteria resistance and anti-inflammation, anti-atherosclerosis and thelike, however, the content of tanshinone in salvia miltiorrhiza is so low as not to meet the demand of medical treatment. According to the invention, SmHMGR and SmGGPPS genes are cloned from salvia miltrorrhiza, expression vectors containing the SmHMGR and SmGGPPS genes are constructed, leaves of salvia miltiorrhiza are subjected to genetic transformation to obtain salvia miltiorrhiza hairy root of SmHMGR and SmGGPPS transgenes, the content of tanshinone is determined by semiquantitative RT-PCR analysis and high performance liquid chromatography, and antioxidant activity of tanshinone in trangene hairy root by DPPH free radical scavenging method. The method has the advantages that: the content of tanshinone in trangene salvia miltiorrhiza hairy root is enhanced dramatically; and the cost of salvia miltiorrhiza is low. The production process causes no environmental pollution.

Description

technical field [0001] The invention belongs to a method for increasing the content of tanshinone in the Chinese medicinal material salvia miltiorrhiza, in particular to a method for improving the content of tanshinone in the hairy root of the salvia miltiorrhiza through gene transformation of double key enzymes. Background technique [0002] Cardiovascular and cerebrovascular diseases are the "number one killer" that threatens human health and life. Around the world, 17 million people die from cardiovascular and cerebrovascular diseases every year, accounting for 1 / 3 of the total global death toll. About 2.6 million people die of cardiovascular and cerebrovascular diseases in my country every year. Actively researching and developing high-efficiency, low-toxic and cheap clinical drugs for treating cardiovascular and cerebrovascular diseases has far-reaching significance for improving human health. [0003] Salvia ( Salvia miltiorrhiza Bunge), also known as purple salvia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/54C12N15/82A01H5/06C12Q1/68G01N30/89G01N21/31
Inventor 开国银许辉廖攀周伟张林
Owner SHANGHAI NORMAL UNIVERSITY
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