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Method for improving content of tanshinone in salvia miltiorrhiza bunge

A technology of tanshinone content and salvia miltiorrhiza, applied in DNA/RNA fragments, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve undiscovered problems

Inactive Publication Date: 2012-07-18
开国银
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

By means of genetic engineering, the above-mentioned key enzyme gene DXS is genetically transformed into Salvia miltiorrhiza, which will break the bottleneck effect of the biosynthetic pathway of tanshinone, obtain the hairy root or plant of tanshinone with high yield, and provide a new way for the commercial production of tanshinone, but it has not been found yet. The key enzyme gene transformation strategy mentioned in the subject of the present invention improves the content of tanshinone in the hairy root of Salvia miltiorrhiza

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Cloning of DXS Gene of Salvia Miltiorrhiza

[0020] 1.1. Extraction of total RNA from Salvia miltiorrhiza

[0021] Take a small amount of young leaves of Salvia miltiorrhiza (produced in Pingyi, Shandong Province with a high tanshinone content) young leaves, quick-frozen with liquid nitrogen, and quickly grind them with a mortar, and then extract total RNA according to the instructions of the RNA preppure plant kit provided by TIANGEN company . The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0022] 1.2. Cloning of Danshen DXS gene

[0023] Using the obtained 0.5 μg of Salvia miltiorrhiza total RNA as the initial amount, the first-strand cDNA was synthesized with reverse transcriptase XL (AMV) (for the operation steps, refer to the relevant instructions provided by Promega Company). According to the coding sequence of the Danshen DXS gene (sequence 1 in the sequen...

Embodiment 2

[0025] Construction of Plant Expression Vector Containing DXS Gene

[0026] 2.1. Construction of intermediate vector pCAMBIA1304+

[0027] Using pBIl21 and pCAMBIA1304 as materials, the plant expression vector pCAMBIA1304+ was constructed. 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 clone colonies were picked to extract plasmids for digestion verification. The results showed that the plant expression vector pCAMBIA1304+ was constructed successfully.

[0028] 2.2. Construction of plant expression vector pCAMBIA1304+-DXS

[0029] On the basis of the successfully constructed pCAMBIA1304+, the GUS gene on it was replaced with the DXS gene cloned from Salvia miltiorrhiza. Specifically, BamHI / SacI double digestion of pMD18T-DXS and pCAMBIA1304+; recovery of the DXS gene and pCAMBIA1304+ large fragments; ligation tr...

Embodiment 3

[0032] Agrobacterium rhizogenes mediated DXS gene genetic transformation of Salvia miltiorrhiza to obtain transgenic Salvia miltiorrhiza hairy roots

[0033] 3.1. Acquisition of engineering bacteria containing plant expression vector pCAMBIA1304+-DXS Agrobacterium rhizogenes

[0034] The plant expression vector pCAMBIA1304+-DXS containing the DXS gene in Example 2 was transformed into Agrobacterium rhizogenes C58C1, and a single clone colony was picked for PCR verification. The results showed that the plant expression vector containing DXS gene had been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0035] 3.2. Agrobacterium rhizogenes mediated DXS gene genetic transformation of Salvia miltiorrhiza

[0036] 3.2.1. Preculture of explants

[0037] Cut the leaves (0.5 cm2) of robust aseptic seedlings of Salvia miltiorrhiza, inoculate them on the pre-cultivation medium (MS), and culture them in the dark at 25°C for 2 days.

[0038] 3.2.2. Co-cultivation of...

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Abstract

The invention relates to a method for improving the content of tanshinone in salvia miltiorrhiza bunge, which is characterized in that a coding frame sequence of a 1-deoxidizing xylulose-5-phosphate synthetase (DXS) gene is cloned from the salvia miltiorrhiza bunge, a high-efficiency plant expression vector containing the DXS gene is constructed, a salvia miltiorrhiza bunge blade is genetically transformed to obtain a salvia miltiorrhiza bunge hairy root for converting the DXS gene, and the expression of DXS in the salvia miltiorrhiza transgenic hairy root is analyzed through semi-quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The content of the total tanshinone (Cryptotanshinone, tanshinone and tanshinone IIA) in the salvia miltiorrhiza bunge transgenic hairy root is determined by adopting a high-efficiency liquid chromatography. The content of the tanshinone in the salvia miltiorrhiza transgenic hairy root obtained by the invention is remarkably improved, and the total content of the tanshinone in a strain with best performance is 2.89 times of a contrast strain. The invention provides the method for improving the content of the tanshinone in the salvia miltiorrhiza bunge and also provides a novel high-quality raw material for the tanshinone having important clinical demands in the production.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for improving the content of tanshinone in hairy roots of Salvia miltiorrhiza by genetic transformation of key enzyme gene DXS. Background technique [0002] Salvia miltiorrhiza Bunge, also known as purple salvia, red root, red ginseng, blood ginseng, etc., belongs to the genus Salvia of the Labiatae family, and is a perennial herb. As a traditional Chinese medicine, Danshen is mainly used clinically in the treatment of cardiovascular diseases. The main medicinal components of Danshen include fat-soluble diterpene quinones (tanshinones) and water-soluble phenolic acids. Modern studies have shown that the tanshinone substances contained in Salvia miltiorrhiza have various pharmacological activities such as anti-tumor, anti-bacterial and anti-inflammatory, anti-oxidation, and anti-atherosclerosis, so there is a huge market demand. At present, the long growth cycl...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/82
Inventor 开国银
Owner 开国银