Method for increasing content of tanshinone in salvia miltiorrhiza bunge hairy roots

A technology of tanshinone content and hairy roots, applied in the biological field, can solve undiscovered problems, achieve the effect of reducing drug prices and alleviating shortages

Active Publication Date: 2015-09-09
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
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Problems solved by technology

Using metabolic engineering means, the Danshen SmWRKY70 transcription factor is transformed into Danshen, and the hairy root of Salvia miltiorrhiza with high yield of tanshinone is obtained, which provides a new high-quality drug source for commercial production of tanshinone. At present, it has not been found that the use of the Danshen SmWRKY70 transcription factor mentioned in the subject of the present invention improves Related reports on the content of tanshinone in the hairy root of Salvia miltiorrhiza

Method used

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  • Method for increasing content of tanshinone in salvia miltiorrhiza bunge hairy roots

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Embodiment 1

[0025] Example 1: Cloning of Salvia miltiorrhiza SmWRKY70 gene

[0026] 1.1. Extraction of total RNA from Salvia miltiorrhiza

[0027] Take a small amount of young leaves of Salvia miltiorrhiza (produced in Pingyi, Shandong Province with a high tanshinone content) young leaves, quickly freeze them with liquid nitrogen, and quickly grind them with a mortar, and then extract total RNA according to the instructions of the RNAprep Pure Plant Kit provided by TIANGEN company . The integrity of the RNA was detected by ordinary agarose gel electrophoresis (electrophoresis conditions: gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min). The purity and concentration were detected with a Nano Drop 2000c ultra-micro spectrophotometer.

[0028] 1.2. Cloning of Danshen SmWRKY70 gene

[0029] 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, ...

Embodiment 2

[0030] Example 2: Cloning of Danshen SmWRKY70 promoter

[0031] 2.1. Extraction of Danshen Genomic DNA

[0032] Genomic DNA of young leaves of Salvia miltiorrhiza was extracted by CTAB method, and the extracted DNA was subjected to gel electrophoresis to preliminarily confirm the integrity of the extracted DNA. The purity and concentration of DNA samples were detected by Nano Drop 2000c ultramicro spectrophotometer.

[0033] 2.2. Cloning of Danshen SmWRKY70 promoter

[0034] The LA PCRTM in vitro Cloning Kit (Code: DRR015) kit from TaKaRa Company was used to clone the SmWRKY70 promoter (for the operation steps, refer to the relevant instructions provided by TaKaRa Company). The extracted DNA was digested with restriction enzymes EcoR I, Pst I, and Bgl II for 6 hours, then recovered by ethanol precipitation, connected to the corresponding adapter in the kit, and recovered by ethanol precipitation 4 hours later. According to the known sequence of SmWRKY70, primer SmWRKY70-SP1 (S...

Embodiment 3

[0037] Example 3: Tissue expression profiling analysis of Salvia miltiorrhiza SmWRKY70

[0038] In order to study the tissue expression pattern of SmWRKY70, the total RNA of five tissues of root, stem, leaf, flower and seed of Salvia miltiorrhiza were extracted, and the purity and concentration were tested respectively. Then it was reverse-transcribed into cDNA and used for QRT-PCR analysis of the tissue expression profile of SmWRKY70. The reaction system used the SuperReal PreMix (SYBR Green) kit provided by TIANGEN Company, Actin was used as an internal reference gene, and the relative expression of the gene was calculated by the comparative CT method quantity. The results showed that SmWRKY70 was highly expressed in leaves and petioles, followed by higher expression in stems, and weaker expression in tap roots, fibrous roots, flowers, filaments, and calyx in all tissues tested (see image 3 ).

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Abstract

The invention provides a method for increasing content of tanshinone in salvia miltiorrhiza bunge hairy roots. According to the method, recombinant plant expression vectors are constructed with salvia miltiorrhiza bunge SmWRKY70 transcription factors, and SmWRKY70 gene modified salvia miltiorrhiza bunge hairy roots are obtained through genetic transformation of salvia miltiorrhiza bunge leaves. According to the method, 789 bp of a coding frame sequence of the SmWRKY70 transcription factors is isolated and cloned from salvia miltiorrhiza bunge, and 918 bp of a promoter sequence of the SmWRKY70 transcription factors is cloned; subcellular localization vectors of the SmWRKY70 transcription factors are constructed; overexpression vectors of SmWRKY70 genes are constructed, and overexpression salvia miltiorrhiza bunge hairy roots with the SmWRKY70 genes are obtained through genetic transformation of the salvia miltiorrhiza bunge leaves. The content of the tanshinone in the gene modified salvia miltiorrhiza bunge hairy roots obtained with the method is significantly increased, a novel high-quality drug resource is provided for production of the tanshinone with important clinical demands, and the active promotion effect on alleviating shortage of the tanshinone drug resource is realized, and the method has the important theoretical significance and the potential application value.

Description

technical field [0001] The invention relates to biotechnology, in particular to a method for transforming the SmWRKY70 transcription factor of Salvia miltiorrhiza to increase the content of tanshinone in the hairy root of Salvia miltiorrhiza by using a metabolic engineering strategy. Background technique [0002] Salvia miltiorrhiza Bunge is a perennial dicotyledonous herb. As a traditional Chinese medicine, Salvia miltiorrhiza Bunge is mainly used clinically for the treatment of cardiovascular and cerebrovascular diseases. The active ingredients in Danshen mainly include two categories, one is fat-soluble tanshinone compounds, mainly including dihydrotanshinone, cryptotanshinone, tanshinone I, tanshinone IIA, etc.; the other is water-soluble salvianolic acid compounds . A large number of studies have proved that tanshinone has a variety of pharmacological activities, including cardiovascular and cerebrovascular protection, anti-tumor, anti-oxidation, anti-inflammatory, ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/84A01H5/00
Inventor 开国银李磊磊郝小龙
Owner SHANGHAI NORMAL UNIVERSITY
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