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Method for increasing content of salvianolic acid B in salvia miltiorrhiza a hairy roots and laccase gene

A technology of middle salvianolic acid and hairy root, applied in the field of genetic engineering, can solve the problems of unsatisfactory salvianolic acid B content, influence on medicinal value, low salvianolic acid B content and the like

Active Publication Date: 2020-10-30
SHANGHAI CHANGZHENG HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the content of salvianolic acid B in the root of Salvia miltiorrhiza is low in the natural state, which makes the content of salvianolic acid B in the root extract of Salvia miltiorrhiza root unsatisfactory, which affects its medicinal value.

Method used

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  • Method for increasing content of salvianolic acid B in salvia miltiorrhiza a hairy roots and laccase gene
  • Method for increasing content of salvianolic acid B in salvia miltiorrhiza a hairy roots and laccase gene
  • Method for increasing content of salvianolic acid B in salvia miltiorrhiza a hairy roots and laccase gene

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: Amplification of the Laccase Gene Sequence

[0024] The purpose of this embodiment is to obtain the gene fragment of the suspected laccase gene from Salvia miltiorrhiza, and amplify it to obtain the amplified related gene fragment for transformation, including the following steps:

[0025] Step 1-1, extracting total RNA from Salvia miltiorrhiza, the operation method refers to the manual of TransZol UP PlusRNA Kit total RNA extraction reagent of TRANSGENE, including the following sub-steps:

[0026] Step 1-1-1, cut off the leaves of the aseptic salvia miltiorrhiza seedlings grown in the culture room for 10 weeks in an ultra-clean bench, and store them in liquid nitrogen;

[0027] Step 1-1-2, add liquid nitrogen to the leaf and quickly grind it into a fine powder, immediately divide the powder into eppendorf tubes with TRNzol-UP added in advance, use 1mL TRNzol-UP for every 50mg-100mg leaf;

[0028] Step 1-1-3, mix with a homogenizer, let stand at room tempe...

Embodiment 2

[0068] Example 2: Construction of overexpression vector

[0069] The purpose of this embodiment is to obtain the target gene from the Escherichia coli containing the target gene obtained in Example 1, and connect the three target genes to the PHB-flag vector respectively to construct an overexpression vector containing the target gene, including the following step:

[0070] Step 2-1, designing primers and cloning the target fragment.

[0071] Among them, the principle of PCR primer design is: select the monoclonal restriction site contained on PHB-flag and the restriction site does not exist in the ORF region of the gene, and remove the stop codon of the target gene at the same time. The designed primer sequences are shown in Table 2-1 below. In Table 2-1, the parts in italics indicate restriction sites.

[0072] Table 2-1 Primers used for cloning target fragments

[0073]

[0074] The PCR reaction system is shown in Table 2-2 below:

[0075] Table 2-2 PCR reaction s...

Embodiment 3

[0090] Example 3: Construction of recombinant Agrobacterium

[0091] The purpose of this embodiment is to transfer the overexpression vectors obtained in Example 2 into Agrobacterium respectively to obtain recombinant Agrobacterium, including the following steps:

[0092] Step 3-1, prepare the required reagents, the formula of each reagent is as follows:

[0093] LB medium: 5g of yeast extract, 5g of sodium chloride, 10g of tryptone dissolved in 1L of deionized water, adjust the pH to 7; add 7.5g of plant agar powder per L of solid medium;

[0094] YEB medium: weigh yeast extract, sucrose, beef extract, peptone, MgSO 4 ·7H 2 O 0.983g, dissolved in 1000mL deionized water, adjust pH=7.2, if preparing solid medium, add 7g agar. Sterilize at 121°C for 20 minutes under high temperature and high pressure, and set aside.

[0095] B5 medium: B5 powder 3.21g, MgSO 4 ·7H 2 O 0.983g, plant agar powder 7.5g, sucrose 20g, high pressure steam sterilization, 121 ℃, 20min;

[0096] K...

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Abstract

The invention provides a method for increasing the content of salvianolic acid B in salvia miltiorrhiza hairy roots. The method is characterized in that the transgenic salviae miltiorrhizae hairy rootwith increased salvianolic acid B content is obtained by overexpressing salviae miltiorrhizae laccase SmLAC20 gene in the salviae miltiorrhizae hairy root, and the method comprises the following steps: S1, constructing an overexpression vector containing the salviae miltiorrhizae laccase SmLAC20 gene; S2, transfecting the overexpression vector obtained in the step S1 into agrobacterium to obtainrecombinant agrobacterium; S3, the recombinant agrobacterium obtained in the step S2 is used for transforming salvia miltiorrhiza bunge tissue and inducing rooting, transgenic salvia miltiorrhiza bunge hairy roots are obtained, and the sequence of the salvia miltiorrhiza bunge laccase SmLAC20 gene is shown as SEQ ID No: 2.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for increasing salvianolic acid B content in hairy roots of Salvia miltiorrhiza, and a laccase gene used in the method. Background technique [0002] Danshen (Salvia miltiorrhiza Bunge) is an important Chinese herbal medicine, which contains a variety of active ingredients. Among them, salvianolic acid B is the one that has been studied more, and has various pharmacological effects such as anti-oxidation, anti-tumor, and protection of the cardiovascular system. [0003] At present, the acquisition of salvianolic acid B mainly depends on the extraction from the dried root of Salvia miltiorrhiza. However, the content of salvianolic acid B in the root of Salvia miltiorrhiza is low in the natural state, which makes the content of salvianolic acid B in the root extract of Salvia miltiorrhiza not ideal, which affects its medicinal value. [0004] Studies have shown that phen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/53A01H5/06A01H6/50
CPCC12N9/0061C12N15/8243C12Y110/03002A01H5/06Y02E50/10
Inventor 陈万生张磊陈亮李卿冯婧娴王芸谭何新陈昊轩
Owner SHANGHAI CHANGZHENG HOSPITAL
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