Method for promoting salvia miltiorrhiza bunge lateral root development and increasing tanshinone content in salvia miltiorrhiza bunge root, salvia miltiorrhiza bunge gene sequence and overexpression vector

A technology of tanshinone content and overexpression vector, applied in the field of genetic engineering, can solve the problems of high cost of breed selection, complex structure of tanshinone, environmental pollution, etc., and achieve the effect of improving medicinal value

Active Publication Date: 2021-03-16
SHANGHAI CHANGZHENG HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the decreasing number of wild resources, Salvia miltiorrhiza is a perennial herb with a long growth cycle and low content of medicinal active ingredients, under the traditional cultivation mode, there are problems such as the degradation of the quality of Salvia miltiorrhiza and the high cost of variety selection.
In addition, the structure of tanshinone is complex, so its chemical synthesis process is very cumbersome, the cost is high and it is easy to cause environmental pollution; while the cells obtained by the method of cell culture have very low content of active ingredients and poor stability

Method used

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  • Method for promoting salvia miltiorrhiza bunge lateral root development and increasing tanshinone content in salvia miltiorrhiza bunge root, salvia miltiorrhiza bunge gene sequence and overexpression vector
  • Method for promoting salvia miltiorrhiza bunge lateral root development and increasing tanshinone content in salvia miltiorrhiza bunge root, salvia miltiorrhiza bunge gene sequence and overexpression vector
  • Method for promoting salvia miltiorrhiza bunge lateral root development and increasing tanshinone content in salvia miltiorrhiza bunge root, salvia miltiorrhiza bunge gene sequence and overexpression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Amplification of the Danshen SmMYC2b gene sequence

[0028] The purpose of this embodiment is to obtain the gene fragment of the suspected transcription factor MYC2 from Salvia miltiorrhiza, and amplify it to obtain the amplified related gene fragment for transformation, including the following steps:

[0029] 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:

[0030] 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;

[0031] 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;

[0032] Step 1-1-3, mix with a homogenizer, le...

Embodiment 2

[0072] Example 2: Construction of overexpression vector

[0073] The purpose of this example is to obtain the target gene from the Escherichia coli containing the target gene obtained in Example 1, and connect the target gene to the PHB-flag vector to construct an overexpression vector containing the target gene. Schematic diagram of the construction of the overexpression vector Such as figure 1 shown, including the following steps:

[0074] Step 2-1, designing primers and cloning the target fragment.

[0075] 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.

[0076] Table 2-1 Primers used for cloning target fragments

[0077]

[0078] The PCR reaction ...

Embodiment 3

[0093] Example 3: Construction of recombinant Agrobacterium

[0094] The purpose of this embodiment is to transfer the overexpression vector obtained in Example 2 into Agrobacterium to obtain recombinant Agrobacterium, including the following steps:

[0095] Step 3-1, prepare the required reagents, the formula of each reagent is as follows:

[0096] 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;

[0097] 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 under high temperature and high pressure at 121°C for 20 minutes, and set aside.

[0098] 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;

[0099] Kanamycin and h...

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Abstract

The invention provides a method for promoting salvia miltiorrhiza bunge lateral root development and increasing tanshinone content in the salvia miltiorrhiza bunge roots, and belongs to the field of gene engineering. The method is characterized in that transgenic salvia miltiorrhiza bunge roots with better lateral root development and increased tanshinone content are obtained by overexpressing salvia miltiorrhiza bunge SmMYC2b genes in salvia miltiorrhiza bunge roots, and the method comprises the steps of S1, constructing an overexpression vector containing the SmMYC2b gene of the salvia miltiorrhiza bunge; S2, transfecting the overexpression vector obtained in the step S1 into agrobacterium to obtain recombinant agrobacterium; and S3, adopting the recombinant agrobacterium obtained in thestep S2 for transforming salvia miltiorrhiza bunge tissue and inducing rooting, and obtaining the transgenic salvia miltiorrhiza bunge roots, wherein the sequence of the SmMYC2b gene of salvia miltiorrhiza bunge is shown as SEQ ID No: 1, and the sequence of the overexpression vector 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 promoting the development of lateral roots of salvia miltiorrhiza and increasing the content of tanshinone in the root of salvia miltiorrhiza, and a gene sequence of salvia miltiorrhiza and an overexpression carrier 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, tanshinone and phenolic acid are its main bioactive components. Modern studies have shown that the tanshinone substances contained in Danshen 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. [0003] However, due to the dwindling wild resources, Salvia miltiorrhiza is a perennial herb with a long growth cycle and low content of medicinal active ing...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H4/00A01H5/06A01H6/50
CPCC07K14/415C12N15/8243C12N15/8261A01H4/00Y02A40/10
Inventor 周阳云冯婧娴陈万生张磊黄豆豆
Owner SHANGHAI CHANGZHENG HOSPITAL
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