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Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2

A technology of hairy root and tanshinone, which is applied in the field of genetic engineering, can solve problems such as undiscovered hairy root tanshinone of Salvia miltiorrhiza, and achieve the effects of low cost, increased content, and reliable effect

Inactive Publication Date: 2015-06-24
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, Arabidopsis does not produce tanshinone and salvianolic acid substances. Whether AtMYC2 can promote the synthesis of tanshinone and salvianolic acid has not been published yet.
At present, no relevant reports have been found to improve the content of tanshinone and salvianolic acid in the hairy root of Salvia miltiorrhiza with the AtMYC2 gene overexpression strategy mentioned in the subject of the present invention

Method used

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  • Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2
  • Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2
  • Method for increasing contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root by using transgene AtMYC2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning of Arabidopsis AtMYC2 Gene

[0055] 1.1. Extraction of Arabidopsis total RNA

[0056] Take a small amount of young leaves of Arabidopsis thaliana, quick-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. 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.

[0057] 1.2. Cloning of Arabidopsis AtMYC2 gene

[0058] Using 0.5 μg of total RNA from Arabidopsis thaliana as the initial amount, reverse transcriptase XL (AMV) was used to synthesize the first-strand cDNA (for the operation steps, refer to the relevant instructions provided by Promega). According to the coding sequence (SEQ ID NO.1) of described AtMYC2 gene, d...

Embodiment 2

[0060] Construction of Plant Overexpression Vector Containing Arabidopsis AtMYC2 Gene

[0061] For the carrier construction model diagram, see figure 1 . pCAMBIA2300 + As an intermediate expression vector, replace pCAMBIA2300 with the AtMYC2 gene cloned from Arabidopsis + on the gus gene. Specifically, Spe I / BstPI double enzyme cut pMD18-T::AtMYC2 and pCAMBIA2300 + ; Recover AtMYC2 gene and pCAMBIA2300 + Large fragments; ligation transformation, picking single clone colony PCR screening positive clones; extracting plasmid for further enzyme digestion verification. The results show that the AtMYC2 gene has been successfully constructed into the plant expression vector pCAMBIA2300 + In order to obtain the plant overexpression vector pCAMBIA2300 containing the AtMYC2 gene + ::AtMYC2.

[0062] In this example, the transcription factor AtMYC2 is operably linked to the expression control sequence to form the plant overexpression vector pCAMBIA2300 containing the AtMYC2 gene ...

Embodiment 3

[0065] Agrobacterium rhizogenes-mediated genetic transformation of Arabidopsis thaliana AtMYC2 gene to obtain transgenic hairy roots of Salvia miltiorrhiza

[0066] 3.1. Acquisition of Agrobacterium rhizogenes Engineering Bacteria Containing Plant Expression Vectors

[0067] The plant overexpression vector pCAMBIA2300 containing AtMYC2 gene in embodiment 2 + ::AtMYC2 was transformed into Agrobacterium rhizogenes C58C1, and a single clone colony was picked for PCR verification. The results showed that the plant overexpression vector pCAMBIA2300 containing AtMYC2 gene + ::AtMYC2 has been successfully constructed into Agrobacterium rhizogenes C58C1.

[0068] 3.2. Agrobacterium rhizogenes mediated genetic transformation of Salvia miltiorrhiza with AtMYC2 gene

[0069] 3.2.1 Preculture of explants

[0070] Cut the healthy aseptic seedling leaves of Salvia miltiorrhiza (0.5cm 2 ), inoculated onto the pre-culture medium (1 / 2MS), and cultured in the dark at 25°C for 2 days.

[0...

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Abstract

The invention relates to a method for increasing the contents of tanshinone and salvianolic acid in a salvia miltiorrhiza hairy root by using a transgene AtMYC2, belonging to the technical field of gene engineering. The method comprises the steps of constructing a high-efficiency expression vector of a plant by using an arabidopsis transcription factor AtMYC2, and carrying out genetic transformation on salvia miltiorrhiza leaves to obtain a gene AtMYC2 overexpressed transgenetic salvia miltiorrhiza hairy root; analyzing the expression of AtMYC2 in the transgenetic salvia miltiorrhiza hairy root and related genes in biosynthetic pathways of tanshinone and salvianolic acid through qRT-PCR; measuring the contents of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a high-performance liquid chromatography (HPLC); and measuring the antioxidant activity of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a DPPH free radical scavenging method. The invention provides the method for simultaneously increasing the contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root and also provides a novel high-quality raw material for producing tanshinone and salvianolic acid with important clinic demands so as to have the positive promoting significance and application value for relieving the problem that the drug resources of tanshinone and salvianolic acid are short.

Description

technical field [0001] The invention relates to a method for improving the contents of tanshinone and salvianolic acid in hairy roots of salvia miltiorrhiza by transgenic AtMYC2 gene, and belongs to the technical field of genetic engineering. Background technique [0002] Cardiovascular and cerebrovascular diseases are currently the "number one killer" that threatens the health and lives of all human beings. According to statistics, about 17 million people die of cardiovascular and cerebrovascular diseases in the world every year, accounting for about 1 / 3 of the total deaths in the world; about 3 million people die of cardiovascular and cerebrovascular diseases in my country every year. Therefore, active research on high-efficiency, low-toxic and cheap clinical drugs for the treatment of cardiovascular and cerebrovascular diseases has far-reaching significance for improving human health. [0003] Salvia miltiorrhiza Bunge is a perennial herb of the genus Salvia in the famil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/06
Inventor 开国银王晓荣
Owner SHANGHAI NORMAL UNIVERSITY
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