Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes

A technology of tanshinone content and hairy roots, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc.

Inactive Publication Date: 2013-06-05
SHANGHAI NORMAL UNIVERSITY
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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 two key enzyme genes SmHMGR and SmDXR are co-transformed into Salvia miltiorrhiza, which will break the bottleneck effect of the biosynthetic pathway of tanshinone, obtain the hairy root of tanshinone with high yield, and provide a new high-quality drug source for commercial production of tanshinone. No relevant reports have been found to improve the content of tanshinone in the hairy root of Salvia miltiorrhiza with the double key enzyme gene co-transformation strategy mentioned in the subject of the present invention

Method used

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  • Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes
  • Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes
  • Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1.1. Salvia miltiorrhiza total RNA extraction and cDNA first-strand synthesis

[0032] Total RNA was extracted from the transgenic hairy roots of Salvia miltiorrhiza using the RNA prep pure plant kit provided by TIANGEN (see the instructions in the kit for the extraction steps). The fresh weight of Salvia miltiorrhiza transgenic hairy root used for extracting total RNA is about 0.1 g, and the DNA in the sample has been removed with DNase I working solution during the extraction process. Measure the relative absorbance value of the extracted RNA on a spectrophotometer, and calculate the purity and concentration of the extracted RNA. After calculation based on the concentration of different RNA samples, the first-strand cDNA was synthesized with reverse transcriptase XL (AMV) using 0.5 μg RNA as the initial amount (for the operation steps, refer to the relevant instructions provided by TaKaRa Company).

[0033] 1.2. Design of SmHMGR and SmDXR coding sequence-specific pri...

Embodiment 2

[0035] Example 2 Construction of plant expression vectors containing Salvia miltiorrhiza SmHMGR and SmDXR genes

[0036] 2.1. Intermediate vector pCAMBIA1304 + build

[0037] Using pBI121 and pCAMBIA1304 as materials, construct the plant expression vector pCAMBIA1304 + . 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 + The construction was successful, including CaMV35S promoter and terminator, multiple cloning site, replication origin and kanamycin resistance site.

[0038] 2.2. Plant expression vector pCAMBIA1304 + - Construction of SmHMGR (see figure 1 )

[0039] The successful pCAMBIA1304 constructed above + Basically, replace the GUS gene with the SmHMGR gene cloned ...

Embodiment 3

[0045] Example 3 Agrobacterium rhizogenes Mediates SmHMGR and SmDXR Gene Genetic Transformation of Salvia Miltiorrhiza to Obtain Transgenic Hairy Roots

[0046] 3.1. Containing plant expression vector pCAMBIA1304 + -Acquisition of SmHMGR / SmDXR Agrobacterium rhizogenes engineering bacteria

[0047] The plant expression vector pCAMBIA1304 containing SmHMGR and / or SmDXR gene in embodiment 2 + -SmHMGR / SmDXR was transferred into Agrobacterium rhizogenes pBiA4, and a single clone colony was picked for PCR verification. The results showed that the plant expression vector containing SmHMGR and SmDXR genes had been successfully constructed in Agrobacterium rhizogenes strain pBiA4.

[0048] 3.2. Genetic transformation of Salvia miltiorrhiza mediated by SmHMGR and SmDXR genes by Agrobacterium rhizogenes

[0049] 3.2.1. Preculture of explants

[0050] Cut the healthy aseptic seedling leaves of Salvia miltiorrhiza (0.5cm 2 ), inoculated onto the pre-culture medium (1 / 2MS), and culture...

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Abstract

The invention relates to the field of biological technology and discloses a method of increasing the content of tanshinone. According to the method, the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene SmHMGR and the 1-deoxy-D-xylosone-5-phosphate reductase gene SmDXR of salvia miltiorrhiza bunge are used to construct a recombinant vector, and genetic transformation is carried out on leaves of salvia miltiorrhiza bunge so as to obtain hairy roots of salvia miltiorrhiza bunge with transgenic SmHMGR and SmDXR genes. The content of tanshinone in the obtained transgenic hairy roots of salvia miltiorrhiza bunge in the invention substantially increases, and total tanshinone content in a line (HD34) of cotransformed SmHMGR and SmDXR double genes is 5.30 times of that of a comparison hairy root of salvia miltiorrhiza bunge. The invention provides the method for increasing content of tanshinone in the hairy roots of salvia miltiorrhiza bunge, providing a novel high-quality drug source for producing tanshinone in critical clinical demand and having a positive promotion effect in alleviating shortage of drug sources for tanshinone.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for improving the content of tanshinone in the hairy root of Salvia miltiorrhiza by utilizing a metabolic engineering strategy and co-transforming double key enzyme genes. 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 2.6 million people die of cardiovascular and cerebrovascular diseases in my country every year. Therefore, active research and development of high-efficiency, low-toxic and cheap clinical drugs for the treatment of cardiovascular and cerebrovascular diseases have far-reaching significance for improving human health. [0003] Salvia miltiorrhi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/82C12Q1/68A01H5/06
Inventor 开国银罗秀芹
Owner SHANGHAI NORMAL UNIVERSITY
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