A key gene gbmyb6 that promotes the synthesis of ginkgo flavonoids and its expressed protein, vector and application

A key gene and expression vector technology, applied in the field of the key gene GbMYB6 and its expressed proteins, can solve the problem of less in-depth research on gene function and so on

Active Publication Date: 2022-04-08
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many genes related to flavonoid synthesis have been cloned, there are few in-depth studies on gene function, so it is of great significance to further study the synthesis mechanism of ginkgo flavonoids

Method used

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  • A key gene gbmyb6 that promotes the synthesis of ginkgo flavonoids and its expressed protein, vector and application
  • A key gene gbmyb6 that promotes the synthesis of ginkgo flavonoids and its expressed protein, vector and application
  • A key gene gbmyb6 that promotes the synthesis of ginkgo flavonoids and its expressed protein, vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of the GbMYB6 gene

[0031] (1) Based on Ginkgo biloba genome and Ginkgo transcriptome data, a MYB gene was screened and named GbMYB6 through sequence alignment and evolution analysis. The ORF primers of GbMYB6 were artificially designed using Primer Premier 5.0 software. Wherein, the GbMYB6 ORF forward primer (ORF F primer) is SEQ ID NO.3: 5'-ATGGGGAGATCACCGTGCTG-3', and the GbMYB6 ORF reverse primer (ORF R primer) is SEQ ID NO.4: 5'-ATTCAAATGGGGGAATTGTGC-3 '.

[0032] (2) Using the high-fidelity enzyme PrimeSTAR Max (Takara, Japan) for PCR amplification, the PCR system is as follows:

[0033]

[0034] Gently mix the above mixture, centrifuge briefly at low speed and place it in an ordinary PCR reaction instrument, set the following program:

[0035]

[0036] Gel running: Take out the gene amplification product in the PCR instrument, use the electrophoresis instrument to spot an appropriate amount of the product on a 1% agarose gel for detection, take ...

Embodiment 2

[0055] Construction of GbMYB6 Gene Plant Expression Vector

[0056] (1) This experiment uses TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) to pCAMBIA2300-35S-OCS vector ( figure 2 ) (Genome-wide identification and analysis of the growth-regulating factor family in Chinese cabbage (Brassica rapa L.ssp.pekinensis), Wang et al. BMC Genomics 2014, 15:807) and the ORF sequence of GbMYB6 were subjected to enzyme digestion experiments, and the specific reactions The system is as follows:

[0057]

[0058] After the solutions in the system were mixed, they were centrifuged instantaneously, incubated in a 37°C water bath for 30 minutes, and then the enzyme digestion reaction was completed. The enzyme-cut bands were observed by agarose gel electrophoresis, and then the target gene and carrier fragments were cut and recovered for subsequent use. The carrier ligation reaction.

[0059] (2) Referring to the operation manual of TaKaRa T4 DNA Ligase (TaKaRa, Japan), connect the ex...

Embodiment 3

[0066] Genetic transformation of the GbMYB6 gene

[0067] 1. Genetic transformation of Arabidopsis

[0068] (1) Planting wild-type Arabidopsis thaliana in a normal growth environment;

[0069] (2) Select the Arabidopsis plants that have just bloomed for about four weeks in the soil, and cut off the flowers that have bloomed and the existing siliques with scissors for Agrobacterium transformation;

[0070] (3) Inoculate the Agrobacterium containing the 35S::GbMYB6 carrier obtained in Example 2 into LB liquid medium with Kana and Rif antibiotics, put it in a shaker at 28°C, and culture it overnight for 18-24h until OD 600 =1.0-1.5;

[0071] (4) Put the bacterial solution that meets the requirements into a centrifuge tube, centrifuge at 4°C, 6000rpm, for 10min, and remove the supernatant;

[0072] (5) Add 50 mL of Arabidopsis transformation solution (5% sucrose+0.02% Silwet L-77) to the precipitate in step (4), and resuspend the precipitate;

[0073] (6) Remove the supernatan...

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Abstract

The invention discloses a key gene GbMYB6 for promoting the synthesis of ginkgo flavonoids and its expressed protein, carrier and application. The nucleotide sequence of the key gene GbMYB6 is shown in SEQ ID NO.1, and the amino acid of the expressed protein is The sequence is shown in SEQ ID NO.2. The present invention uses Ginkgo biloba leaves as materials, clones the GbMYB6 gene, constructs an overexpression vector 35S::GbMYB6 containing the gene GbMYB6 by enzyme cleavage, and transfers it into Ginkgo callus and Arabidopsis thaliana, GbMYB6 transgenic Ginkgo callus and transgene The content of flavonoids in Arabidopsis thaliana was significantly increased, which indicated that GbMYB6 can promote the synthesis of flavonoids. Therefore, regulating the expression of GbMYB6 has important application value in improving the medicinal quality of Ginkgo biloba leaves.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a key gene GbMYB6 for promoting the synthesis of ginkgo flavonoids and its expressed protein, carrier and application. Background technique [0002] Ginkgo biloba (Gingo biloba L.) is a perennial tree of the genus Ginkgo. It is an ancient tree species that survived the Quaternary Glacier. It is a famous relic plant in the world. Its leaves, kernels and outer testa contain medicinal ingredients. "The whole body is a living fossil of treasure". Ginkgo biloba has been used as medicine for more than 600 years, and its efficacy was first recorded in "Shen Nong's Herbal Classic". Ginkgo biloba Extract (GbE) is the raw material of many medicines, and it has certain effects on the prevention and treatment of early Alzheimer's disease and cardiovascular diseases. Flavonoids are the main active components of GbE. More than 40 flavonoids have been isolated fr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20A01H6/00
CPCC07K14/415C12N15/8243
Inventor 刘思安操萌贾志超王莉
Owner YANGZHOU UNIV
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