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Key gene GbMYB4 for regulating synthesis of ginkgo flavonoids, protein expressed by key gene GbMYB4, vector and application

A technology for expressing vectors and key genes, used in applications, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2020-12-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the genes that negatively regulate the synthesis of ginkgo flavonoids. Therefore, it is of great significance to develop and study the genes related to the negative regulation of ginkgo flavonoids. To provide technical support for the popularization and application of bioengineering in the production of secondary metabolites of Ginkgo biloba

Method used

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  • Key gene GbMYB4 for regulating synthesis of ginkgo flavonoids, protein expressed by key gene GbMYB4, vector and application
  • Key gene GbMYB4 for regulating synthesis of ginkgo flavonoids, protein expressed by key gene GbMYB4, vector and application
  • Key gene GbMYB4 for regulating synthesis of ginkgo flavonoids, protein expressed by key gene GbMYB4, vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of the GbMYB4 gene

[0031] (1) Based on Ginkgo biloba genome and Ginkgo transcriptome data, a MYB gene was screened and named GbMYB4 through sequence alignment and evolution analysis. The ORF primers of GbMYB4 were artificially designed using Primer Premier 5.0 software. Among them, the GbMYB4 ORF forward primer (ORF F primer) is seq id no.3: 5'-atgggtcggtctccttgctg-3', and the gbmyb4 orf reverse primer (orf r primer) is seq id no.4: 5'-tacccgcagttgcctgtaat-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, tak...

Embodiment 2

[0056] Construction of GbMYB4 Gene Plant Expression Vector

[0057] (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-regulatingfactor family in Chinese cabbage (Brassica rapa L.ssp.pekinensis), Wang et al.BMC Genomics 2014, 15:807) and the ORF sequence of GbMYB4 were subjected to enzyme digestion experiments, and the specific reactions The system is as follows:

[0058]

[0059] 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.

[0060] (2) Referring to the operation manual of TaKaRa T4 DNA Ligase (TaKaRa, Japan), connect the expr...

Embodiment 3

[0067] Genetic transformation of the GbMYB4 gene

[0068] 1. Genetic transformation of Arabidopsis

[0069] (1) Planting wild-type Arabidopsis thaliana in a normal growth environment;

[0070] (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;

[0071] (3) Inoculate the Agrobacterium containing the 35S::GbMYB4 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;

[0072] (4) Put the bacterial solution that meets the requirements into a centrifuge tube, centrifuge at 4°C, 6000rpm, for 10min, and remove the supernatant;

[0073] (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;

[0074] (6) Remove the supernata...

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Abstract

The invention discloses a key gene GbMYB4 for regulating synthesis of ginkgo flavonoids, protein expressed by the key gene GbMYB4, a vector and application. The nucleotide sequence of the key gene GbMYB4 is shown as SEQ ID NO. 1, and the amino acid sequence of the protein expressed by the key gene GbMYB4 is shown as SEQ ID NO. 2. Ginkgo leaves are used as materials, the gene GbMYB4 is cloned, andan overexpression vector containing the gene GbMYB4 is constructed and transferred into ginkgo callus and arabidopsis thaliana. The content of flavonoids in the GbMYB4 transgenic ginkgo callus and thetransgenic arabidopsis thaliana is remarkably reduced, that the GbMYB4 can inhibit synthesis of the flavonoids, has a negative regulation effect on the flavonoids and can be matched with a promotinggene for flavonoid synthesis to effectively control the synthesis amount of the ginkgo flavonoids is indicated, and therefore regulation of expression of the GbMYB4 has important application value inthe aspects of improving the medicinal quality of the ginkgo leaves and the like.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a key gene GbMYB4 for regulating the synthesis of ginkgo flavonoids and its expressed protein, carrier and application. Background technique [0002] Ginkgo (Ginkgo biloba L.), also known as Gongsun tree, is a perennial tree belonging to the family Ginkgoceae of the gymnosperm family Ginkgoceae. It is an important economic forest tree species endemic to China. Ginkgo biloba has high medicinal value, and the medicinal value of Ginkgo biloba is the most prominent. Studies have found that Ginkgo biloba extract (Extraet of Ginkgo biloba, EGB761) can be used to treat cardiovascular and cerebrovascular diseases and nervous system diseases, and has little toxic and side effects. Flavonoids are an important substance to control the quality of Ginkgo biloba extract. At present, more than 40 flavonoids have been isolated from Ginkgo biloba extract, mainly inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8243
Inventor 王莉毛欣雨贾志超刘思安
Owner YANGZHOU UNIV
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