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SmbZIP1 gene in increasing content of salvianolic acid in salvia miltiorrhiza and application thereof

A salvianolic acid and gene technology, applied in the field of genetic engineering, can solve problems such as low salvianolic acid content, and achieve the effect of reliable effect and low cost

Active Publication Date: 2020-02-21
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a SmbZIP1 gene and its application in increasing the salvianolic acid content in Salvia miltiorrhiza to solve the problem of low salvianolic acid content in wild salvia miltiorrhiza

Method used

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  • SmbZIP1 gene in increasing content of salvianolic acid in salvia miltiorrhiza and application thereof
  • SmbZIP1 gene in increasing content of salvianolic acid in salvia miltiorrhiza and application thereof
  • SmbZIP1 gene in increasing content of salvianolic acid in salvia miltiorrhiza and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Acquisition of SmbZIP1 gene from Salvia miltiorrhiza

[0031] 1.1. Establishment of yeast two-hybrid library of Salvia miltiorrhiza mixed tissue cDNA

[0032] The library was established by Shanghai Ouyi Biomedical Co., Ltd.

[0033] 1.2. Yeast two-hybrid screening library using SmMYB1 as bait protein

[0034] For the screening process, refer to the screening instructions provided by Shanghai Ouyi Biomedical Co., Ltd., and carry out screening in strict accordance with the steps. The positive clones were sequenced after PCR amplification. DNA sequence determination was completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The sequence result is shown in SEQ ID NO.1.

[0035] 1.3. Verify the screening results

[0036] In order to verify the accuracy of the screening library, the obtained SmbZIP1 gene was rebuilt into the yeast two-hybrid vector pGADT7, and the yeast two-hybrid experiment was carried out again. Specific steps: preparation of compet...

Embodiment 2

[0039] Construction and Transformation of Plant Expression Vector of Salvia SmbZIP1 Gene

[0040] 2.1. Construction of pHB-SmbZIP1-YFP vector

[0041]According to the sequence of the Danshen SmbZIP1 obtained by cloning, the primers (see SEQ ID NO.2 and SEQ ID NO.3 for the sequence) constructed by the plant overexpression vector were designed to construct the pHB-SmbZIP1-YFP vector, and the pHB-SmbZIP1-YFP plasmid The transformation of Agrobacterium rhizogenes C58C1 was purchased from Shanghai Weidi Biotechnology Co., Ltd. in this example, but not limited thereto, and a single clone colony was picked for PCR verification. The results showed that the plant overexpression vector pHB-SmbZIP1-YFP containing SmbZIP1 had been successfully transformed into Agrobacterium rhizogenes C58C1, which could be used for subsequent genetic transformation and hair root infection experiments.

[0042] Primer name sequence serial number pHB-SmbZIP1-HindIII-KF: 5'-CAAGCTTATGCA...

Embodiment 3

[0046] Analysis of Subcellular Localization of SmbZIP1 Gene in Salvia Miltiorrhiza

[0047] 3.1. pHB-SmbZIP1-YFP vector transformation of Agrobacterium tumefaciens EHA105

[0048] Take the pHB-SmbZIP1-YFP plasmid constructed in 2.1 and the empty vector pHB-YFP to transform Agrobacterium tumefaciens EHA105, and pick a single clone colony for PCR verification. The results showed that the plant overexpression vector pHB-SmbZIP1-YFP containing SmbZIP1 had been successfully transformed into Agrobacterium tumefaciens EHA105.

[0049] 3.2. Transient expression in tobacco

[0050] Use a sterile syringe to absorb the Agrobacterium tumefaciens EHA105 containing the carrier pHB-SmbZIP1-YFP and the empty vector pHB-YFP constructed in 3.1 to perform transient transformation on the well-grown tobacco leaves. First, dark culture for 24 hours and then light for 24 hours, take Leaves were placed on a glass slide with the back facing up, soaked with double distilled water, fixed with a cover ...

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Abstract

The invention provides a SmbZIP1 gene of the SmbZIP1 gene in increasing the content of salvianolic acid in salvia miltiorrhiza and an application thereof. The SmbZIP1 gene with a full length of 474bpis screened from a salvia miltiorrhiza yeast library by a yeast two-hybrid screening method; the content of salvianolic acid in salvia miltiorrhiza can be increased by overexpression of the SmbZIP1 gene in salvia miltiorrhiza, and the specific method comprises the following steps: constructing the SmbZIP1 gene in a plant overexpression regulation sequence according to a salvia miltiorrhiza SmbZIP1gene sequence to form a plant overexpression vector containing the SmbZIP1 gene; converting the obtained plant expression vector containing the SmbZIP1 gene into agrobacterium rhizogenes; using the transformed agrobacterium rhizogenes strain for genetic transformation and infection of the salvia miltiorrhiza aseptic seedling explant, and taking the root system after infection and detecting to bepositive through PCR, namely overexpression; the invention provides a method for increasing the content of salvianolic acid in hairy roots of salviae miltiorrhizae, a new gap can be opened for solvingthe market situation that salvianolic acid supply is short, and the method has high application value and research value.

Description

technical field [0001] The invention relates to a SmbZIP1 gene, overexpressing the gene SmbZIP1 can increase the content of salvianolic acid in salvia miltiorrhiza, and belongs to the technical field of genetic engineering. Background technique [0002] Danshen (Salvia miltiorrhiza Bunge) was first recorded in "Shen Nong's Materia Medica" and was listed as top grade. It is a commonly used Chinese herbal medicine and belongs to the genus Salvia of the family Lamiaceae. It is a perennial dicotyledon The herbaceous plant, named Danshen for its red-like ginseng root, its dried roots and rhizomes are often used for medicine and have high medicinal value. As a traditional Chinese medicine, it is a strong menstruation agent, mostly used as a compound drug, and has been included in the Pharmacopoeia of the People's Republic of China. [0003] According to WHO statistics, cardiovascular and cerebrovascular diseases have become the "number one killer" of human health, and the number ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/82A01H6/50
CPCC07K14/415C12N15/8243
Inventor 开国银邓昌平王瑶时敏付蓉张毅郝小龙
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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