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Application of SmbZIP2 gene in increasing content of salvianolic acid in salvia miltiorrhizae

A salvianolic acid and gene technology, applied in the field of genetic engineering, can solve the problems that have not yet been discovered, and achieve the effect of reliable effect and low cost

Active Publication Date: 2021-04-23
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Have not found the related report of the method for improving salvianolic acid in salvia miltiorrhiza by knocking out the SmbZIP2 gene mentioned in the subject of the present invention

Method used

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  • Application of SmbZIP2 gene in increasing content of salvianolic acid in salvia miltiorrhizae
  • Application of SmbZIP2 gene in increasing content of salvianolic acid in salvia miltiorrhizae
  • Application of SmbZIP2 gene in increasing content of salvianolic acid in salvia miltiorrhizae

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1 Screening and cloning of Salvia miltiorrhiza SmbZIP2 gene

[0041] 1.1. ABA-induced Salvia transcriptome database establishment

[0042] The prepared ABA was added to C58C1 hairy roots (the hairy roots were obtained by Agrobacterium C58C1 infecting Salvia miltiorrhiza explants), so that the final working concentration of ABA was 50 μM. Samples were taken at 0h, 0.5h, 1h, 2h, 4h, and 8h. The transcriptome was sequenced using the Illumina sequencing platform, and the Illumina PE library was constructed for 2×150bp sequencing. After quality control and assembly, software was used to quantitatively analyze the expression levels of genes / transcripts. The difference analysis software is: DESeq2, and the screening threshold is: |log2FC|>=1&padjust<0.05.

[0043] 1.2. Screening of SmbZIP2 gene in Salvia miltiorrhiza

[0044] Set the screening condition as FPKM value>200 and the expression is 5 times higher than the control, a total of 26 transcription factors were...

Embodiment 2

[0048] Example 2 The target of Danshen SmbZIP2 gene

[0049] 2.1. Dual-luciferase (Dual-LUC) assay

[0050] The promoters of the key enzyme genes of the salvianolic acid pathway were amplified and constructed on the pGreen0800 vector to obtain the pGreen0800-promotor vector, which was transformed into Agrobacterium GV3101 (containing psoup19). The pHB-SmbZIP2-YFP vector (primers: pHB-bZIP2-YFP-KF, pHB-bZIP2-YFP-KR) was constructed and transformed into Agrobacterium GV3101. Suspend the cells with the permeate, mix 1:1, and inject tobacco. Dual-LUC detects the effect of SmbZIP2 on the promoter. the result shows( image 3 B), SmbZIP2 transcription factor can repress SmPAL at the transcriptional level. To further determine the target of SmbZIP2, the promoter of PAL gene was analyzed for subsequent experiments.

[0051] 2.2. Yeast one-hybrid experiment

[0052] Such as figure 2 As shown, the phylogenetic tree results showed that SmbZIP2 had a close evolutionary relationship...

Embodiment 3

[0054] Example 3 Subcellular localization of Salvia miltiorrhiza SmbZIP2 protein

[0055] The GV3101 strain containing the pHB-SmbZIP2-YFP plasmid in 2.1 was expanded and cultured, and the empty vector pHB-YFP was transferred into GV3101 as a control. The engineered strains suspended in the infiltrate were sucked into a sterile syringe and injected into tobacco leaves in good growth state. After the injection, cultured in dark for 1 day, and then cultured in light for 1 day. Take the leaves on a glass slide with the back facing up, infiltrate with double distilled water, fix the leaves with a cover glass (to avoid air bubbles), and observe under a laser confocal microscope. result( Figure 4 ) showed that the SmbZIP2 protein was localized in the nucleus, which was consistent with its function as a transcription factor.

[0056]

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Abstract

The invention provides an application of a transcription factor SmbZIP2 in increasing the content of salvianolic acid in salvia miltiorrhizae. According to the application, the SmbZIP2 gene with the full length of 1386bp is screened from an abscisic acid induced salvia miltiorrhizae transcriptome database. A SmbZIP2 gene knockout vector is constructed by using a CRISPR-Cas9 technology, a hairy root strain with SmbZIP2 gene knockout is obtained by genetic transformation of a salvia miltiorrhizae explant, and whether the knockout succeeds or not is identified by sequencing. The content of salvianolic acid in the transgenic salvia miltiorrhizae hairy roots is determined by using high performance liquid chromatography (HPLC). The content of salvianolic acid produced by the knockout strain obtained by the invention is remarkably improved and is 1.56 times that of a control strain. QRT-PCR analysis shows that the expression of the salvianolic acid biosynthesis key enzyme gene is also significantly up-regulated. The invention provides a method for increasing the content of salvianolic acid in hairy roots of salviae miltiorrhizae, a new thought can be provided for production of salvianolic acid, and the method has high application value and research value.

Description

technical field [0001] The invention relates to a SmbZIP2 gene, which can increase the content of salvianolic acid in salvia miltiorrhiza by knocking out the SmbZIP2 gene, and belongs to the technical field of genetic engineering. Background technique [0002] Salvia miltiorrhiza Bunge is a perennial herb of Lamiaceae. It was first recorded in "Shen Nong's Materia Medica", "Materia Medica" and "Compendium of Materia Medica". As a traditional Chinese medicine, it has been used in my country for a long time. Salvia miltiorrhiza is widely used in the treatment of cardiovascular and cerebrovascular diseases, irregular menstruation and various inflammations, and has therefore been included in the Pharmacopoeia of the People's Republic of China. At present, the dosage forms produced with Danshen as raw materials on the market include injections, granules, syrups, tablets, etc., including Compound Danshen Tablets, Compound Danshen Dropping Pills, Danshen Injection, Tanshinone Capsu...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/06A01H6/50
CPCC07K14/415C12N15/8218C12N15/8243
Inventor 开国银时敏
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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