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Fusion protein, its coding gene and its application in biosynthesis

A technology encoding gene and chalcone synthase, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of explosion, low yield, long time consumption, etc., and achieve the effect of good application prospects

Inactive Publication Date: 2020-12-29
刘春生
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Predatory excavation of wild licorice has not only caused a substantial reduction in licorice production, but also led to environmental damage and desertification in licorice planting areas. It is difficult to recover licorice populations after being destroyed
In order to alleviate the shortage of drug sources, there are a large number of ways to synthesize isoliquiritigenin by chemical methods, but the chemical synthesis method needs to use a large amount of organic solvents, which is not good for environmental protection, and there is a certain risk of explosion
Therefore, methods such as tissue cell culture have also been developed to obtain isoliquiritigenin, but the yield is too low and the time-consuming is too long

Method used

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  • Fusion protein, its coding gene and its application in biosynthesis
  • Fusion protein, its coding gene and its application in biosynthesis
  • Fusion protein, its coding gene and its application in biosynthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Cloning of Isoliquiritigenin Biosynthetic Pathway Enzyme Genes in Glycyrrhizae Ural

[0032] 1. Primer Design

[0033] The full-length sequence fragment of the gene was screened according to the transcriptome data annotation of Ural licorice, and the upstream and downstream cloning primers were designed. The primer sequences are as follows:

[0034]

[0035] 2. PCR amplification

[0036] Ural licorice RNA was reverse transcribed into cDNA using QuantScript RT Kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China).

[0037] Using cDNA as a template, PCR amplification was carried out.

[0038] The amplification system was: 25 μL of 2×KAPA HiFi Hotstart ReadyMix (Kapa Biosystems, Wilmington, USA), 1.5 μL of primers P1 and P2, 2 μL of template, and 50 μL of double distilled water. Reaction conditions: pre-denaturation at 98°C for 3min, 98°C for 20s, annealing at 62°C for 15s, extension at 72°C for 1.5min, extension at 72°C for 5min after 35 cycles, a...

Embodiment 2

[0041] Example 2 Prokaryotic expression and in vitro enzymatic reaction of PAL, CHS and CHR genes

[0042] 1. Prokaryotic expression

[0043] PAL, CHS and CHR were respectively inserted between KpnI and XhoI of pET-32a(+) using EasyGeno Assembly Cloning kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China), and transferred into E.coli BL21(DE3). Transformants were screened on LB plates containing 100 mg / mL ampicillin and single clones were selected for sequencing verification. Recombinant expression cells were cultured in 200 mL LB medium containing 100 mg / mL ampicillin with shaking at 37° C. to OD600 = 0.6-1.0, and induced with 0.2 mM IPTG at 16° C. for 10 h. The cells were collected by centrifugation at 5000 rpm at 4°C. Resuspend the bacteria in 3mL PBS buffer (pH 8.0), break the bacteria by ultrasonic in an ice bath, and collect the supernatant by centrifugation. The recombinant protein was purified with (Kangwei Century Biotechnology Co., Ltd., Beijing, China),...

Embodiment 3

[0053] Example 3 Yeast expression and characterization of C4H and 4CL genes

[0054] Yeast expression vectors were constructed by using the EasyGeno Assembly Cloning kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) to insert C4H or CHS into the yeast expression vector pESC-His (Agilent Technologies, Santa Clara, USA) (or It can also be inserted between the SpeI and NotI sites of pESC-Leu (Agilent Technologies, Santa Clara, USA), respectively, and 4CL can be inserted between the NheI and BamHI sites of the recombinant vector that has been linked with CHS. The recombinant plasmid was transformed into the host strain WAT11 using a yeast transformation kit (Zymo Research Corporation, Irvine, USA), and the transformants were screened at 30°C on the corresponding deficient medium (SC-His or -Leu, 2% glucose and 2% agar) Cultured for 4 days. Positive clones were stored in corresponding liquid deficient medium (2% glucose) and shaken at 30°C until OD600 was about 0.8. ...

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Abstract

The invention relates to a fusion protein, a gene used for encoding the fusion protein and a recombinant engineering bacterium containing the gene. The fusion protein comprises chalcone synthase and chalcone reductase, and the chalcone synthase is connected with the chalcone reductase through a connexin GGGS. The fusion protein can be applied to biosynthesis of chalcone and isoliquiritigenin.

Description

technical field [0001] The invention relates to a fusion protein, a gene encoding the fusion protein, an expression vector containing the gene encoding the fusion protein, a recombinant bacterium, and the application of the fusion protein or the recombinant in the synthesis of isoliquiritigenin, which belongs to medicinal The field of compositional synthetic biology. Background technique [0002] As an ancient herbal medicine with a history of thousands of years, licorice (Glycyrrhizae Radix et Rhizoma) is one of the most commonly used bulk rare and endangered medicinal materials. It has shown good and safe preventive and therapeutic effects on many diseases, and it appears in almost all traditional Chinese medicine Prescriptions, over-the-counter medicines, and more and more health products and even food. Isoliquiritigenin is a kind of chalcone with high content in licorice. Allergic, anti-viral and estrogen-like, and other significant activities, among which, the anti-ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N9/02C12N15/54C12N15/53C12N15/81C12N1/19
CPCC12N9/0004C12N9/1037C12N15/81C12Y203/01074
Inventor 刘春生李妍芃尹艳高伟姜丹
Owner 刘春生
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