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Zymoprotein, encoding gene thereof and application

A gene and protein technology, applied in the field of enzyme protein and its coding gene and application, can solve problems such as less research

Active Publication Date: 2019-09-10
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on Andrographis paniculata flavone glycosyltransferase at home and abroad

Method used

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  • Zymoprotein, encoding gene thereof and application
  • Zymoprotein, encoding gene thereof and application
  • Zymoprotein, encoding gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Discovery of a flavone glycosyltransferase protein ApUGT2 and its coding gene from Andrographis paniculata

[0031] Transcriptome sequencing was performed using MEJA (methyl jasmonate) induction, and nine candidate glycosyltransferases were found in the leaf transcriptome, all of which were induced by methyl jasmonate. Further prokaryotic expression and functional verification of nine genes revealed that the protein ApUGT2 is a glycosyltransferase that can exclusively participate in the biosynthesis of flavonoid glycosides, but cannot catalyze the biosynthesis of diterpenoids in Andrographis paniculata.

[0032] The amino acid of the glycosyltransferase protein ApUGT2 is shown in sequence 1 of the sequence listing, and its corresponding coding region in cDNA is shown in sequence 2 of the sequence listing.

Embodiment 2

[0034] Functional Verification

[0035] 1. Construction of recombinant plasmids

[0036] 1. Synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing, namely the ApUFGT2 gene.

[0037] 2. Using the DNA molecule obtained in step 1 as a template, a primer pair composed of F1 and R1 is used for PCR amplification, and the PCR amplification product is recovered.

[0038] F2: 5'-TCCAGGGGCCCGAATTCGGAATGTCGGCCGCCACCGCC-3';

[0039] R2: 5'-AGTGCGGCCGCAAGCTTGTTATTGTAACGATACAGCTC-3'.

[0040] 3. Take the vector HIS-MBP-pET28a, perform double digestion with restriction endonucleases BamHI and SalI, and recover the linearized vector backbone. (Recorded in the following documents: Probing the Single Key Amino Acid Responsible for the Novel Catalytic Function of ent-Kaurene Oxidase Supported by NADPH-CytochromeP450 Reductases in Tripterygium Wilfordii; Frontiers in PlantScience, www.frontiersin.org; October 2017, Volume 8, Article 6).

[0041]4. Take the PCR...

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Abstract

The invention relates to the technical field of biology, in particular to zymoprotein, an encoding gene thereof and application. The protein has the amino acid sequence shown as the sequence 1 of (a1)or (a2):(a1). The (a2) is the protein which is formed by substituting and / or deleting and / or adding one or more amino acid residues of (a1), has a glycosyl transferase function and is derived from (a1). The protein can conduct glycosylation on flavonoids compounds of different structures and different hydroxyl groups of the flavonoids compounds through sequencing, authenticating and separating oftranscriptome.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzyme protein, its coding gene and its application. Background technique [0002] Andrographis paniculata, also known as Yijianxi, gall grass, Indian grass, Lamane, and square lotus. Happiness at first sight, bitter gall grass, Indian grass, Larvae, and quadrangular lotus. Contains diterpene lactones and is rich in flavonoids in its rhizomes. A variety of andrographolides and various injections have antipyretic effects; they also have anti-pathogenic microorganisms, anti-inflammation, and enhance immune function; total flavonoids can inhibit ADP-induced platelet aggregation in vivo and in vitro, and have pharmacological effects such as anti-platelet and anti-proliferation . Glycosylation is a key modification step that occurs in various biological processes, especially in secondary metabolic pathways, where it alters the stability, polarity, solubility, biological activity, to...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/60C12P19/18C12R1/19
CPCC12N9/1048C12N15/70C12P19/18C12P19/60
Inventor 黄璐琦高伟李媛
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI