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Preparation and application of AtAGM2 and AtAGM3 coding gene and enzyme

A coding gene and coding technology, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of expensive hexose phosphate and cumbersome production steps

Inactive Publication Date: 2018-10-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The price of hexose phosphate is very expensive. At present, it is mainly synthesized by chemical methods, and the production steps are cumbersome.

Method used

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  • Preparation and application of AtAGM2 and AtAGM3 coding gene and enzyme
  • Preparation and application of AtAGM2 and AtAGM3 coding gene and enzyme
  • Preparation and application of AtAGM2 and AtAGM3 coding gene and enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Cloning of full-length genes of acetylglucosamine phosphate mutase AtAGM2 and AtAGM3

[0076] After analyzing the hexose phosphate mutase genes of Arabidopsis in The National Center for Biotechnology Information (NCBI) database, two genes in the hexose mutase family whose functions have not been clarified were selected. After sequence analysis, the primers were designed as follows:

[0077] Agm2-F: 5'-GCGTCCATGGAAGGAAAGGTTTTCCAAAAAC-3'; Agm2-R: 5'-ATATATGCGGCCGCGGAGGAGTGAGTG-3' to amplify the gene sequence of Atagm2. Agm3-F: 5'-AACTCCATGGCGTCGACTTCAACATCATC-3'; Agm3-R: 5'-ACTGCTCGAGAGATTGACCTCCGATGTAA-3' to amplify the gene sequence of Atagm3.

[0078] The mRNA of Arabidopsis leaves was extracted according to the operation steps of the RNA extraction kit (Bomad Biotechnology, Cat. No. RN0112). PCR amplification was carried out using the reversed cDNA of the extracted Arabidopsis RNA as a template. The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 9...

Embodiment 2

[0079] Example 2 Gene sequence analysis of acetylglucosamine phosphate mutase AtAGM2 and AtAGM3

[0080] The results of the sequencing were analyzed using the Basic Local Alignment Search Tool (BLAST) in the GenBank database, and the Vector NTI Suite 8.0 software was used for multiple sequence alignment and sequence information analysis.

[0081] The coding region of the obtained acetylglucosamine phosphate mutase gene 2 (named AtAGM2) is 1878 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. AtAGM2 encodes 625 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 3, the theoretical protein molecular weight is 67.0kDa, and the predicted isoelectric point is 6.1. The nucleotide sequence of AtAGM2 is located on chromosome 5 of Arabidopsis thaliana (locus-tag="AT5G17530") in the Arabidopsis genome.

[0082] The coding region of the obtained acetylglucosamine phosphate mutase gene 3 (named AtAGM3) is 1872 bp long, and its nucleotide sequence is s...

Embodiment 3

[0086] Example 3 Recombinant expression and purification of AtAGM2 and AtAGM3 genes in Escherichia coli

[0087] Sequencing results showed that the AtAGM2 gene shown in SEQ ID NO 1 was inserted into pET28a, and the insertion direction was correct, which proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET28a-AtAGM2.

[0088] At the same time, the recombinant plasmid of AtAGM3 was also constructed successfully. The AtAGM3 gene shown in SEQ ID NO 3 was inserted into pET28a, and the insertion direction was correct. The recombinant plasmid was named pET28a-AtAGM3.

[0089] Transform pET28a-AtAGM2 (or pET28a-AtAGM3) into Escherichia coli strain BL21(DE3) for induced expression. Add the overnight cultured seed solution to fresh LB medium with 1% inoculum size for expansion at 37°C, add IPTG when the OD600nm of the bacterial solution is 0.6-0.8, make the final concentration 0.5mM, and carry out at 16°C Induce overnight. After the cell...

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Abstract

The invention discloses two gene sequences of Arabidopsis thaliana N-acetylpHosphoglucosamine mutase 2.3; AtAGM2, AtAGM3 derived from Arabidopsis thaliana and the application thereof. The invention provides a preparation method for arabidopsis thaliana N-acetylpHeophorbide mutase 2.3; AtAGM2, AtAGM3, which comprises the steps that the gene of the enzyme is cloned into the expression vector of theescherichia coli, a recombinant escherichia coli strain with heterologous expression mutaseis is obtained, and AtAGM2 and AtAGM3 are prepared by heterologous expression of recombinant strains. The twoAGM proteins have allozyme activity of catalytic hexose phosphate isomer, and can be applied to the field of enzymatic synthesis of uridine diphosphate acetylglucosamine (UDP-GlcNAc) or the production of hexose phosphoric acid isomer.

Description

technical field [0001] The invention relates to gene sequences of two acetylglucosamine phosphate mutases AtAGM2 and AtAGM3 and a preparation method thereof, in particular to the application of these two enzymes in the production of nucleotide sugar and hexose phosphate isomers. The invention also provides the recombinant plasmids and recombinant genetic engineering strains of the two acetylglucosamine phosphate mutases, as well as the enzymatic properties of the two enzymes. Background technique [0002] Acetylglucosamine (GlcNAc, N-acetyl-β-D-glucosamine) is an amino sugar derivative of glucose. In organisms, it is a monosaccharide with important physiological and biochemical functions after glucose. Often in the form of UDP-GlcNAc, it participates in many biological reaction processes including the nervous system and immune system and the structural composition of various glycoconjugates in organisms. In organisms, the main pathway for generating UDP-GlcNAc is the Hexos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/02C12P19/38
CPCC12N9/90C12P19/02C12P19/38C12Y504/02
Inventor 尹恒贾晓晨李悝悝曹海龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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