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Preparation and application of glutamine-fructose-6-phosphate aminotransferase coding gene and glutamine-fructose-6-phosphate aminotransferase enzyme

A technology of aminotransferase and fructose phosphate, applied in the direction of transferase, application, genetic engineering, etc., can solve the problem that GFAT has no relevant transformation research

Pending Publication Date: 2021-05-28
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

There have been studies on modifying GFA in prokaryotic systems to overproduce UDP-GlcNAc, but there is no related modification research on GFAT in eukaryotes

Method used

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  • Preparation and application of glutamine-fructose-6-phosphate aminotransferase coding gene and glutamine-fructose-6-phosphate aminotransferase enzyme
  • Preparation and application of glutamine-fructose-6-phosphate aminotransferase coding gene and glutamine-fructose-6-phosphate aminotransferase enzyme
  • Preparation and application of glutamine-fructose-6-phosphate aminotransferase coding gene and glutamine-fructose-6-phosphate aminotransferase enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Cloning of embodiment 1 phosphofructose aminotransferase OsGFAT full-length gene

[0051] The mRNA of rice leaves was extracted according to the operating steps of the RNA extraction kit (Bomad Biology, Cat. No. RN0112). After analyzing the sequence of phosphofructose aminotransferase in The National Center for Biotechnology Information (NCBI) database, design primers GFAT-F: 5'-CATTCCATGGATATGTGCGGGATCTTCGCG-3'; GFAT-R: 5'-CGGTAAGCTTTTGGGTAGTCACACTCTTTGCC-3' to amplify the phosphate The gene sequence of the mature protein of fructose aminotransferase OsGFAT was amplified by PCR using the reversed cDNA of the extracted rice RNA as a template. The PCR reaction conditions were: 94°C for 2 min, 1 cycle; 94°C for 30 s, 58°C for 30 s, 72°C for 3 min, 35 cycles; 72°C for 5 min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (eg figure 1 (a)), the target fragment was recovered by gel cutting, ligated to the prokaryotic expression vector pET28a after doubl...

Embodiment 2

[0052] Embodiment 2 Phosphofructose aminotransferase OsGFAT gene sequence analysis

[0053]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.

[0054] The coding region of the obtained phosphofructose aminotransferase gene (named OsGFAT) is 2043bp long, and its nucleotide sequence is shown in SEQ ID NO 1. OsGFAT encodes 679 amino acids and a stop codon, its amino acid sequence is shown in SEQID NO 2, the theoretical protein molecular weight is 74.41kDa, and the predicted isoelectric point is 6.1. It is predicted by bioinformatics that the protease expressed by this gene may have the function of phosphofructose aminotransferase, but it has not been studied so far.

Embodiment 3

[0055] Example 3 Recombinant expression and purification of OsGFAT gene in Escherichia coli

[0056] Sequencing results showed that the OsGFAT 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-OsGFAT.

[0057] Transform pET28a-OsGFAT into Escherichia coli strain BL21(DE3) for induced expression. Add the overnight cultured seed solution to fresh LB medium at 37°C with an inoculation amount of 1% to carry out expanded cultivation. When the OD600nm of the bacterial solution = 0.6-0.8, add IPTG to make the final concentration 0.25mM, and carry out at 10°C 72h induction. After the cells were broken, the supernatant was collected by high-speed centrifugation and purified by a nickel column, and eluted with gradient imidazole (20-250mM imidazole, 20mM Tris-HCl, pH7.1). Polyacrylamide gel electrophoresis was used to detect the exp...

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Abstract

The invention discloses a gene sequence of Oryza sativa Japonica Group glutamine-fructose-6-phosphate aminotransferase (OsGFAT) derived from Oryza sativa Japonica Group, and application of the gene sequence. The invention provides a method for preparing the OsGFAT. The method comprises the following steps: cloning the gene of the OsGFAT to an escherichia coli expression vector to obtain an escherichia coli recombinant strain capable of heterologously expressing the OsGFAT, and preparing OsGFAT by heterologously expressing the strain. The invention also provides the application of the OsGFAT. The OsGFAT is used for catalytic synthesis of GlcN-6-P and Glc-6-P by an enzymic method.

Description

technical field [0001] The invention relates to a gene sequence of phosphofructose aminotransferase OsGFAT and a preparation method thereof, in particular to the application of the enzyme in the production of glucose-6-phosphate and glucosamine-6-phosphate. The invention also provides the recombinant plasmid and recombinant genetically engineered strain of the phosphofructose aminotransferase, as well as a method for producing and isolating glucose-6-phosphate and glucosamine-6-phosphate by using the enzyme. 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 g...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/02C12P19/26
CPCC12N9/1096C12Y206/01C12N15/70C12P19/02C12P19/26
Inventor 尹恒刘俊杰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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