CN108611344A - The preparation and application of AtAGM2 and AtAGM3 encoding genes and enzyme - Google Patents

Abstract

The present invention discloses two phosphoacetylglucosamine mutase (Arabidopsis thaliana N acetylpHospHoglucosamine mutase 2,3 for deriving from arabidopsis (Arabidopsis thaliana)；AtAGM2, AtAGM3) gene order and its application.The present invention provides a kind of methods preparing acetyl glucosamine transphosphorylase, it i.e. will be in the gene cloning to coli expression carrier of enzyme, obtain can heterogenous expression mutase E. coli recombinant stain, the mutase activity that AtAGM2 all has with AtAGM3. two kinds of AGM albumen catalysis pbosphohexose isomers is prepared with recombinant bacterial strain heterogenous expression, can be applied to enzyme law catalysis synthesis uridine 5'-diphosphate acetylglucosamine (UDP GlcNAc) or production pbosphohexose isomers field.

Description

Preparation and Application of AtAGM2 and AtAGM3 Encoding Genes and Enzymes

technical field

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

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 Hexosamine biosynthesis pathway. This pathway is a branch of glycolysis, which uses the intermediate product of hexose metabolism pathway, fructose-6-phosphate (Fructose-6-P) as the initial substrate, and finally synthesizes UDP under the synergistic catalysis of various enzymes. -GlcNAc. According to the sequence of catalytic reactions in the synthesis process and the sources of enzymes involved in the synthesis pathway, it can be divided into eukaryotic, prokaryotic and mimivirus UDP-GlcNAc synthesis pathways. At present, the hexosamine pathway has been extensively studied, but the third enzyme acetylglucosamine phosphate mutase in the plant hexosamine pathway has not been studied and applied so far, so the hexosamine pathway in plants has not yet been studied. get through.

In addition, UDP-GlcNAc, as an important active nucleoside sugar in the body, can be used in large quantities for the production of oligosaccharides, and then be developed into pharmaceuticals or functional materials. However, due to the limitations of its production methods, its supply is still small and its price is high. At present, there have been studies using a cell-free catalyzed multi-step enzyme-catalyzed reaction system, coupled with three enzymes, glucokinase, AGM and GlcNAc-1-P uridine transferase (GlmU), to produce UDP-GlcNAc with GlcNAc as a substrate. Regardless of the expression system in vivo or in vitro, the soluble expression level of AGM 1 from yeast is very low, which affects the efficiency and cost of product synthesis. Therefore, it is of great significance to provide a highly active and highly expressed AGM.

The price of hexose phosphate is very expensive. At present, it is mainly synthesized by chemical methods, and the production steps are cumbersome. Companies such as Sigma and Santa Cruz have little or no stock. Due to the limitation of the synthesis method, the price difference between the different isomers of hexose phosphate is huge (tens or even hundreds of times); for example, the price of GlcN-6-P is 29 yuan/mg, while the price of GlcN-1-P is 29 yuan/mg. It is 460/mg. Therefore, it is imperative to explore new preparation methods of hexose phosphates, especially expensive hexose phosphates.

In view of the research status of the above-mentioned acetylglucosamine phosphate mutase and the problems existing in its application, the present invention discloses two acetylglucosamine phosphate mutases (Arabidopsis thaliana N-acetylpHospHoglucosamine) derived from Arabidopsis thaliana (Arabidopsis thaliana) mutase, AtAGM) gene sequence and preparation method thereof. These two enzymes both have the activity of catalyzing different hexose phosphate isomers. Compared with the other one and the acetylglucosamine phosphate mutase AtAGM1 in Arabidopsis thaliana, the efficiency of the three catalytic reactions is not much different, and they have Similar enzymatic properties can be applied to the cell-free enzymatic synthesis of UDP-GlcNAc, or the production of hexose phosphate isomers.

Contents of the invention

The first object of the present invention is to provide two plant-derived acetylglucosamine phosphate mutases AtAGM2 and AtAGM3 and their coding genes.

The second object of the present invention is to provide a method for preparing acetylglucosamine phosphomutases AtAGM2 and AtAGM3.

The third object of the present invention is to provide recombinant expression plasmids and recombinant genetic engineering strains containing the acetylglucosamine phosphate mutases AtAGM2 and AtAGM3.

The acetylglucosamine phosphate mutase AtAGM2 provided by the present invention is derived from Arabidopsis thaliana (Arabidopsisthaliana), and its amino acid sequence has one or both of the following characteristics:

1) The 1-625th amino acid residue sequence of SEQ ID NO.2 in the sequence listing starting from the amino terminal is the amino acid sequence of the active acetylglucosamine phosphomutase AtAGM, and 620-625 is the amino acid residue of His-Tag sequence.

2) Substituting, deleting or adding one or more amino acids to amino acid residues 1-625 of SEQ ID NO.2 starting from the amino terminal of SEQ ID NO.2 in the sequence listing to form an acetylglucosamine phosphomutase activity unchanged amino acid sequence.

The present invention also provides a gene encoding the above-mentioned acetylglucosamine phosphate mutase AtAGM2, which is derived from Arabidopsis thaliana, and its nucleotide sequence has one or more than two of the following characteristics:

1) the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing;

2) the deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 in the sequence listing;

3) A nucleotide encoding acetylglucosamine phosphate mutase activity obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing sequence.

The acetylglucosamine phosphate mutase AtAGM3 provided by the invention is derived from Arabidopsis thaliana (Arabidopsisthaliana), and its amino acid sequence has one or both of the following characteristics:

1) The sequence of amino acid residues 1-623 from the amino terminal of SEQ ID NO.4 in the sequence listing is the amino acid sequence of the active acetylglucosamine phosphomutase AtAGM, and 518-623 is the amino acid of His-Tag sequence.

2) Substituting, deleting or adding one or more amino acids to amino acid residues 1-623 of SEQ ID NO.4 starting from the amino terminus in the sequence listing to form an acetylglucosamine phosphomutase activity unchanged amino acid sequence.

The present invention also provides a gene encoding the above-mentioned acetylglucosamine phosphate mutase AtAGM3, which is derived from Arabidopsis thaliana, and its nucleotide sequence has one or more than two of the following characteristics:

1) the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.3 in the sequence listing;

2) the deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.4 in the sequence listing;

3) The nucleotide encoding the acetylglucosamine phosphate mutase activity obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.3 in the sequence listing sequence.

The amino acid sequences and nucleotide coding sequences of the acetylglucosamine phosphate mutases AtAGM2 and AtAGM3 of the present invention can also be artificially synthesized according to the predicted amino acid sequences and nucleotide coding sequences of AtAGM2 and AtAGM3.

The method for preparing recombinant enzymes AtAGM2 and AtAGM3 is to clone the coding gene of acetylglucosamine phosphate mutase AtAGM2 or AtAGM3 into a recombinant expression vector, introduce it into host cells, and obtain recombinantly expressed acetylglucosamine phosphate mutase

The nucleotide sequence of the gene encoding the above-mentioned acetylglucosamine phosphate mutase AtAGM2 or AtAGM3 has one or more than two of the following characteristics:

1) have the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 or SEQ ID NO.3 in the sequence listing, 2) the deoxyribonucleic acid (DNA) sequence of encoding SEQ ID NO.2 or SEQ ID NO.4 aminoacid sequence ,

3) The code obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 or SEQ ID NO.3 in the sequence listing has acetylglucosamine phosphate shift Nucleotide sequence for enzymatic activity.

The expression vector for the recombinant expression of acetylglucosamine phosphate mutase or AtAGM2 or AtAGM3 can be Escherichia coli expression vector, yeast expression vector, Bacillus subtilis expression vector, lactic acid bacteria expression vector, streptomyces expression vector, phage vector, filamentous fungus Expression vectors, plant expression vectors, insect expression vectors, or mammalian cell expression vectors, etc.

Recombinant bacteria or transgenic cell lines for recombinant expression of acetylglucosamine phosphate mutase AtAGM2 or AtAGM3 can be Escherichia coli host cells (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5α, etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyces lactis, etc.), Bacillus subtilis host cells (such as Bacillus subtilis R25, Bacillus subtilis 9920, etc.), lactic acid bacteria host cells (such as Lactic acid bacteria COCC101, etc.), actinomyces host cells (such as Streptomyces spp., etc.) , filamentous fungal host cells (such as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans, etc.), insect cells (such as Bombyx mori, Antharaea eucalypti, etc.) or mammalian cells (such as Chinese hamster ovary cells CHO, young hamster kidney cells BHK , Chinese hamster lung cells CHL, etc.).

The above-mentioned acetylglucosamine phosphate mutase can be applied in the production of hexose phosphate and nucleotide sugar, including one or more of the following applications:

1) Application in realizing the transformation of the 1,6-position isomer of hexose phosphate and producing the corresponding isomer;

2) Application in the production of nucleotide sugars such as UDP-GlcNAc (UDP-GlcN; UDP-Glc);

Description of drawings

Figure 1: Agarose gel electrophoresis detection map of N-acetylglucosamine phosphate mutase gene Atagm2 and Atagm3.

Figure 2: SDS-PAGE diagram of the expression and purification of acetylglucosamine phosphate mutase AtAGM2. The added samples are: 1-E.coli BL21(DE3)/pET28a-AtAGM2 induced bacteria; 2-E.coli BL21(DE3)/pET28a-AtAGM2 uninduced bacteria; M-pre-stained protein molecular weight standard; The rest are eluents of different concentrations of imidazole. The band indicated by the arrow is the AtAGM2 protein band.

Figure 3: SDS-PAGE diagram of the expression and purification of acetylglucosamine phosphomutase AtAGM3. The added samples are: 1-E.coli BL21(DE3)/pET28a-AtAGM3 uninduced cells; 2,3-E.coli BL21(DE3)/pET28a-AtAGM3 induced cells; M-prestained protein Molecular weight standards; the rest are different concentrations of imidazole eluents. The band indicated by the arrow is the AtAGM3 protein band.

Figure 4: The relative activity of AtAGM2 enzyme at different reaction system pH.

Figure 5: Line graph of relative activity of AtAGM2 enzyme at different reaction temperatures.

Figure 6: The relative activity of AtAGM3 enzyme at different reaction system pH.

Figure 7: Line graph of relative activity of AtAGM3 enzyme at different reaction temperatures.

Figure 8: Relative activity diagram of AtAGM2 enzyme under the catalysis of different metal ions.

Figure 9: Relative activity diagram of AtAGM3 enzyme under the catalysis of different metal ions.

Detailed ways

Information on SEQ ID NO.1

(a) Sequential features

Length: 1878 nucleotides

Type: Nucleotide

Chain type: single chain

(b) Molecule type: DNA

Sequence description: SEQ ID NO.1

ATGGAAGGAAAGGTTTTCCAAAACTTTAATGTAGTACAGAGCTGCTACCGACAAAATAAGCAGTTTAAGACACGATACCAAAGAGAACCTGACCTGTTCATGTCTACTTTACTTCCCTGTCCAAGAGAGAAGATGGCATTTAACCTCAACTCTTCCATGCGTGCCCACACTTTGTCTAAATACCAGTTTGTTCTTTCAAAGCAAAGAACTTTTTACTGCAATGCTACTTCGTCAAGTGCTACTGTGCCATCTCTTGACAAAAATGATTTTCTGAAGCTCCAAAACGGCAGTGATATTCGGGGTGTAGCGGTCACTGGGGTTGAGGGGGAACCTGTAAGCCTTCCTGAACCAGTGACTGAAGCCATAGCTGCTGCTTTTGGGCAATGGCTGTTACACAAGAAGAAGGCTGAATCCCGGCGTTTGAGAGTATCTGTTGGCCATGACTCTCGCATCTCTGCACAAACTTTGCTGGAGGCGGTTTCTCGAGGTCTTGGTGTTTCTGGATTAGATGTTGTTCAGTTTGGATTAGCATCAACACCAGCAATGTTTAATAGCACATTGACTGAAGATGAGTCATTCTTGTGCCCAGCTGATGGGGCTATTATGATAACAGCAAGCCATCTTCCTTACAACAGGAACGGTTTCAAGTTCTTTACCAGTGATGGAGGACTTGGGAAGGTTGATATCAAGAACATTTTGGAGCGAGCTGCAGATATTTACAAGAAGCTTTCTGATGAAAATTTGAGGAAATCACAAAGAGAAAGTTCTTCTATTACAAAGGTTGACTACATGTCAGTATACACCTCTGGTCTTGTAAAGGCAGTCCGGAAAGCAGCAGGAGATTTGGAGAAGCCTCTAGAGGGATTTCATATAGTTGTTGATGCTGGAAATGGAGCTGGAGGATTTTTTGCTGCCAAGGTGCTTGAGCCTTTAGGAGCAATTACTTCTGGCAGTCAATTTCTGGAACCAGATGGTATGTTCCCAAATCATATCCCTAATC CGGAAGATAAGGCGGCAATGGAAGCTATAACCAAGGCTGTTCTTGATAATAAGGCTGATTTGGGTATCATCTTTGATACTGATGTTGATAGGTCTGCTGCTGTGGATTCATCTGGCCGTGAATTCAACCGTAATCGTCTTATTGCCTTGCTATCAGCCATTGTTCTAGAGGAACACCCTGGCACAACTATAGTTACGGATAGTGTCACTTCGGACGGTCTGACCTCATTTATTGAGAAGAAGCTTGGCGGAAAGCATCACAGGTTCAAAAGAGGTTACAAGAATGTCATTGACGAAGCTATTCGCTTGAACTCGGTTGGGGAAGAATCACATCTGGCTATAGAAACCAGTGGTCATGGAGCTCTAAAGGAAAACCATTGGCTCGACGATGGGGCCTATCTCATGGTAAAAATCCTGAACAAACTAGCTGCGGCCCGAGCTGCTGGTCAAGGGAGTGGCAGCAAAGTTTTAACAGATCTTGTTGAAGGTCTGGAAGAGCCCAAAGTGGCTTTAGAACTGAGGCTTAAAATCGACAAGAATCACCCTGACCTTGAAGGAAGTGATTTCCGGGAGTATGGAGAGAAGGTCCTGCAACACGTGTCGAACTCAATAGAAACAAATCCAAATCTTATAATAGCTCCAGTTAACTACGAAGGGATCCGCGTTTCGGGCTTTGGTGGATGGTTTCTTCTCAGACTTTCTCTCCATGATCCTGTTCTTCCCCTTAACATCGAGGCACAGAGTGAGGATGATGCTGTGAAATTAGGCCTTGTGGTTGCTACGACAGTGAAGGAGTTCAATGCTTTGGACACCTGTGCCTTGTCCAACCTCACTCACTCCTCCGCGGCCGCACTCGAGCACCACCACCACCACCACTGA

Information on SEQ ID NO.2

(a) Sequential features

Length: 625 amino acids

Type: amino acid

Chain type: single chain

(b) Molecule type: protein

Sequence description: SEQ ID NO.2

MEGKVFQNFNVVQSCYRQNKQFKTRYQREPDLFMSTLLPCPREKMAFNLNSSMRAHTLSKYQFVLSKQRTFYCNATSSSATVPSLDKNDFLKLQNGSDIRGVAVTGVEGEPVSLPEPVTEAIAAAFGQWLLHKKKAESRRLRVSVGHDSRISAQTLLEAVSRGLGVSGLDVVQFGLASTPAMFNSTLTEDESFLCPADGAIMITASHLPYNRNGFKFFTSDGGLGKVDIKNILERAADIYKKLSDENLRKSQRESSSITKVDYMSVYTSGLVKAVRKAAGDLEKPLEGFHIVVDAGNGAGGFFAAKVLEPLGAITSGSQFLEPDGMFPNHIPNPEDKAAMEAITKAVLDNKADLGIIFDTDVDRSAAVDSSGREFNRNRLIALLSAIVLEEHPGTTIVTDSVTSDGLTSFIEKKLGGKHHRFKRGYKNVIDEAIRLNSVGEESHLAIETSGHGALKENHWLDDGAYLMVKILNKLAAARAAGQGSGSKVLTDLVEGLEEPKVALELRLKIDKNHPDLEGSDFREYGEKVLQHVSNSIETNPNLIIAPVNYEGIRVSGFGGWFLLRLSLHDPVLPLNIEAQSEDDAVKLGLVVATTVKEFNALDTCALSNLTHSSAAALEHHHHHH-

Information on SEQ ID NO.3

(a) Sequential features

Length: 1872 nucleotides

Type: Nucleotide

Chain type: single chain

(b) Molecule type: DNA

Sequence description: SEQ ID NO.3

ATGGCGTCGACTTCAACATCATCTTTAATGGCTTCTAAAACTGTAATCTCCAAAACAGCTCTGTTTTCTTCCTTACCGGGAATAGTCAGCCGGAGTTTTTTAACATTCGCACCGGCTTCTCCTTCCGTTAAACCCCTTAGGATAAGATCTTCAAATGTTACTAAGTTCGACGAAGTAACCAACAGTCTTGACGAAGACATGGACCAGATTCGACGGTTACAAAACGGTTCTGACGTGAGAGGAGTCGCATTGGAAGGAGAGAAAGGTCGAACAGTTGACCTAACGCCTGCAGCTGTTGAAGCAATCGCAGAGAGCTTTGGAGAATGGGTTGCAGCAACGGAGAGTAACGGAAACGGCGTCATTAAGATTTCTCTCGGACGGGATCCACGTGTTTCCGGTGGGAAGCTAAGCACGGCAGTGTTTGCCGGCTTAGCTCGTGCAGGCTGTTTAGCTTTTGACATGGGTTTAGCTACAGCGCCAGCTTGCTTCATGAGCACGTTACTCTCTCCATTCGAATACGACGCTTCAATTATGATGACAGCTTCTCATTTACCGTATACAAGAAACGGACTCAAGTTCTTTACCAAGAGAGGAGGATTAACGTCTCCTGAAGTGGAGAAGATATGCGATTTAGCTGCGCGAAAGTACGCTACTAGGCAGACTAAAGTCTCTACATTGATCAGAACGCGACCGCAGCAAGTTGATTTTATGAGCGCTTACTCTAAGCACCTTAGAGAAATCATTAAAGAGAGAATCAATCACCCTGAACACTATGACACTCCTCTCAAAGGATTTCAGATAGTTGTGAATGCGGGTAATGGGTCAGGAGGCTTCTTTACGTGGGACGTTCTAGACAAGTTAGGAGCCGATACATTCGGTTCGCTCTATCTAAACCCTGACGGGATGTTCCCTAATCACATTCCTAATCCGGAAAACAAAATCGCAATGCAACACACCCGAGCCGCGGTTCTTGAGAACTCAGCAGATCTCGGGGTTGTGT TTGATACGGATGTTGACAGGAGTGGAGTGGTGGATAACAAAGGAAATCCTATCAACGGAGATAAGCTTATTGCGCTTATGTCAGCTATAGTGCTTAAAGAACATCCAGGAAGTACAGTAGTGACTGACGCAAGAACGAGTATGGGGCTAACTAGGTTTATAACGGAGCGAGGAGGGAGGCATTGTTTGTATAGAGTAGGGTATAGAAACGTGATTGACAAGGGAGTAGAGCTGAACAAAGACGGCATCGAGACTCATCTCATGATGGAAACTTCAGGACATGGTGCGGTTAAGGAGAATCACTTCTTGGATGATGGTGCATACATGGTGGTGAAGATCATAATTGAAATGGTGAGAATGAGACTCGCGGGATCAAATGAAGGTATCGGTAGTTTGATCGAAGATCTTGAGGAGCCGTTAGAAGCGGTTGAGCTTCGGTTGAATATTTTATCAGAGCCAAGAGATGCCAAAGCAAAAGGCATTGAAGCCATTGAGACTTTCAGGCAATACATTGAGGAAGGAAAACTGAAAGGGTGGGAATTGGGCACGTGTGGGGATTGTTGGGTTACTGAAGGTTGCTTGGTGGACTCAAATGATCATCCATCTGCTATTGATGCTCACATGTACAGGGCAAGAGTGAGTGATGAAGAGAGTGGGGAAGAGTATGGTTGGGTGCATATGAGGCAGAGTATTCATAACCCTAACATCGCACTTAATATGCAATCAATGCTTCCTGGTGGATGTCTCTCCATGACAAGAATCTTCAGAGACCAGTTTCTTGAAGCTAGTGGGGTGGCTAGATTCCTGGATATAAGTGACTTCGACAATTACATCGGAGGTCAATCTCTCGAGCACCACCACCACCACCACTGA

Information on SEQ ID NO.4

(a) Sequential features

Length: 623 amino acids

Type: amino acid

Chain type: single chain

(b) Molecule type: protein

Sequence description: SEQ ID NO.4

MASTSTSSLMASKTVISKTALFSSLPGIVSRSFLTFAPASSPVKPLRIRSSNVTKFDEVTNSLDEDMDQIRRLQNGSDVRGVALEGEKGRTVDLTPAAVEAIAESFGEWVAATESNGNGVIKISLGRDPRVSGGKLSTAVFAGLARAGCLAFDMGLATAP

ACFMSTLLSPFEYDASIMMTASHLPYTRNGLKFFTKRGGLTSPEVEKICDLAARKYATRQTKVSTLIRTRPQQVDFMSAYSKHLREIIKERINHPEHYDTPLKGFQIVVNAGNGSGGFFTWDVLDKLGADTFGSLYLNPDGMFPNHIPNPENKIAMQHTRAAVLENSADLGVVFDTDVDRSGVVDNKGNPINGDKLIALMSAIVLKEHPGSTVVTDARTSMGLTRFITERGGRHCLYRVGYRNVIDKGVELNKDGIETHLMMETSGHGAVKENHFLDDGAYMVVKIIIEMVRMRLAGSNEGIGSLIEDLEEPLEAVELRLNILSEPRDAKAKGIEAIETFRQYIEEGKLKGWELGTCGDCWVTEGCLVDSNDHPSAIDAHMYRARVSDEESGEEYGWVHMRQSIHNPNIALNMQSMLPGGCLSMTRIFRDQFLEASGVARFLDISDFDNYIGGQSLEHHHHHH-

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

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:

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.

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; 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min, 1 cycle. After the PCR product was analyzed by agarose gel electrophoresis, the target fragment was recovered by gel cutting (as shown in Figure 1), and after double enzyme digestion, it was connected to the prokaryotic expression vector pET28a and then sequenced.

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

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.

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.

The coding region of the obtained acetylglucosamine phosphate mutase gene 3 (named AtAGM3) is 1872 bp long, and its nucleotide sequence is shown in SEQ ID NO 2. AtAGM encodes 623 amino acids and a stop codon, its amino acid sequence is shown in SEQ ID NO 4, the theoretical protein molecular weight is 67.3kDa, and the predicted isoelectric point is 5.62. The nucleotide sequence of AtAGM3 is located on chromosome 1 of Arabidopsis thaliana (locus-tag="AT1G70820") in the Arabidopsis genome.

Both AtAGM2 and AtAGM3 are putative hexose phosphate mutase genes, which belong to the family of α-D-phosphohexomutases.

There is another acetylglucosamine phosphate mutase AtAGM1 in Arabidopsis thaliana. The coding region of the coding gene (AT5G18070) of this enzyme is 1710bp long and encodes 556 amino acids. The isoelectric point is 5.35. The nucleotide sequence of AtAGM1 is located on chromosome 5 of Arabidopsis (locus-tag="AT5G18070"), and its activity has been identified.

The gene similarity between AtAGM2 and AtAGM3 is as high as 36.39%, while their sequence similarity with AtAGM1 is lower, only 15.45% and 12.28%, but some sequences are highly conserved. And the protein molecular weight and isoelectric point of the three are not much different.

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

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.

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.

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 cells were broken, the supernatant was collected by high-speed centrifugation for nickel column purification, and gradient imidazole elution (20-500mM imidazole, 20mM Tris-HCl, pH7.6). Polyacrylamide gel electrophoresis was used to detect the expression and purification of acetylglucosamine phosphate mutase AtAGM2 (or AtAGM3). The electrophoresis used 12% separation gel, and after the 80V voltage was flattened, the separation gel was run with 120V voltage. The results are shown in Figure 2 (or 3), the position of the purified acetylglucosamine phosphate mutase AtAGM2 (or AtAGM3) on the electrophoresis gel coincides with the predicted molecular weight.

Example 4 Enzymatic properties of acetylglucosamine phosphate mutases AtAGM2 and AtAGM3

(1) Activity determination of acetylglucosamine phosphate mutase AtAGM

The general system for the determination of AtAGM enzyme activity is as follows: different hexose phosphate substrates (GlcNAc-1-P, GlcNAc-6-P, GlcN-1-P, GlcN-6-P, Glc-1-P, Glc-6 -P) As substrates, each group of substrates was added to the reaction system (300 μL): 20 mMPBS, pH 7.6, 5 mM MgSO ₄ , 10 μM Glc-1,6-2P, an appropriate amount of recombinant enzyme AtAGM was added, and reacted for 10 min. After the reaction system was boiled immediately, protein was removed, and 200 μL of 200 mM NaOH was added to each group of substrates. The consumption of substrate and the formation of product in the system were detected by HAPEC-PAD method. Since the reaction catalyzed by AtAGM is a reversible reaction, and there is also the production of the intermediate product hexose-1,6-2P, the enzyme activity (nmol/min/mg) is expressed by the amount of substrate consumed by 1 mg protein per minute (nmol) . Protein concentration was determined using Beyontian BCA Protein Concentration Assay Kit.

The ion exchange chromatographic system used in HAPEC-PAD enzyme activity detection includes Diane Bio-LC gradient mixing pump, GM-3 (4mm) gradient mixer, ion exchange column CarboPac PA-100column (4×250mm), AgCl Reference electrode and electrochemical detector. The pulse potential change used for detection is: t=0s, E=0.10v; t=0.20s, E=0.10v; t=0.40s, E=0.10v; t=0.41s, E=-2.00v; t =0.42s, E=-2.00v; t=0.43s, E=0.60v; t=0.44s, E=-0.10v; t=0.50s, E=-0.10v. Mobile phase used: A, 100mM NaOH in water; B, 800 mM sodium acetate with 100 mM NaOH in water. The mobile phase elution conditions are 0-5min, 90%A+10%B; 6-15min, 10%-90%B; 16-18min, 10%A+90%B; 19-20min, 90%A+10 %B. The total flow rate is 0.5ml/min, the detection column temperature is 30°C, and the injection volume is 20μL

(2) Substrate specificity of acetylglucosamine phosphomutase AtAGM

Due to the wide substrate selectivity of AtAGM, it can catalyze several pairs of isomerization of GlcNAc-6-P and GlcNAc-1-P; GlcN-6-P and GlcN-1-P; Glc-6-P and Glc-1-P conversion between entities. And AtAGM catalyzed a reversible reaction, so the above six substrates were used to test the substrate specificity of AtAGM. Using the general system mentioned in Example 4(1), different hexose phosphate substrates (GlcNAc-1-P, GlcNAc-6-P, GlcN-1-P, GlcN-6-P, Glc -1-P, Glc-6-P) as substrates, add 0.5 μg AtAGM2 (or AtAGM3 or AtAGM1) to each group of substrates, and react in a water bath at 30°C for 10 minutes. Protein concentration was determined using Beyontian BCA Protein Concentration Assay Kit.

The results are shown in Table 1. Under the above reaction conditions and the same substrate concentration, AtAGM has different activities to different substrates. Compared with the previously identified AtAGM1, AtAGM2 and AtAGM3, although the three AGM enzymes showed different catalytic rates for the six substrates, there were still some commonalities. The general rule is that the activity of the forward reaction is greater than that of the reverse reaction (hexose-6-P is more active than the corresponding hexose-1-P). But in general, the activity of AtAGM2 and AtAGM3 is lower than that of AtAGM1, especially on its main substrates GlcNAc-1-P and GlcNAc-6-P. And the catalytic rate of AtAGM1 and AtAGM3 to Glc-1-P is higher than that of Glc-6-P, which is different from AtAGM2.

Table 1: Substrate specificity of acetylglucosamine phosphomutases AtAGM2 and AtAGM3.

(3) Effect of pH on the recombinant enzyme AtAGM

Using the general system mentioned in Example 4(1), under the condition of 30°C, 30 μM GlcNAc-6-P was used as the substrate respectively, at pH 3.6-10.6 (pH 3.6-5.6 HAc-NaAc, Ph 6.6 -7.6Na ₂ HPO ₄ -NaH ₂ PO ₄ , pH 8.6 Tris-HCl, pH 9.6-10.6 Gly-NaOH) reaction system, the activity of 5 μg enzyme was determined according to the standard detection method. According to the relative activity of the enzyme at different pH, the histogram and the line graph were drawn to determine the optimum reaction pH of the enzyme. The relative activity of the enzyme at each reaction pH was measured with the inactivated enzyme as a control, with the highest activity being 100%.

The results are shown in Figure 4. When AtAGM2 uses GlcNAc-6-P as the substrate, the optimum pH range is in the neutral to acidic range, and the optimum pH is 5.6. As shown in Figure 6, when AtAGM3 uses GlcNAc-6-P as a substrate, the optimum pH range is within neutral, and the optimum pH is 7.0. Compared with the optimum pH of AtAGM1 which was 7.6, the optimum pH of the three were not much different, and they were all in the neutral range.

(4) The influence of temperature on the recombinant enzyme AtAGM

Using the general system mentioned in Example 4(1), using 30 μM GlcNAc-1-P or GlcNAc-6-P as a substrate, the activity of the recombinant enzyme was determined according to the standard method at 4-80° C., 5 μg enzyme The relative activities at different temperatures are shown in Figures 5 and 7. Taking the inactivated enzyme as a control, the relative enzyme activity was calculated with the highest enzyme activity as 100%. The optimum reaction temperature of AtAGM2 with GlcNAc-6-P as substrate is 30°C (Figure 5), and the optimum reaction temperature of AtAGM2 with GlcNAc-6-P as substrate is 20°C (Figure 7). Compared with AtAGM1, which has an optimum temperature range of 25-30°C, the requirements for the reaction temperature of the three are roughly the same.

(5) Effects of EDTA, SDS and metal ions on the activity of AtAGM

The general system mentioned in Example 4 (1) was adopted, 30 μM GlcNAc-6-P was used as the substrate, the concentration of various metal ions was set at 10 mM in the reaction system, and the enzyme activity was detected according to standard methods. The inactivated enzyme was used as a control, and the value with the highest activity was 100%. The results are shown in Figure 8, Mg ²⁺ can obviously improve the enzyme activity of AtAGM2, and some ions such as Ca ^{2 +} and Zn ²⁺ can obviously inhibit the reaction. As shown in Figure 9, Mg ²⁺ can also significantly improve the enzyme activity of AtAGM3, and some ions such as Fe ²⁺ can obviously inhibit the reaction. For AtAGM1, the presence of Mg ²⁺ can also promote the reaction. It shows that the phenomenon of Mg ²⁺ activation reaction is common to all AtAGMs.

SEQUENCE LISTING

<110> Dalian Institute of Chemical Physics, Chinese Academy of Sciences

<120> Preparation and application of AtAGM2 and AtAGM3 coding genes and enzymes

<130>

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1878

<212>DNA

<213> Arabidopsis thaliana

<220>

<221> DNA

<222> (1)..(1878)

<400> 1

atggaaggaa aggttttcca aaactttaat gtagtacaga gctgctaccg acaaaataag 60

cagtttaaga cacgatacca aagagaacct gacctgttca tgtctacttt acttccctgt 120

ccaagagaga agatggcatt taacctcaac tcttccatgc gtgccccacac tttgtctaaa 180

taccagtttg ttctttcaaa gcaaagaact ttttactgca atgctacttc gtcaagtgct 240

actgtgccat ctcttgacaa aaatgatttt ctgaagctcc aaaacggcag tgatattcgg 300

ggtgtagcgg tcactggggt tgagggggaa cctgtaagcc ttcctgaacc agtgactgaa 360

gccatagctg ctgcttttgg gcaatggctg ttacacaaga agaaggctga atcccggcgt 420

ttgagagtat ctgttggcca tgactctcgc atctctgcac aaactttgct ggaggcggtt 480

tctcgaggtc ttggtgtttc tggattagat gttgttcagt ttggattagc atcaacacca 540

gcaatgttta atagcacatt gactgaagat gagtcattct tgtgcccagc tgatggggct 600

attatgataa cagcaagcca tcttccttac aacaggaacg gtttcaagtt ctttaccagt 660

gatggaggac ttgggaaggt tgatatcaag aacattttgg agcgagctgc agatattac 720

aagaagcttt ctgatgaaaa tttgaggaaa tcacaaagag aaagttcttc tattacaaag 780

gttgactaca tgtcagtata cacctctggt cttgtaaagg cagtccggaa agcagcagga 840

gatttggaga agcctctaga gggatttcat atagttgttg atgctggaaa tggagctgga 900

ggattttttg ctgccaaggt gcttgagcct ttaggagcaa ttacttctgg cagtcaattt 960

ctggaaccag atggtatgtt cccaaatcat atccctaatc cggaagataa ggcggcaatg 1020

gaagctataa ccaaggctgt tcttgataat aaggctgatt tgggtatcat ctttgatact 1080

gatgttgata ggtctgctgc tgtggattca tctggccgtg aattcaaccg taatcgtctt 1140

attgccttgc tatcagccat tgttctagag gaacaccctg gcacaactat agttacggat 1200

agtgtcactt cggacggtct gacctcattt attgagaaga agcttggcgg aaagcatcac 1260

aggttcaaaa gaggttacaa gaatgtcatt gacgaagcta ttcgcttgaa ctcggttggg 1320

gaagaatcac atctggctat agaaaccagt ggtcatggag ctctaaagga aaaccattgg 1380

ctcgacgatg gggcctatct catggtaaaa atcctgaaca aactagctgc ggcccgagct 1440

gctggtcaag ggagtggcag caaagtttta acagatcttg ttgaaggtct ggaagagccc 1500

aaagtggctt tagaactgag gcttaaaatc gacaagaatc accctgacct tgaaggaagt 1560

gatttccggg agtatggaga gaaggtcctg caacacgtgt cgaactcaat agaaacaaat 1620

ccaaatctta taatagctcc agttaactac gaagggatcc gcgtttcggg ctttggtgga 1680

tggtttcttc tcagactttc tctccatgat cctgttcttc cccttaacat cgaggcacag 1740

agtgaggatg atgctgtgaa attaggcctt gtggttgcta cgacagtgaa ggagttcaat 1800

gctttggaca cctgtgcctt gtccaacctc actcactcct ccgcggccgc actcgagcac 1860

caccaccacc accactga 1878

<210> 2

<211> 625

<212> PRT

<213> Arabidopsis thaliana

<220>

<221> PRT

<222> (1)..(625)

<400> 2

Met Glu Gly Lys Val Phe Gln Asn Phe Asn Val Val Gln Ser Cys Tyr

1 5 10 15

Arg Gln Asn Lys Gln Phe Lys Thr Arg Tyr Gln Arg Glu Pro Asp Leu

20 25 30

Phe Met Ser Thr Leu Leu Pro Cys Pro Arg Glu Lys Met Ala Phe Asn

35 40 45

Leu Asn Ser Ser Met Arg Ala His Thr Leu Ser Lys Tyr Gln Phe Val

50 55 60

Leu Ser Lys Gln Arg Thr Phe Tyr Cys Asn Ala Thr Ser Ser Ser Ser Ala

65 70 75 80

Thr Val Pro Ser Leu Asp Lys Asn Asp Phe Leu Lys Leu Gln Asn Gly

85 90 95

Ser Asp Ile Arg Gly Val Ala Val Thr Gly Val Glu Gly Glu Pro Val

100 105 110

Ser Leu Pro Glu Pro Val Thr Glu Ala Ile Ala Ala Ala Phe Gly Gln

115 120 125

Trp Leu Leu His Lys Lys Lys Ala Glu Ser Arg Arg Leu Arg Val Ser

130 135 140

Val Gly His Asp Ser Arg Ile Ser Ala Gln Thr Leu Leu Glu Ala Val

145 150 155 160

Ser Arg Gly Leu Gly Val Ser Gly Leu Asp Val Val Gln Phe Gly Leu

165 170 175

Ala Ser Thr Pro Ala Met Phe Asn Ser Thr Leu Thr Glu Asp Glu Ser

180 185 190

Phe Leu Cys Pro Ala Asp Gly Ala Ile Met Ile Thr Ala Ser His Leu

195 200 205

Pro Tyr Asn Arg Asn Gly Phe Lys Phe Phe Thr Ser Asp Gly Gly Leu

210 215 220

Gly Lys Val Asp Ile Lys Asn Ile Leu Glu Arg Ala Ala Asp Ile Tyr

225 230 235 240

Lys Lys Leu Ser Asp Glu Asn Leu Arg Lys Ser Gln Arg Glu Ser Ser

245 250 255

Ser Ile Thr Lys Val Asp Tyr Met Ser Val Tyr Thr Ser Gly Leu Val

260 265 270

Lys Ala Val Arg Lys Ala Ala Gly Asp Leu Glu Lys Pro Leu Glu Gly

275 280 285

Phe His Ile Val Val Asp Ala Gly Asn Gly Ala Gly Gly Phe Phe Ala

290 295 300

Ala Lys Val Leu Glu Pro Leu Gly Ala Ile Thr Ser Gly Ser Gln Phe

305 310 315 320

Leu Glu Pro Asp Gly Met Phe Pro Asn His Ile Pro Asn Pro Glu Asp

325 330 335

Lys Ala Ala Met Glu Ala Ile Thr Lys Ala Val Leu Asp Asn Lys Ala

340 345 350

Asp Leu Gly Ile Ile Phe Asp Thr Asp Val Asp Arg Ser Ala Ala Val

355 360 365

Asp Ser Ser Gly Arg Glu Phe Asn Arg Asn Arg Leu Ile Ala Leu Leu

370 375 380

Ser Ala Ile Val Leu Glu Glu His Pro Gly Thr Thr Ile Val Thr Asp

385 390 395 400

Ser Val Thr Ser Asp Gly Leu Thr Ser Phe Ile Glu Lys Lys Leu Gly

405 410 415

Gly Lys His His Arg Phe Lys Arg Gly Tyr Lys Asn Val Ile Asp Glu

420 425 430

Ala Ile Arg Leu Asn Ser Val Gly Glu Glu Ser His Leu Ala Ile Glu

435 440 445

Thr Ser Gly His Gly Ala Leu Lys Glu Asn His Trp Leu Asp Asp Gly

450 455 460

Ala Tyr Leu Met Val Lys Ile Leu Asn Lys Leu Ala Ala Ala Arg Ala

465 470 475 480

Ala Gly Gln Gly Ser Gly Ser Lys Val Leu Thr Asp Leu Val Glu Gly

485 490 495

Leu Glu Glu Pro Lys Val Ala Leu Glu Leu Arg Leu Lys Ile Asp Lys

500 505 510

Asn His Pro Asp Leu Glu Gly Ser Asp Phe Arg Glu Tyr Gly Glu Lys

515 520 525

Val Leu Gln His Val Ser Asn Ser Ile Glu Thr Asn Pro Asn Leu Ile

530 535 540

Ile Ala Pro Val Asn Tyr Glu Gly Ile Arg Val Ser Gly Phe Gly Gly

545 550 555 560

Trp Phe Leu Leu Arg Leu Ser Leu His Asp Pro Val Leu Pro Leu Asn

565 570 575

Ile Glu Ala Gln Ser Glu Asp Asp Ala Val Lys Leu Gly Leu Val Val

580 585 590

Ala Thr Thr Val Lys Glu Phe Asn Ala Leu Asp Thr Cys Ala Leu Ser

595 600 605

Asn Leu Thr His Ser Ser Ala Ala Ala Leu Glu His His His His His His His

610 615 620

His

625

<210> 3

<211> 1872

<212>DNA

<213> Arabidopsis thaliana

<220>

<221> DNA

<222> (1)..(1872)

<400> 3

atggcgtcga cttcaacatc atctttaatg gcttctaaaa ctgtaatctc caaaacagct 60

ctgttttctt ccttaccgggg aatagtcagc cggagttttt taacattcgc accggcttct 120

ccttccgtta aaccccttag gataagatct tcaaatgtta ctaagttcga cgaagtaacc 180

aacagtcttg acgaagacat ggaccagatt cgacggttac aaaacggttc tgacgtgaga 240

ggagtcgcat tggaaggaga gaaaggtcga acagttgacc taacgcctgc agctgttgaa 300

gcaatcgcag agagctttgg agaatgggtt gcagcaacgg agagtaacgg aaacggcgtc 360

attaagattt ctctcggacg ggatccacgt gtttccggtg ggaagctaag cacggcagtg 420

tttgccggct tagctcgtgc aggctgttta gcttttgaca tgggtttagc tacagcgcca 480

gcttgcttca tgagcacgtt actctctcca ttcgaatacg acgcttcaat tatgatgaca 540

gcttctcatt taccgtatac aagaaacgga ctcaagttct ttaccaagag aggaggatta 600

acgtctcctg aagtggagaa gatatgcgat ttagctgcgc gaaagtacgc tactaggcag 660

actaaagtct ctacattgat cagaacgcga ccgcagcaag ttgattttat gagcgcttac 720

tctaagcacc ttagagaaat cattaaagag agaatcaatc accctgaaca ctatgacact 780

cctctcaaag gatttcagat agttgtgaat gcgggtaatg ggtcaggagg cttctttacg 840

tgggacgttc tagacaagtt aggagccgat acattcggtt cgctctatct aaaccctgac 900

gggatgttcc ctaatcacat tcctaatccg gaaaacaaaa tcgcaatgca acacacccga 960

gccgcggttc ttgagaactc agcagatctc ggggttgtgt ttgatacgga tgttgacagg 1020

agtggagtgg tggataacaa aggaaatcct atcaacggag ataagcttat tgcgcttatg 1080

tcagctatag tgcttaaaga acatccagga agtacagtag tgactgacgc aagaacgagt 1140

atggggctaa ctaggtttat aacggagcga ggagggaggc attgtttgta tagagtaggg 1200

tatagaaacg tgattgacaa gggagtagag ctgaacaaag acggcatcga gactcatctc 1260

atgatggaaa cttcaggaca tggtgcggtt aaggagaatc acttcttgga tgatggtgca 1320

tacatggtgg tgaagatcat aattgaaatg gtgagaatga gactcgcggg atcaaatgaa 1380

ggtatcggta gtttgatcga agatcttgag gagccgttag aagcggttga gcttcggttg 1440

aatattttat cagagccaag agatgccaaa gcaaaaggca ttgaagccat tgagactttc 1500

aggcaataca ttgaggaagg aaaactgaaa gggtgggaat tgggcacgtg tggggattgt 1560

tgggttactg aaggttgctt ggtggactca aatgatcatc catctgctat tgatgctcac 1620

atgtacaggg caagagtgag tgatgaagag agtggggaag agtatggttg ggtgcatatg 1680

aggcagagta ttcataaccc taacatcgca cttaatatgc aatcaatgct tcctggtgga 1740

tgtctctcca tgacaagaat cttcagagac cagtttcttg aagctagtgg ggtggctaga 1800

ttcctggata taagtgactt cgacaattac atcggaggtc aatctctcga gcaccaccac 1860

caccaccact ga 1872

<210> 4

<211>623

<212> PRT

<213> MASTSTSSLMASKTVISKTALFSSLPGIVSRSFLTFAPASSPVKPLRIRSSNVTKFDEVTNSLDEDMDQIRRLQNGSDVRGVALEGEKGRTVDLTPAAVEAIAESFGEWVAATESNGNGVIKISLGRDPRVSGGKLSTAVFAGLARAGCLAFDMGLATAP

<220>

<221> PRT

<222> (1)..(623)

<400> 4

Met Ala Ser Thr Ser Thr Ser Ser Leu Met Ala Ser Lys Thr Val Ile

1 5 10 15

Ser Lys Thr Ala Leu Phe Ser Ser Leu Pro Gly Ile Val Ser Arg Ser

20 25 30

Phe Leu Thr Phe Ala Pro Ala Ser Pro Ser Val Lys Pro Leu Arg Ile

35 40 45

Arg Ser Ser Asn Val Thr Lys Phe Asp Glu Val Thr Asn Ser Leu Asp

50 55 60

Glu Asp Met Asp Gln Ile Arg Arg Leu Gln Asn Gly Ser Asp Val Arg

65 70 75 80

Gly Val Ala Leu Glu Gly Glu Lys Gly Arg Thr Val Asp Leu Thr Pro

85 90 95

Ala Ala Val Glu Ala Ile Ala Glu Ser Phe Gly Glu Trp Val Ala Ala

100 105 110

Thr Glu Ser Asn Gly Asn Gly Val Ile Lys Ile Ser Leu Gly Arg Asp

115 120 125

Pro Arg Val Ser Gly Gly Lys Leu Ser Thr Ala Val Phe Ala Gly Leu

130 135 140

Ala Arg Ala Gly Cys Leu Ala Phe Asp Met Gly Leu Ala Thr Ala Pro

145 150 155 160

Ala Cys Phe Met Ser Thr Leu Leu Ser Pro Phe Glu Tyr Asp Ala Ser

165 170 175

Ile Met Met Thr Ala Ser His Leu Pro Tyr Thr Arg Asn Gly Leu Lys

180 185 190

Phe Phe Thr Lys Arg Gly Gly Leu Thr Ser Pro Glu Val Glu Lys Ile

195 200 205

Cys Asp Leu Ala Ala Arg Lys Tyr Ala Thr Arg Gln Thr Lys Val Ser

210 215 220

Thr Leu Ile Arg Thr Arg Pro Gln Gln Val Asp Phe Met Ser Ala Tyr

225 230 235 240

Ser Lys His Leu Arg Glu Ile Ile Lys Glu Arg Ile Asn His Pro Glu

245 250 255

His Tyr Asp Thr Pro Leu Lys Gly Phe Gln Ile Val Val Asn Ala Gly

260 265 270

Asn Gly Ser Gly Gly Phe Phe Thr Trp Asp Val Leu Asp Lys Leu Gly

275 280 285

Ala Asp Thr Phe Gly Ser Leu Tyr Leu Asn Pro Asp Gly Met Phe Pro

290 295 300

Asn His Ile Pro Asn Pro Glu Asn Lys Ile Ala Met Gln His Thr Arg

305 310 315 320

Ala Ala Val Leu Glu Asn Ser Ala Asp Leu Gly Val Val Phe Asp Thr

325 330 335

Asp Val Asp Arg Ser Gly Val Val Asp Asn Lys Gly Asn Pro Ile Asn

340 345 350

Gly Asp Lys Leu Ile Ala Leu Met Ser Ala Ile Val Leu Lys Glu His

355 360 365

Pro Gly Ser Thr Val Val Thr Asp Ala Arg Thr Ser Met Gly Leu Thr

370 375 380

Arg Phe Ile Thr Glu Arg Gly Gly Arg His Cys Leu Tyr Arg Val Gly

385 390 395 400

Tyr Arg Asn Val Ile Asp Lys Gly Val Glu Leu Asn Lys Asp Gly Ile

405 410 415

Glu Thr His Leu Met Met Glu Thr Ser Gly His Gly Ala Val Lys Glu

420 425 430

Asn His Phe Leu Asp Asp Gly Ala Tyr Met Val Val Lys Ile Ile Ile

435 440 445

Glu Met Val Arg Met Arg Leu Ala Gly Ser Asn Glu Gly Ile Gly Ser

450 455 460

Leu Ile Glu Asp Leu Glu Glu Pro Leu Glu Ala Val Glu Leu Arg Leu

465 470 475 480

Asn Ile Leu Ser Glu Pro Arg Asp Ala Lys Ala Lys Gly Ile Glu Ala

485 490 495

Ile Glu Thr Phe Arg Gln Tyr Ile Glu Glu Gly Lys Leu Lys Gly Trp

500 505 510

Glu Leu Gly Thr Cys Gly Asp Cys Trp Val Thr Glu Gly Cys Leu Val

515 520 525

Asp Ser Asn Asp His Pro Ser Ala Ile Asp Ala His Met Tyr Arg Ala

530 535 540

Arg Val Ser Asp Glu Glu Ser Gly Glu Glu Tyr Gly Trp Val His Met

545 550 555 560

Arg Gln Ser Ile His Asn Pro Asn Ile Ala Leu Asn Met Gln Ser Met

565 570 575

Leu Pro Gly Gly Cys Leu Ser Met Thr Arg Ile Phe Arg Asp Gln Phe

580 585 590

Leu Glu Ala Ser Gly Val Ala Arg Phe Leu Asp Ile Ser Asp Phe Asp

595 600 605

Asn Tyr Ile Gly Gly Gln Ser Leu Glu His His His His His His His His

610 615 620

<210> 5

<211> 30

<212>DNA

<213> Synthetic

<220>

<221> DNA

<222> (1)..(30)

<400> 5

gcgtccatgg aaggaaaggt tttccaaaac 30

<210> 6

<211> 27

<212>DNA

<213> Synthetic

<220>

<221> DNA

<222> (1)..(27)

<400> 6

atatatgcgg ccgcggagga gtgagtg 27

<210> 7

<211> 29

<212>DNA

<213> Synthetic

<220>

<221>DNA

<222> (1)..(29)

<400> 7

aactccatgg cgtcgacttc aacatcatc 29

<210> 8

<211> 29

<212>DNA

<213> Synthetic

<220>

<221> DNA

<222> (1)..(29)

<400> 8

actgctcgag agattgacct ccgatgtaa 29

Claims (8)

1. A gene encoding acetylglucosamine phosphate mutase AtAGM2, its nucleotide sequence has one or more than two of the following characteristics: 1) have the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing; 2) a deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2; 3) A nucleotide encoding acetylglucosamine phosphate mutase activity obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence listing sequence. 2. A gene encoding acetylglucosamine phosphate mutase AtAGM3, the nucleotide sequence of which has one or more than two of the following characteristics: 1) have the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.3 in the sequence listing; 2) a deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.4; 3) The nucleotide encoding the acetylglucosamine phosphate mutase activity obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.3 in the sequence listing sequence. 3. the acetylglucosamine phosphate mutase of the coding gene coding of a kind of acetylglucosamine phosphate mutase AtAGM2 described in claim 1, it is characterized in that: its amino acid sequence has one or two kinds in the following characteristics: 1) The sequence of amino acid residues 1-625 starting from the amino terminal of SEQ ID NO.2 in the sequence listing; 2) An amino acid sequence having acetylglucosamine phosphomutase activity formed by performing one or more than two amino acid substitutions, deletions or additions to the amino acid sequence shown in SEQ ID NO.2 in the sequence listing. 4. the acetylglucosamine phosphate mutase of the coding gene coded of a kind of acetylglucosamine phosphate mutase AtAGM3 described in claim 2, it is characterized in that: its amino acid sequence has one or two kinds in the following characteristics: 1) The amino acid residue sequence at positions 1-623 of SEQ ID NO.4 starting from the amino terminus in the sequence listing; 2) An amino acid sequence having acetylglucosamine phosphomutase activity formed by performing one or more than two amino acid substitutions, deletions or additions to the amino acid sequence shown in SEQ ID NO.4 in the sequence listing. 5. an application of the acetylglucosamine phosphate mutase described in claim 3 or 4, is characterized in that: this enzyme can be to acetylglucosamine-6-phosphate (GlcNAc-6-P), acetylglucosamine-1 -phosphate (GlcNAc-1-P), glucosamine-6-phosphate (GlcN-6-P), glucosamine-1-phosphate (GlcN-1-P) and glucose-6-phosphate (Glc-6-P) , Glucose-1-phosphate (Glc-1-P) and one or more hexose phosphates have mutagenic activity. 6. A preparation method of acetylglucosamine phosphate mutase described in claims 3 and 4, characterized in that: the coding gene of acetylglucosamine phosphate mutase AtAGM2 or AtAGM3 is cloned into a recombinant expression vector, and introduced into the host cells, obtaining recombinantly expressed acetylglucosamine phosphomutase; The nucleotide sequence of the gene encoding the above-mentioned acetylglucosamine phosphate mutase AtAGM has one or more than two of the following characteristics: 1) have the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 or SEQ ID NO.3 in the sequence listing, 2) a deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 or SEQ ID NO.4, 3) The code obtained by performing one or more than two nucleotide substitutions, deletions or additions to the deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 or SEQ ID NO.3 in the sequence listing has acetylglucosamine phosphate shift Enzymatically active nucleotide sequence; The expression vector for the recombinant expression of acetylglucosamine phosphate mutase refers to an E. coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, One or more of plant expression vectors, insect expression vectors or mammalian cell expression vectors. 7. according to the described method of claim 6, it is characterized in that: the recombinant bacterium or transgenic cell line that is used for recombinant expression acetylglucosamine phosphate mutase refers to Escherichia coli host cell Escherichia coli BL21, Escherichia coliJM109, Escherichia coli DH5α, Yeast host cells Saccharomyces cerevisiae, Pichiapastoris, Kluyveromyces lactis, Bacillus subtilis host cells Bacillus subtilis R25, Bacillus subtilis 9920, lactic acid bacteria host cells Lactic acid bacteria COCC101, actinomycete host cells Streptomyces spp, filamentous fungal host cells Trichoderma viride, Trichoderma rees Aspergillus niger, Aspergillus nidulans, insect cells Bombyx mori, Antharaeaeucalypti, mammalian cells Chinese hamster ovary cells CHO, young hamster kidney cells BHK, Chinese hamster lung cells CHL one or more. 8. An application of the acetylglucosamine phosphate mutase described in claim 3 or 4 in the production of hexose phosphate and nucleotide sugar, characterized in that it includes one or more than two of the following applications: 1) Application in realizing the transformation of the 1,6-position isomer of hexose phosphate and producing the corresponding isomer; 2) Application in the production of nucleotide sugars such as UDP-GlcNAc (UDP-GlcN; UDP-Glc).