Platycodon grandiflorum phosphomannose isomerase gene PgPMI as well as encoded product and application thereof

Abstract

The invention discloses a platycodon grandiflorum phosphomannose isomerase gene PgPMI as well as an encoded product and application thereof, and belongs to the technical field of platycodon grandiflorum genes. According to the invention, the platycodon grandiflorum phosphomannose isomerase gene PgPMI is cloned from platycodon grandiflorum, and the nucleotide sequence of the platycodon grandiflorum phosphomannose isomerase gene PgPMI is shown as SEQ ID NO. 1. According to the invention, the expression of the PgPMI gene in escherichia coli is successfully realized, and an enzymatic experiment verifies that the PgPMI has catalytic activity, so that mannose-6-phosphoric acid and fructose-6-phosphoric acid can be mutually converted, and a foundation is laid for revealing biosynthesis of platycodon grandiflorum polysaccharide. In addition, the platycodon grandiflorum phosphomannose isomerase can also be applied to biosynthesis pathways of plant ascorbic acid, cell walls, glycosylated proteins, lipoproteins, glycosyl phosphate inositol ankyrin and the like.

Description

Technical Field

[0001] This invention belongs to the field of Platycodon grandiflorum gene technology, and specifically relates to a Platycodon grandiflorum mannose phosphate isomerase gene PgPMI and its encoded products and applications. Background Technology

[0002] Platycodon grandiflorum (Jacq.) A.DC., a plant in the Campanulaceae family, is the dried root of the plant. It is primarily used to treat cough with excessive phlegm, chest tightness, sore throat, hoarseness, and lung abscess with purulent sputum. The Compendium of Materia Medica explains its name as follows: "The root of this herb is firm and straight, hence the name Platycodon grandiflorum." Platycodon grandiflorum is a commonly used and widely distributed medicinal herb, belonging to the category of "food and medicine of the same origin," and has significant development value in other fields of medicine. Studies have found that Platycodon grandiflorum contains triterpenoid saponins, polysaccharides, flavonoids, phenols, polyacetylenes, fatty acids, and trace elements. The root of Platycodon grandiflorum is used medicinally, and its main active ingredients include saponins, polysaccharides, and flavonoids, with polysaccharides being one of its effective components.

[0003] In polysaccharide biosynthesis, the biosynthesis of sucrose into GDP-mannose and GDP-fucose begins with SUS (sucrose synthase) catalyzing the conversion of sucrose to fructose, followed by HK (hexokinase) converting it to fructose-6-phosphate. Then, PMI (phosphogannase isomerase) catalyzes the conversion to mannose-6-phosphate, which is then converted to mannose-1-phosphate by PMM (phosphogannase mutant enzyme). Finally, GMPP (GDP-mannose pyrophosphorylase) forms GDP-mannose. PMI, as an important enzyme in polysaccharide biosynthesis, can catalyze the conversion of fructose-6-phosphate to mannose-6-phosphate. However, the platycodon phosphogannase isomerase has not been reported, and its gene has not yet been isolated and identified. Summary of the Invention

[0004] To address the aforementioned problems, in a first aspect, the present invention proposes a platycodon phosphate mannose isomerase gene PgPMI, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0005] Secondly, the present invention proposes a product encoded by the aforementioned Platycodon grandiflorum phosphate mannose isomerase gene PgPMI, wherein the product is a polypeptide or a protein.

[0006] Furthermore, the amino acid sequence of the product is shown in SEQ ID NO.2.

[0007] Thirdly, this invention proposes a recombinant expression vector containing the aforementioned Platycodon grandiflorum phosphate-mannose isomerase gene PgPMI. Specifically, the PgPMI gene can be linked to an expression vector that can guide the expression of exogenous genes in plants to prepare a recombinant expression vector for the PMI gene. When constructing the expression vector, any strong or inducible promoter can be added before the transcription initiation nucleotide, and it must be identical to the reading frame of the coding sequence to ensure the translation of the entire sequence. To facilitate the identification and screening of transgenic plant cells or plants, the expression vector can be modified during its construction, for example, by adding selectable markers. Commonly used markers include genes for antibiotic resistance enzymes and biosafety markers, as well as genes for color-changing enzymes or luminescent compounds such as GUS and GFP. Further, the recombinant expression vector is plasmid pET-30a.

[0008] Fourthly, the present invention proposes a specific primer pair for constructing the recombinant expression vector, wherein the nucleotide sequence of the upstream primer of the specific primer pair is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.6.

[0009] Fifthly, the present invention proposes recombinant engineered bacteria containing the aforementioned mannose phosphate isomerase gene PgPMI or containing the aforementioned recombinant expression vector.

[0010] In a sixth aspect, the present invention proposes a host cell containing the aforementioned platycodon phosphate mannose isomerase gene PgPMI or containing the aforementioned recombinant expression vector.

[0011] Furthermore, the host cells include E. coli TransBL21(DE3) cells.

[0012] In a seventh aspect, the present invention proposes the application of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI, the recombinant expression vector, the recombinant engineered bacteria, or the host cell in the preparation of polysaccharide compounds.

[0013] Furthermore, the preparation of polysaccharide compounds includes the preparation of mannose-6-phosphate or fructose-6-phosphate.

[0014] The beneficial effects of this invention are:

[0015] This invention cloned a mannose-phosphoamyl isomerase gene, PgPMI, from Platycodon grandiflorus and successfully expressed it in Escherichia coli. Enzymatic experiments verified that PgPMI possesses catalytic activity, enabling the interconversion of mannose-6-phosphate and fructose-6-phosphate, laying the foundation for elucidating the biosynthesis of Platycodon grandiflorus polysaccharides. Furthermore, this enzyme can also be applied to the biosynthetic pathways of plant ascorbic acid, cell wall, glycosylated proteins, lipoproteins, and glycosylphosphoinositol ankylosing proteins.

[0016] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures pointed out in the description, claims and drawings. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This shows an agarose gel electrophoresis image of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI in an embodiment of the present invention;

[0019] Figure 2 This diagram shows the predicted functional domain analysis of the PgPMI structure of the Platycodon grandiflorum phosphate isomerase gene in an embodiment of the present invention.

[0020] Figure 3 The diagram shows the secondary structure analysis results of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI in an embodiment of the present invention;

[0021] Figure 4 This invention illustrates a predicted phosphorylation site map of the PgPMI phosphorylation site in the Platycodon grandiflorum phosphate isomerase gene, as shown in an embodiment of the invention.

[0022] Figure 5 The diagram shows the tertiary structure of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI in an embodiment of the present invention;

[0023] Figure 6 The phylogenetic tree of the platycodon phosphate mannose isomerase gene PgPMI in an embodiment of the present invention is shown;

[0024] Figure 7 The following is an example of the SDS-polyacrylamide gel electrophoresis results of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI protein in this invention embodiment.

[0025] Figure 8A The UPLC-ELSD analysis chromatogram of fructose-6-phosphate reference standard in an embodiment of the present invention is shown.

[0026] Figure 8B The UPLC-ELSD analysis chromatogram of the blank control (mannose-6-phosphate as the reaction substrate) in the embodiments of the present invention is shown.

[0027] Figure 8C The UPLC-ELSD analysis diagram of the enzymatic product of the reverse reaction of PgPMI recombinant protein in an embodiment of the present invention is shown.

[0028] Figure 8D The UPLC-ELSD chromatogram of the mannose-6-phosphate reference standard in an embodiment of the present invention is shown.

[0029] Figure 8E The UPLC-ELSD analysis chromatogram of the blank control (fructose-6-phosphate as the reaction substrate) in the embodiments of the present invention is shown.

[0030] Figure 8F The UPLC-ELSD analysis diagram of the enzymatic product of the forward reaction of PgPMI recombinant protein in an embodiment of the present invention is shown. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Rapid Universal Plant RNA Extraction Kit was purchased from Beijing Huayueyang Biotechnology Co., Ltd.; PrimeScript Reverse Transcription Kit TMThe 1st Strand cDNA Synthesis Kit was purchased from Takara Bio; the plasmid extraction kit EasyPureHiPurePlasmidMiniPrepKit was purchased from Beijing TransGen Biotech Co., Ltd.; the non-toxic 4S GreenPlus nucleic acid dye was purchased from Shanghai Sangon Biotech Co., Ltd.; the gel extraction kit EasyPureQuickGelExtractionKit, the T vector pEASY-BluntZeroCloningKit, and E. coli Transl-T1 were purchased from Beijing TransGen Biotech Co., Ltd.; high-fidelity enzymes such as Phusion and BamHI restriction endonucleases were purchased from NEB Biotechnology (Beijing) Co., Ltd.; primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; other reagents were imported or domestically produced analytical grade reagents. Unless otherwise specified, the techniques used in the examples are conventional methods well known to those skilled in the art, and all raw materials used are commercially available products.

[0033] Example 1

[0034] Cloning of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI: Using forward primers, the primer sequences are shown in Table 1.

[0035] Table 1

[0036] Primer name sequence name Base sequence (5'→3') upstream primer P1 SEQ ID NO.3 ATGGGGCTCAACTCAGGGGCTGAGA Downstream primer P2 SEQ ID NO.4 TCAATGGTCCAAGAAAAACCTGGTG

[0037] PCR amplification was performed using the full-length sequence of the PgPMI gene encoding platycodon phosphate isomerase as a template. The PCR reaction system (50 μL) consisted of: 1 μL template cDNA, 2.5 μL each of upstream primer P1 and downstream primer P2, 25 μL high-fidelity enzyme Phusion, and the remaining reaction volume was made up with sterile double-distilled water. PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, followed by a final extension at 72℃ for 5 min, and storage at 4℃.

[0038] The amplification products were subjected to agarose gel electrophoresis, and the agarose gel electrophoresis image of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI is shown below. Figure 1 As shown, the size of the PCR amplification product was determined to be between 1000bp and 2000bp, which is consistent with the length of the target gene. Thus, the clone of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI was obtained.

[0039] Example 2

[0040] Bioinformatics analysis of the PgPMI gene: The full-length cDNA of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI obtained in this invention has an open reading frame (ORF) length of 1203 bp, and the detailed sequence is shown in SEQ ID NO.1 in the sequence listing. The PgPMI gene sequence was subjected to nucleotide homology searches in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDStranslation+PDB+Swissprot+Superdate+PIR databases using the BLAST program in the NCBI database. The amino acid sequence of the PgPMI gene is shown in SEQ ID NO.2. The PgPMI gene shows high homology at the amino acid level with GPIs from other species and also possesses a typical PMI domain, such as... Figure 2 As shown; protein secondary structure analysis using online software NPS revealed that the secondary structure of the PgP MI protein consists of α-helices, extended chains, β-turn amino acids, and random coils. Figure 3 Phosphorylation sites of the PgPMI protein were predicted using the online software NetPhos, revealing three phosphorylation sites: serine, threonine, and tyrosine phosphate sites. Figure 4 The tertiary structure of the PgPMI protein was predicted using the online software SwissModel. Figure 5 A Neighbor-joining phylogenetic tree was constructed using the MEGA6 software, with the bootstrap iteration repeated 1000 times. Figure 6 As shown, PgPMI is located at the same branch point as glutinous rice, tomato, and wolfberry, indicating a high degree of kinship.

[0041] Example 3

[0042] Construction of the prokaryotic expression vector for the PgPMI gene: Using the cDNA of the PgPMI gene as a template, BamHI was selected as the single restriction enzyme site. Specific upstream primer P3 and downstream primer P4 were designed (as shown in Table 2) and PCR amplification was performed. The underlined parts in the primers are the restriction enzyme sites.

[0043] Table 2

[0044] Primer name sequence name Base sequence (5'→3') upstream primer P3 SEQ ID NO.5 AGGCCATGGCTGATATCGGAATGGGGCTCAACTCAGGGGCTGAGA Downstream primer P4 SEQ ID NO.6 CGACGGAGCTCGAATTCGGATCAATGGTCCAAGAAAAACCTGGTG

[0045] The PCR reaction system (50 μL) consisted of: 1 μL template cDNA, 2.5 μL each of upstream primer P3 and downstream primer P4, 25 μL high-fidelity enzyme Phusion, and the remaining reaction volume was made up with sterile double-distilled water. PCR reaction conditions were: 98℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles followed by 72℃ extension for 5 min, and storage at 4℃.

[0046] The amplified product was detected by 1% agarose gel electrophoresis (1 g agarose per 100 mL gel solution), and the gel was then excised and recovered. The expression vector pET-30a plasmid was digested with BamHI, and the excised and recovered gel was then performed. The recovered target fragment was ligated to the expression vector pET-30a using a seamless splicing kit at 50°C for 50 min. The ligation product was transformed into *E. coli* Trans1-T1 competent cells. Single clones were picked for colony PCR testing, sequencing, and plasmid extraction. The recombinant plasmid pET-PgPMI with the correct target sequence was preserved for expression transformation.

[0047] Example 4

[0048] Induction of recombinant engineered bacterial expression: *E. coli* Trans-BL21(DE3) competent cells were transformed with the target plasmid pET-PgPMI, and positive strains containing the pET-PgPMI plasmid, *E. coli* Trans-BL21(DE3)-PgPMI, were cultured and screened. This strain contains a highly efficient expression system capable of inducing the PgPMI recombinant gene. Positive clones *E. coli* Trans-BL21(DE3)-PgPMI were inoculated at a volume ratio of 1:100 in LB medium containing Amp resistance and cultured at 37°C with shaking at 200 rpm until A600 = 0.4–0.6 (i.e., optical density at 600 nm). IPTG (isopropyl-β-D-thiogalactoside) was added to the appropriate final concentration and induced overnight in a shaker at a suitable temperature. The pET-30a empty vector was treated under the same conditions as a blank control. Centrifuge 1 mL of bacterial culture to obtain the precipitate as whole bacteria. Centrifuge the remaining bacterial culture to remove the supernatant and obtain bacterial cells. Add 3-5 mL of Buffer A, resuspend, and transfer to a 15 mL centrifuge tube. Place the tube in an ultrasonic homogenizer and sonicate for 5 min (5 s intervals) at 25% efficiency. Insert the centrifuge tube into a beaker containing ice and operate on ice. Centrifuge the sonicated lysate at 4°C for 15 min to obtain PgPMI supernatant and precipitate. Perform 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polyacrylamide accounting for 12% of the total volume) electrophoresis analysis. The electrophoresis results are as follows (e.g., Figure 7At a molecular weight of approximately 52 kDa, a distinct specific protein expression band appeared, consistent with the theoretical value. Lane 1 (M) was the marker; lane 2 (A1) was the empty sample; lane 3 (A2) was the uninduced sample; lane 4 (A3) was the induced whole bacterial sample; lane 5 (A4) was the induced supernatant protein sample; lane 6 (A5) was the induced precipitate sample; and lanes 7-10 (A6-A9) were purified samples eluted at 20 mM, 100 mM, 300 mM, and 500 mM Hirsch Bufffer A, respectively.

[0049] Example 5

[0050] In vitro enzyme function verification:

[0051] 1. Preparation of purified protein: Take 1 mL of the supernatant of PgPMI obtained in Example 4 and mix it with 500 μL of washed Ni-NTAResin (His-tagged purification resin) in a 2 mL centrifuge tube; place it in an ice box and shake it at 120 rpm for 90 min on a fixed-track shaker; after the end, centrifuge at 500×g for 5 min at 4 °C and discard the supernatant; add 1 mL of Buffer A to resuspend, centrifuge at 500×g for 5 min at 4 °C and discard the supernatant, repeat three times; add 200 μL of Buffer B of different concentrations to elute, centrifuge at 500×g for 5 min at 4 °C and store in a -80 °C freezer.

[0052] 2. PgPMI enzyme function verification: In vitro enzyme function was verified using mannose-6-phosphate / fructose-6-phosphate as substrates. The reaction system consisted of 200 μL of reaction mixture, 80 mM Borate Buffer (pH 9.0), 100 mM mannose-6-phosphate (reverse substrate), 100 mM fructose-6-phosphate (forward substrate), 0.5 mM MgCl2, and pET-30a-PgPMI protein to make up the remaining volume. The mixture was thoroughly mixed, incubated overnight at 40°C, and then dried using a nitrogen evaporator. The pET-30a-PgPMI protease-catalyzed product sample was obtained by reconstitution with 200 μL ddH2O and filtered through a 0.22 μm microporous membrane. The pET-30a empty vector was treated under the same conditions as a blank control sample. 1 mg of mannose-6-phosphate and fructose-6-phosphate were accurately weighed as standards, dissolved in 1 mL ddH2O, and filtered through a 0.22 μm microporous membrane. These were used as standard samples and analyzed by GC-MS.

[0053] GC-MS analysis conditions were as follows: column temperature: 60℃; injection volume: 5 μL; mobile phase A: acetonitrile, mobile phase B: water; flow rate: 0.25 mL / min. Gradient elution conditions: 0–3 min, 10% B; 3–8 min, 10%–20% B; 8–15 min, 20% B; 15–18 min, 20%–10% B (all percentages of mobile phase are volume percentages). Nebulizer mode: cooling; drift tube temperature: 55℃; gas pressure: 207 kPa (30 psi).

[0054] Qualitative analysis of the product revealed that, using mannose-6-phosphate as a substrate, the product fructose-6-phosphate standard showed a chromatographic peak at approximately 13.48 min. Figure 8A The PgPMI recombinant protease-catalyzed product showed a corresponding chromatographic peak at 13.53 min. Figure 8C Furthermore, the blank control group pET-30a showed no chromatographic peaks at or near the 13.5 min position. Figure 8B When fructose-6-phosphate was used as a substrate, the product mannose-6-phosphate standard showed a chromatographic peak at approximately 9.164 min. Figure 8D The PgPMI recombinant protease-catalyzed product showed a corresponding chromatographic peak at 9.141 min. Figure 8F Furthermore, the blank control group pET-30a showed no chromatographic peaks at or near the 9.1 min position. Figure 8E Therefore, the experimental results show that the target product was present in the products of both groups of enzymatic reactions, and the PgPMI recombinant protein has catalytic activity.

[0055] The SEQ ID NO.1 and SEQ ID NO.2 mentioned in Example 2 are as follows:

[0056] SEQ ID NO.1:

[0057] ATGGGGCTCAACTCAGGGGCTGAGATTGACGAGACTAAGCCTTATGCTGAGTTCTGGATG

[0058] GGAACACATGATTCCGGACCCTCGTTCATTGTGGAAAGCTTTGAGAATGGTGGGCCGCCA

[0059] GCAACTACTGGCTCGGGGTCGGTTAAGGTTAGCTTAAAGTCTTGGATTGCTCGAAACCCC

[0060] AAGGTTTTAGGCGATAAGGTTGTCGAGAAATGGGGTAATAACCTCCCTTTCTTGTTCAAG

[0061] GTGCTTTCAGTTGCAAAAGCGTTGTCGATACAGGCACACCCAGATAAGAAATTGGCCGAG

[0062] GTTCTCCATAAGTTGGAACCAAATGTCTATAAAGATGATAATCACAAGCCTGAGATGGTA

[0063] TTGGCGTTGACTGAGTTTGAGGCCTTGTGTGGCTTCATTAGTTTTAAGGAACTTAAGGGT

[0064] GTTCTTCAAGATGTTCCTGAGATTGAGGAAGTGGTTGGCGCTGCATGTGCAAACCAAGTG

[0065] TTACATGTTGATGAGCAAGATGATAAGGATAAAGTAAAGGCAGTTTTGCGGTTACTATTC

[0066] ACCCAACTCATGGCAGCTAGCAAGGAGGTGGTTTCCGAAGTGTTATCAAAACTGATTAGT

[0067] CGTCTAAACATGGAAAGCAAGGCAAGGCAGTTAACTGTTAAGGAAGAACTAGCCCTGCGA

[0068] CTGGAAAAGCAGTATCCAGATGATGTTGGGGTCATAGCAGCCTTCCTCTTTAACTATGTG

[0069] AAGCTAAATCCTGGTGAAGCTTTGTTTCTAGGGGCAAATGAACTTCATGCATATTTGTTT

[0070] GGCGAGTGTATCGAGTGTATGGCTACTTCAGACAATGTAGTTCGAGCTGGCCTTACTCCA

[0071] AAGAACCGGGATGTCAAAATCCTTAGTTCCATGCTCACTTACAAACAGGGATTTCCTGAA

[0072] ATATTGCAAGGGGTACTTTTGAATCCGCATATCCGAAGATACACTCCTCCTTTTGAGGAA

[0073] TTTGAGGTTGATAGTTGTATTCTTGACGAAGGGGCAACAGTTGAGTTCCCTGCATTGGAG

[0074] GGCCCGTCCATTTTTGTGGTTGTAACGGGGGAAGGAGCAATGCATACAACATCATCAGAG

[0075] GATGTTGTTAGTGAAGGTGATGTCTTATTTGTACCTGCGGGAACCGGGATTAGTGTCAGC

[0076] ACGGGATCATATTTGCACTTGTGTAGAGCCGGAGTGAACACCAGGTTTTTCTTGGACCAT

[0077] TGA

[0078] SEQ ID NO.2:

[0079] MGLNSGAEIDETKPYAEFWMGTHDSGPSFIVESFENGGPPATTGSGSVKVSLKSWIARNP

[0080] KVLGDKVVEKWGNNLPFLFKVLSVAKALSIQAHPDKKLAEVLHKLEPNVYKDDNHKPEMV

[0081] LALTEFEALCGFISFKELKGVLQDVPEIEEVVGAACANQVLHVDEQDDKDKVKAVLRLLF

[0082] TQLMAASKEVVSEVLSKLISRLNMESKARQLTVKEELALRLEKQYPDDVGVIAAFLFNYV

[0083] KLNPGEALFLGANELHAYLFGECIECMATSDNVVRAGLTPKNRDVKILSSMLTYKQGFPE

[0084] ILQGVLLNPHIRRYTPPFEEFEVDSCILDEGATVEFPALEGPSIFVVVTGEGAMHTTSSE

[0085] DVVSEGDVLFVPAGTGISVSTGSYLHLCRAGVNTRFFLDH

[0086] Those skilled in the art can, based on the amino acid sequence disclosed in the embodiments of the present invention, substitute, delete, or add one or more amino acids without affecting its activity to obtain a mutant sequence of the protein, and such mutant sequence also falls within the protection scope of the present invention.

[0087] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A platycodon phosphate mannose isomerase gene PgPMI, characterized in that, The nucleotide sequence of the gene PgPMI is shown in SEQ ID NO.

1.

2. A product encoded by the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI as described in claim 1, characterized in that, The product is a polypeptide or a protein.

3. The product encoded by the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI according to claim 2, characterized in that, The amino acid sequence of the product is shown in SEQ ID NO.

2.

4. A recombinant expression vector containing the platycodon phosphate mannose isomerase gene PgPMI as described in claim 1.

5. A specific primer pair for constructing the recombinant expression vector of claim 4, characterized in that, The nucleotide sequence of the upstream primer of the specific primer pair is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

6.

6. Recombinant engineered bacteria containing the platycodon phosphate mannose isomerase gene PgPMI as described in claim 1 or containing the recombinant expression vector as described in claim 4.

7. A host cell, characterized in that, It contains the platycodon phosphate mannose isomerase gene PgPMI as described in claim 1 or the recombinant expression vector as described in claim 4.

8. The host cell according to claim 7, characterized in that, The host cells include E. coli TransBL21(DE3) cells.

9. The application of the Platycodon grandiflorum phosphate mannose isomerase gene PgPMI as described in claim 1, the recombinant expression vector as described in claim 4, the recombinant engineered bacteria as described in claim 6, or the host cell as described in claim 7 or 8 in the preparation of polysaccharide compounds.

10. The application according to claim 9, characterized in that, The preparation of polysaccharide compounds includes the preparation of mannose-6-phosphate or fructose-6-phosphate.

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