Huoxuandan chalcone synthase gene GlCHS and the product and application thereof

Abstract

The application discloses a blood-activating herb naringenin synthase gene GlCHS, a product coded by the gene and application thereof, and belongs to the field of genetic engineering. The blood-activating herb naringenin synthase gene GlCHS is cloned from a blood-activating herb, the nucleotide sequence of the gene is shown as SEQ ID No. 1, and the amino acid sequence of the product coded by the gene is shown as SEQ ID No. 2. The blood-activating herb naringenin synthase is expressed heterogeneously in a host cell, and the enzyme has the function of catalyzing naringenin to be generated by taking p-coumaroyl coenzyme A and malonyl coenzyme A as substrates. By using the application, the content of naringenin in the blood-activating herb can be improved and naringenin can be synthesized in vitro in a medicinal plant by using genetic engineering technology.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a Glechoma longituba chalcone synthase gene GlCHS, its encoded product and application. Background Art

[0002] Glechoma longituba (Nakai) Kupr. is a perennial herb of the genus Glechoma in the family Lamiaceae. In the 2020 edition of the Chinese Pharmacopoeia, the dried above-ground part of Glechoma longituba is used as medicine and called Lysimachiae Herba, which has the effects of promoting diuresis and relieving stranguria, clearing heat and detoxifying, removing stasis and detumescence. As a commonly used traditional Chinese medicine, Lysimachiae Herba has various pharmacological effects such as diuretic, cholagogic, lipid-lowering, litholytic, anti-inflammatory and antibacterial. Its whole herb or stems and leaves are used to treat symptoms such as heat strangury, stone strangury, damp-heat jaundice, sores and carbuncles, swelling and pain, and traumatic injuries. Clinically, compound preparations containing Lysimachiae Herba are often used, such as Paishi Keli, Jinqian Dantong Keli, Niaoganning Keli, Qingre Lidan Keli, Huoluo Zhitong Pills, Tianqi Dieda Fengshi Ointment, etc., for the treatment of various diseases such as cholelithiasis, urolithiasis, urinary tract infection, gout, arthritis, etc. caused by damp-heat.

[0003] The whole plant of Glechoma longituba contains rich secondary metabolites, including flavonoids, phenolic acids, terpenoids, lignins and other compounds. Flavonoid compounds are an important class of secondary metabolites in Glechoma longituba, with high economic value and medical value. The biosynthesis of flavonoid substances originates from the phenylpropanoid biosynthetic pathway. Phenylalanine is catalyzed by phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), and p-coumaryl-CoA ligase (4CL) to generate p-coumaryl-CoA, and then enters the flavonoid biosynthetic pathway. P-coumaryl-CoA is catalyzed by chalcone synthase (CHS) to generate naringenin chalcone, and then naringenin is generated under the catalysis of chalcone isomerase (CHI). Naringenin is the precursor substance of various flavonoid compounds. Chalcone synthase is the first key rate-limiting enzyme in the plant flavonoid biosynthetic pathway, and its function and activity are closely related to the synthesis and accumulation of various flavonoid compounds. However, at present, the chalcone synthase gene in the flavonoid biosynthetic pathway of Glechoma longituba has not been isolated and identified. Summary of the Invention

[0004] In view of the above problems, one of the purposes of the present invention is to provide a Glechoma longituba chalcone synthase gene GlCHS, and the nucleotide sequence of the gene GlCHS is shown in SEQ ID NO.1.

[0005] A second objective of this invention is to propose a product encoded by the *GlCHS* gene of *Dendrobium nobile*, wherein the product comprises amino acids, polypeptides, or proteins.

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

[0007] A third objective of this invention is to provide a recombinant expression vector containing the *GlCHS* gene of *Haloxylon ammodendron*. Further, the target gene in the recombinant expression vector is amplified by PCR using the cDNA of the *GlCHS* gene as a template and SEQ ID No. 5 and SEQ ID No. 6 as primers. The *GlCHS* gene is inserted into the BamHI single-restriction site of the pET-30a expression vector, and then ligated and transformed into *E. coli* to obtain the recombinant expression vector (pET-30a-GlCHS).

[0008] The fourth objective of this invention is to provide a specific primer pair for constructing the recombinant expression vector described above; further, 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 of the specific primer pair is shown in SEQ ID NO.6.

[0009] The fifth objective of this invention is to provide a recombinant engineered bacterium containing the aforementioned *GlCHS* chalcone synthase gene or the aforementioned recombinant expression vector.

[0010] The sixth objective of this invention is to provide a host cell containing the aforementioned chalcone synthase gene GlCHS or the aforementioned recombinant expression vector.

[0011] Furthermore, the host cells include BL21(DE3) cells.

[0012] The seventh objective of this invention is to propose the application of the aforementioned *GlCHS* chalcone synthase gene, the recombinant expression vector, the recombinant engineered bacteria, or the host cell in the preparation of flavonoid compounds.

[0013] Furthermore, the application is in the preparation of naringenin using p-coumaroyl-CoA and malonyl-CoA as substrates.

[0014] The beneficial effects of this invention are:

[0015] This invention marks the first time that the *Glechoma heliotropium* chalcone synthase gene *GlCHS* has been cloned and prepared from the medicinal plant *Glechoma heliotropium*. This gene can be used to prepare naringenin using p-coumaroyl-CoA and malonyl-CoA as substrates. The gene provided by this invention can be used to increase the content of flavonoid components in *Glechoma heliotropium* through genetic engineering. This technology can be used to subsequently produce large quantities of flavonoid compounds in vitro from medicinal plants through genetic engineering, providing an effective method to meet the huge market demand for flavonoid compounds.

[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 The image shown is an agarose gel electrophoresis diagram of the charone synthase gene GlCHS from *Hypericum perforatum* in an embodiment of the present invention.

[0019] Figure 2 This invention illustrates the predictive analysis of the functional domain structure of the GluCHS structure of the chalcone synthase in *Dendrobium nobile* in an embodiment of the present invention.

[0020] Figure 3 This invention illustrates the predictive analysis of the transmembrane domain of ghrelin chalcone synthase GlCHS in an embodiment of the present invention.

[0021] Figure 4 The present invention illustrates the predicted structure analysis of the GlCHS homodimer of the blood-activating pill chalcone synthase in an embodiment of the present invention;

[0022] Figure 5 The present invention illustrates the monomeric structure prediction analysis of the ghrelin synthase GlCHS protein in an embodiment of the present invention;

[0023] Figure 6 The phylogenetic tree of the ghrelin charone synthase GlCHS system in an embodiment of the present invention is shown;

[0024] Figure 7 This shows an SDS-polyacrylamide gel electrophoresis image of the chalcone synthase GlCHS protein from an embodiment of the present invention.

[0025] Figure 8 The chromatograms shown are those of the reactions of pET-30a empty carrier and cloxacillin chalcone synthase GlCHS catalyzed by coumaroyl coenzyme A and malonyl coenzyme A, respectively, in embodiments of the present invention. Detailed Implementation

[0026] 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.

[0027] The techniques used in these embodiments are conventional methods well-known to those skilled in the art, and the raw materials used are all commercially available products. Some of the raw materials are as follows:

[0028] Rapid high-fidelity DNA polymerase FastPfu Fly DNA Polymerase, Quick Gel Extraction Kit, HiPure Plasmid MiniPrep Kit, Seamless Splicing Kit Seamless Cloning and Assembly Kit, Restriction Endonuclease BamHI, protein purification Ni-NTA Resin, cloned competent cells Trans1-T1, expression-competent cells BL21(DE3), protein molecular weight standard Blue II Protein Marker (14-120kDa) was purchased from Beijing TransGen Biotechnology Co., Ltd.

[0029] DNA Marker molecular weight standards (100-2000bp), isopropyl-β-D-thiogalactoside (IPTG), and SDS-PAGE denaturing acrylamide gel rapid preparation kit were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0030] Potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0031] Lithium malonyl-CoA salt was purchased from Shanghai Myriel Biochemical Technology Co., Ltd.

[0032] The standards p-Coumaroyl-CoA and Naringenin were purchased from Sichuan Jingcui Tiancheng Pharmaceutical Technology Co., Ltd.

[0033] All other reagents are either imported or domestically produced analytical grade reagents.

[0034] Example 1

[0035] Cloning of the chalcone synthase gene GlCHS from *Hypericum perforatum*:

[0036] Primers were designed based on the GluCHS sequence from the transcriptome of *Hedyotis diffusa*, and PCR amplification was performed using *Hedyotis diffusa* cDNA as a template. The primer sequences are shown in Table 1.

[0037] Table 1

[0038] Primer name Serial Number Base sequence (5'→3') upstream primer SEQ ID NO.3 ATGGTGACCGTGGAGGAAATCCGCC Downstream primer SEQ ID NO.4 TCAATTAATGTGCGGCACACTGTGC

[0039] The amplification system is as follows: rapid high-fidelity DNA polymerase FastPfu Fly DNAPolymerase 1μL, 25 μL FastPfu Fly Reaction Mix, 1 μL template cDNA, 1 μL each of upstream and downstream primers, and 21 μL sterile water.

[0040] Reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 50 s, 55±5℃ annealing for 1 min, 72℃ extension for 1 min, 30 cycles followed by 72℃ extension for 10 min, and storage at 4℃.

[0041] The clone (amplification product) of the *Hypericum perforatum* chalcone synthase gene GlCHS was obtained according to the above amplification system and conditions. The agarose gel electrophoresis result of the *Hypericum perforatum* chalcone synthase gene GlCHS is shown below. Figure 1 As shown, Figure 1 In the text, M stands for Marker (i.e., DNA Marker, molecular weight standard (100-2000bp)). It can be seen that the size of the target gene GlCHS fragment is about 1100bp, which is consistent with the number of bases in the GlCHS gene sequence.

[0042] Bioinformatics analysis of the charone synthase gene GlCHS from *Hypericum perforatum*:

[0043] The open reading frame (ORF) of the obtained *GlCHS* chalcone synthase gene from *GlCHS* is 1176 bp in length, and its detailed sequence is shown in SEQ ID No. 1. This sequence encodes 391 amino acids, as shown in SEQ ID No. 2. The *GlCHS* chalcone synthase gene sequence was used to perform nucleotide homology searches in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank+CDStranslation+PDB+Swissprot+Superdate+PIR databases using the BLAST program in the NCBI database. This gene shows high homology at the amino acid level with CHS from other species. *GlCHS* lacks a transmembrane domain and is an extramembrane protein, such as... Figure 2 As shown.

[0044] The GLCHS protein contains an active site, a product binding site, and a malony-CoA binding site, and belongs to the chalcone synthase superfamily. Figure 3 As shown.

[0045] Homology modeling of the Arabidopsis thaliana CHS protein was performed using the crystal structure (PDB ID: 6dxb.1.A) as a model. The GlCHS sequence showed 85.49% sequence identity with the template protein. The GlCHS protein model was a homodimeric structure, as shown below. Figure 4 As shown, the monomer structure is as follows Figure 5 As shown.

[0046] A Neighbor-Joining phylogenetic tree was constructed using MEGA 6.0 software and the neighbor-joining method based on the GLCHS amino acid sequences, such as... Figure 6 As shown, the amino acid sequence of *GlCHS* from *Huoxuedan* and the amino acid sequence of *Agastache rugosa* chalcone synthase are located at the same branch point, indicating that they are most closely related.

[0047] Example 2

[0048] Construction of a recombinant expression vector system for the chalcone synthase gene GluCHS from *Hypericum perforatum*:

[0049] Using the nucleotide sequence of the cloned chalcone synthase gene GlCHS as a template, restriction endonucleases were selected. Using BamHI (hereinafter referred to as BamHI enzyme) as the restriction site, upstream and downstream primers with restriction sites were designed (as shown in Table 2) for PCR amplification. The underlined parts in the primers represent the base sequences on the vector.

[0050] Table 2. Base sequences of specific upstream and downstream primers.

[0051] Primer name Serial Number Base sequence (5'-3') GlCHS_BamHI_F SEQ ID NO.5 AGGCCATGGCTGATATCGGAATGGTGACCGTGGAGGAAATCCGCC GlCHS_BamHI_R SEQ ID NO.6 CGACGGAGCTCGAATTCGGATCAATTAATGTGCGGCACACTGTGC

[0052] The *GlCHS* gene of *Hypericum perforatum* and the pET30a vector were digested with BamHI, and the resulting fragments were recovered by agarose gel electrophoresis using a Quick Gel Extraction Kit. The recovered target fragment was then joined to the prokaryotic expression vector pET30a using a seamless splicing kit. The Seamless Cloning and Assembly Kit was used for ligation at 50°C for 30 min. The ligation product was transformed into Trans1-T1 competent E. coli cells, and single clones were picked for colony PCR detection. The positive bacterial cultures with correct sequencing were expanded and extracted using the HiPure Plasmid MiniPrep Kit to obtain the constructed recombinant expression vector pET30a-GlCHS.

[0053] Example 3

[0054] Induction and protein purification of genetically engineered bacteria:

[0055] The recombinant expression vector pET30a-GlCHS was transformed into competent BL21(DE3) cells. Transformed cells were plated on LB solid medium containing 50 mg / L kanamycin to screen for positive clones. Single positive colonies were selected and inoculated into LB liquid medium containing 50 mg / L kanamycin and cultured overnight. The culture was then scaled up at a 1:100 ratio until the bacterial culture reached OD500. 600 When the concentration reached between 0.4 and 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.8 mM, and the mixture was slowly induced for 12 h in a shaker at 16 °C. The empty pET30a vector was treated in the same manner as a blank control.

[0056] The induced bacterial culture was centrifuged at 5000×g for 10 min at 4℃, the supernatant was discarded, and the cells were washed three times with 5 mL PBS. After washing, the cells were centrifuged again at 5000×g for 10 min, the supernatant was discarded, and the cells were immediately cooled on ice. The bacterial cells were resuspended in 5 mL PBS and sonicated on ice. After sonication, the cells were centrifuged at 10000×g for 15 min at 4℃. 50 μL of the supernatant was used for 10% SDS-PAGE electrophoresis analysis (the SDS-PAGE gel used for analysis was prepared using an SDS-PAGE denaturing acrylamide gel rapid preparation kit). The electrophoresis results are shown below. Figure 7 As shown, the recombinant expression vector pET30a-GlCHS, after induction, expressed a soluble protein with a band at approximately 45 kDa, consistent with the molecular weight of the GlCHS protein (using Blue). II Protein Marker (14-120kDa) is used as a protein molecular weight standard.

[0057] Pipe 1 mL of protein supernatant and mix it with 500 μL of washed protein for purification. Mix Ni-NTA Resin and incubate with shaking in an ice bath for 2 hours. After incubation, centrifuge at 4°C and 500×g for 5 minutes, and discard the supernatant. Add 200 μL of imidazole buffer at different concentrations (20 mM, 100 mM, 200 mM, 300 mM, 500 mM) sequentially to elute the protein. Centrifuge at 4°C and 500×g for 5 minutes, collect the supernatant, and obtain the purified protein. The purified protein is analyzed by 10% SDS-PAGE electrophoresis, and the results are as follows: Figure 7 As shown, M represents Protein Marker, lane 1 contains the pET-30a empty vector, lane 2 contains whole bacterial culture containing GluCHS protein after induction, lane 3 contains GluCHS protein supernatant, and lanes 4-8 contain GluCHS protein samples eluted with 20mM, 100mM, 200mM, 300mM, and 500mM imidazole solutions, respectively. The arrows indicate the target protein. Recombinant GluCHS protein can be efficiently eluted with 200mM, 300mM, and 500mM imidazole solutions, with the highest amount of purified protein (purified GluCHS protein) and the best purification effect obtained from elution in 300mM imidazole solution.

[0058] Example 4

[0059] In vitro enzyme function verification:

[0060] 1. Using p-coumaroyl-CoA as a substrate and pET-30a empty vector protein as a negative control, the in vitro function of GLCHS purified protein was identified. The 500 μL enzymatic reaction system included 360 μL of 0.1 M potassium phosphate buffer (0.05 mol / L K₂HPO₄:0.05 mol / L KH₂PO₄ = 94:6, pH = 8.0), 20 μL of 230 μM lithium malonyl-CoA, 20 μL of 280 μM p-coumaroyl-CoA, and 100 μL of enzyme solution (purified protein supernatant). The reaction was incubated at 30 °C for 1 h. The reaction was terminated by adding 250 μL of ethyl acetate, and the reaction product was extracted (repeated three times). After drying the solvent with nitrogen, the product was dissolved in 100 μL of mass spectrometry grade methanol and filtered through a 0.22 μm microporous membrane for later use.

[0061] 2. Using naringenin as a standard, the formation of enzymatic products was detected by UPLC. UPLC analysis was performed using an Agilent 1290 ultra-high performance liquid chromatograph with an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm). Gradient elution was performed using 0.1% phosphoric acid (phase A) and acetonitrile (phase B) as the mobile phase. The flow rate was 0.1 mL / min, the detection wavelength was 290 nm, the column temperature was 30 °C, and the injection volume was 5 μL.

[0062] The elution program is as follows: 0-3 min, 20%-22.5% B; 3-8 min, 22.5%-25% B; 8-10 min, 25%-75% B; 10-12 min, 75%-85% B; 12-18 min, 85%-88% B; 18-20 min, 90% B.

[0063] 3. CHS can catalyze the formation of naringenin chalcone from coumaroyl-CoA and malonyl-CoA in vitro. Naringenin chalcone is unstable in aqueous solution and can slowly and spontaneously cyclize to naringenin. Therefore, in this example, the activity of the GluCHS-purified protein was determined by detecting the presence or absence of naringenin in the enzymatic system. The catalytic product was detected under the above conditions, and the results are as follows: Figure 8 As shown, compared with the empty pET30a carrier protein, the purified GlCHS protein catalyzed a new chromatographic peak for coumaroyl-CoA substrate at 13.495 min, which coincides with the peak time of the standard naringenin (13.549 min). Therefore, it can be concluded that pET30a-GlCHS has the function of chalcone synthase.

[0064] As can be seen from the above embodiments, this invention clones the gene encoding chalcone synthase (GlCHS) from *Hedyotis diffusa*, and by transforming the GlCHS gene into a prokaryotic expression vector, the purified GlCHS protein expressed in large quantities in host cells can promote the synthesis of the flavonoid compound naringenin. Using the gene and technology provided by this invention, naringenin can be synthesized in large quantities in vitro, and the content of naringenin in *Hedyotis diffusa* can also be increased through genetic engineering techniques.

[0065] The SEQ ID NO.1 and SEQ ID NO.2 mentioned in Example 1 are as follows:

[0066] SEQ ID NO.1:

[0067]

[0068] SEQ ID NO.2:

[0069] MVTVEEIRRAQRAEGPATVLAIGTAVPTNCVDQSAYPDYYFRITNSEHKTDLKEKFVRMCEKSMIKKRYMHLTEEYLKENPNITAYMAPSLDARQDIVVVEVPKLGKEAAQKAIKEWGQSKSKITHLVFCTTSGVDMPGADYQLTKLLGLRASVKRFMMYQQGCFAGGTVLRMAKDLAENNAGARVLVVCSEITA VTFRGPSESHLDSLVGQALFGDGAAAVIVGSDPVLGVERPLFQLVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIEKSLKEAFAPLGISDWNSVFWIAHPGGPAILDQVEAKLGLKPEKLRSTRHVLSEYGNMSSACVLFILDEMRKSSAKEGMTTTGEGLDWGVLFGFGPGLTVETVVLHSVPHIN

[0070] 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 chalcone synthase gene GlCHS from *Hypericum perforatum*, characterized in that, The nucleotide sequence of the gene GlCHS is shown in SEQ ID NO.

1.

2. A product encoded by the ghrelin chalcone synthase gene GlCHS as described in claim 1, characterized in that, The product includes amino acids, polypeptides, or proteins.

3. The product encoded by the chalcone synthase gene GlCHS of the blood-activating herb 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, characterized in that, The expression vector is pET-30a and contains the ghrelin synthase gene GlCHS as described in claim 1.

5. A recombinant engineered bacterium, characterized in that, It contains the ghrelin chalcone synthase gene GlCHS as described in claim 1 or the recombinant expression vector as described in claim 4.

6. A host cell, characterized in that, It contains the ghrelin chalcone synthase gene GlCHS as described in claim 1 or the recombinant expression vector as described in claim 4.

7. The host cell according to claim 6, characterized in that, The host cells include BL21 (DE3) cells.

8. The application of the ghrelin charone synthase gene GlCHS as described in claim 1, the recombinant expression vector as described in claim 4, the recombinant engineered bacteria as described in claim 5, or the host cell as described in claim 6 in the preparation of flavonoid compounds, wherein the application is the application in the preparation of naringenin using p-coumaroyl-CoA and malonyl-CoA as substrates.

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