Astragalus root calyx heterophylla methyltransferase protein AmOMT1, coding gene, primer set and application thereof

Abstract

The application provides a astragalus membranaceus calyx heterophylla isoflavone methyltransferase protein AmOMT1, a coding gene, a primer set and application thereof, and belongs to the technical field of biotechnology.The amino acid sequence of the astragalus membranaceus calyx heterophylla isoflavone methyltransferase protein AmOMT1 is shown as SEQ ID No.1.Based on astragalus membranaceus genomic and second-generation transcriptomic data, a methyltransferase AmOMT1 with 4' and 7 position methylation functions for 3', 4', 7-trihydroxyisoflavone is found and identified by a reverse genetics method.Experiments show that AmOMT1 can catalyze the methylation of 3', 4', 7-trihydroxyisoflavone to generate calyx heterophylla isoflavone and 3', 4'-dihydroxy-7-methoxyisoflavone.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an astragalus isoflavone methyltransferase protein AmOMT1, its encoding gene, primer set, and its applications. Background Technology

[0002] 3',4',7-Trihydroxyisoflavone, also known as 3'-hydroxydaidzein, has the chemical name 3-(3,4-dihydroxyphenyl)-7-hydroxychromen-4-one, and its molecular formula is C2. 15 H 10 O5, slightly soluble in methanol and DMSO. 3',4',7-Trihydroxyisoflavone is the main metabolite of genistein, which can inhibit UV-B-induced skin cancer by targeting Cot and MKK4, and also has anti-angiogenic, chemoprotective and free radical scavenging activities.

[0003] Calycosin, chemically named (7-hydroxy-3-(3-hydroxy-4-methoxyphenyl)chromen-4-one), has the molecular formula C2. 16 H 12 O5, a white crystalline solid, is soluble in organic solvents such as methanol, ethanol, and DMSO, and is mainly derived from the rhizome of Astragalus membranaceus. Verbena isoflavones are also phytoestrogens with broad pharmacological activity. By influencing signaling pathways such as PI3K / Akt / mTOR, WDR7-7-GPR30, and Rab27B-β-catenin-VEGF, verbena isoflavones also exhibit anticancer, anti-inflammatory, anti-osteoporosis, neuroprotective, and hepatoprotective effects. Its glycosylated product, verbena isoflavone-7-O-glucoside, is the index component of Astragalus membranaceus as specified in the Chinese Pharmacopoeia.

[0004] 3',4',7-Trihydroxyisoflavones have hydroxyl groups at the 7-position of ring A and at the 3' or 4' position of ring B, both of which can undergo O-methylation. Versicolor isoflavone is the product of methylation of 3',4',7-trihydroxyisoflavone at the 4' position of ring B. Studies have reported isoflavone 7-O-methyltransferases (I7OMTs), 3'-O-methyltransferases (HI3'OMTs), and 4'-O-methyltransferases (HI4'OMTs) in various legumes, including soybean (Glycine max), alfalfa (Medicago sativa), licorice (Glycyrhizza echinata), kudzu (Pueraria lobata), lotus japonicus, and medicago truncatula. The I7OMT recombinase of alfalfa can catalyze the methylation of daidzein 7-OH in vitro, generating isomandibularin. HI4'OMTs from licorice, bird's eye root, and alfalfa can recognize 2,7,4'-trihydroxydihydroisoflavones, but cannot directly use daidzein as a methyl acceptor. In contrast, PlOMT9 from kudzu chooses daidzein instead of 2,7,4'-trihydroxyisoflavones as a substrate, and PlOMT4 possesses enzymatic activity for methylating the 3'-OH of isoflavone substrates. These findings suggest that isoflavone methylation products may be synthesized through different pathways. Some studies have also reported methyltransferases with both 4' and 7' methylation functions, such as GmOMT5 from soybean and MtIOMT3 from alfalfa. Summary of the Invention

[0005] The purpose of this invention is to provide an Astragalus membranaceus isoflavone methyltransferase protein AmOMT1, its encoding gene, primer set, and its application. Based on the Astragalus genome and second-generation transcriptome, the isoflavone methyltransferase sequence was obtained, and the recombinant protein was expressed in prokaryotes. It was found that AmOMT1 can catalyze the methylation of 3',4',7-trihydroxyisoflavone to generate vernix isoflavone and 3',4'-dihydroxy-7-methoxyisoflavone.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an astragalus isoflavone methyltransferase protein AmOMT1, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] This invention also provides the application of the Astragalus melanoleucine methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to produce verbascoflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0009] The present invention also provides a catalyst comprising the aforementioned astragalus verbena isoflavone methyltransferase protein AmOMT1.

[0010] The present invention also provides a gene encoding the astragalus isoflavone methyltransferase protein AmOMT1, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0011] The present invention also provides a primer set for amplifying the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1, including an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0012] The present invention also provides the application of the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1 in the preparation of recombinant expression vectors or recombinant bacteria.

[0013] Preferably, the recombinant expression vector further includes the initial expression vector pEASY-Blunt.

[0014] Preferably, the recombinant bacteria also include NovaBlue Escherichia coli.

[0015] The present invention also provides the application of the gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0016] The present invention also provides the application of the gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in the preparation of products that catalyze the methylation of 3',4',7-trihydroxyisoflavone to produce verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0017] The beneficial effects of this invention compared to the prior art are as follows:

[0018] Although numerous genes related to isoflavone methylation biosynthesis have been reported, no methyltransferases with simultaneous 4' and 7' methylation functions have been reported in Astragalus membranaceus. Based on Astragalus membranaceus genome and second-generation transcriptome data, this invention uses reverse genetics to identify and characterize a methyltransferase, AmOMT1, which simultaneously performs 4' and 7' methylation functions on 3',4',7-trihydroxyisoflavone. Its protein sequence was compared with the NCBI database; sequences with a similarity greater than 80% all originated from legumes. Among them, a sequence from Astragalus alpinus var. arcticus was predicted to contain an "AdoMet_MTases" domain, but its function has not been reported. Another sequence from Sesbania bispinosa was classified as belonging to the "Winged helix-like DNA-binding domain superfamily," providing the methyltransferase protein and its coding sequence for the biosynthesis of verrucoside and 3',4'-dihydroxy-7-methoxyisoflavone. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The molecular structural formulas of 3',4',7-trihydroxy-7-methoxyisoflavone and its methylated products are shown from left to right as follows: verbascoflavonoid, 3'-methyldaidzein, and 3',4'-dihydroxy-7-methoxyisoflavone.

[0021] Figure 2 The image shows an agarose gel electrophoresis image of the AmOMT1 gene clone, where M represents the maker.

[0022] Figure 3 This is an SDS-Page gel image of the AmOMT1 recombinant protein, where M represents the maker.

[0023] Figure 4 UPLC chromatograms for identifying the catalytic activity of AmOMT1 on 3',4',7-trihydroxyisoflavone, where I and II represent methylated products of 3',4',7-trihydroxyisoflavone;

[0024] Figure 5The images show the MS and MS / MS identification of the catalytic products of 3',4',7-trihydroxyisoflavone catalyzed by AmOMT1. Peaks I and II both represent the methylated products of 3',4',7-trihydroxyisoflavone. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] This invention provides an astragalus isoflavone methyltransferase protein AmOMT1, the amino acid sequence of which is shown in SEQ ID No. 1.

[0031] In this invention, the amino acid sequence of the Astragalus membranaceus isoflavone methyltransferase protein AmOMT1 is: MASSTNGRKASEIFQGQALLYKHLYAFIDSMCLKWTVELDIPNIIHNHGKPITLQELVSILKVPQTKVGNVQRLMRYLAHNGFFEIVRIKEDNQEDKEAYSLTAASELLVKGTDFCLAPLVEFVLDPTLSGSYHQLKKWIYAEDLTLFGLSLGSHFYEFLNRNPENSRSFDEAMASDSQIINLALRDCNLVFEGLDSIVDVGGGIGITAKLISEIFPELKCIVFDRPQVVENLSGTNNVSYVGGDMFISVPKADAVLLKYILHNWTDKECIKILEKCKEAISRDGKSGKVIIVDVVINGNKDEHGLTQLKLLLDLTMASLNGKERNEEEWKKLFTEAGFQDYKISPFTGFLSLIEIYP (SEQ ID) No. 1).

[0032] This invention also provides the application of the Astragalus melanoleucine methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to produce verbascoflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0033] The present invention also provides a catalyst comprising the aforementioned astragalus verbena isoflavone methyltransferase protein AmOMT1.

[0034] The present invention also provides a gene encoding the astragalus isoflavone methyltransferase protein AmOMT1, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0035]

[0036] The present invention also provides a primer set for amplifying the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1, including an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4.

[0037] In this invention, the nucleotide sequence of the upstream primer is: ATGGCTTCTTCAACCAATGG (SEQ ID No. 3), and the nucleotide sequence of the downstream primer is: TTAAGGATAGATCTCAATAAGAG (SEQ ID No. 4).

[0038] The present invention also provides the application of the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1 in the preparation of recombinant expression vectors or recombinant bacteria.

[0039] In this invention, the recombinant expression vector preferably further includes the initial expression vector pEASY-Blunt, and the recombinant bacteria preferably further includes NovaBlue Escherichia coli.

[0040] The present invention also provides the application of the gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0041] The present invention also provides the application of the gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in the preparation of products that catalyze the methylation of 3',4',7-trihydroxyisoflavone to produce verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone.

[0042] Example 1: Methods and Procedures for Gene Cloning

[0043] Based on Astragalus genome and second-generation transcriptome data, combined with the analysis of conserved OMTs domains, 60 AmOMTs sequences ranging from 232aa to 407aa were obtained. Through combined analysis of functional gene phylogenetic tree and flavonoid-targeting metabolome, AmOMT1 was the focus.

[0044] Mongolian Astragalus (extracted separately) Astragalus membranaceus (Fisch.) Bge.var. mongholicusRNA from the roots, stems, and leaves of *B. ge.* Hsiao* was reverse transcribed into cDNA. Primer sequences were designed (as shown in Table 1). Using the mixed cDNA from roots, stems, and leaves as a template, an AmOMT gene fragment was cloned using KOD high-fidelity enzyme (KMM-101, purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.). The total volume of the KOD high-fidelity enzyme PCR system was 50 μL: 5 μL 10×Buffer, 3 μL MgSO4, 5 μL dNTP (2 mM), 2 μL each of forward and reverse primers (10 mM), 1 μL template, and 32 μL water. The procedure is shown in Table 2. Using the Blunt gene cloning kit (CB101, purchased from Beijing TransGen Biotech Co., Ltd.), the AmOMT1 fragment was successfully ligated into a vector (the total volume of the ligation system was 2.5 μL: 0.5 μL pEASY-Blunt cloning vector and 2 μL template, reacted at 25℃ for 2 h). The ligation system was directly transformed into Trans1-T1 competent cells (purchased from Beijing TransGen Biotech Co., Ltd.). Positive clones were selected for sequencing (the total volume of the colony PCR system was 20 μL: 13 μL Mix Buffer, 1 μL template, 1 μL primer and 5 μL water, the procedure is shown in Table 3). The sequences were compared with the CDS sequence. The nucleotide sequence was 99% similar to the original data. The actual sequencing results shall prevail.

[0045]

[0046] Sequencing revealed that AmOMT1 contains 1077 nucleotides, encoding a protein of 358 amino acids. Its amino acid sequence is shown in SEQ ID No. 1, and its nucleotide sequence is shown in SEQ ID No. 2.

[0047] Example 2: Verification of gene function

[0048] Gene function was validated using a prokaryotic system. The pMAL-c2X-AmOMT1 vector was constructed, and after sequencing confirmed its correctness, the vector was successfully transformed into the prokaryotic expression strain Novablue for in vitro validation (e.g., Figure 2 (As shown). The specific verification method is as follows:

[0049] First, an enzyme restriction site adapter was added to the AmOMT1 fragment (the system and procedure were the same as the gene cloning method described above, and the primer information is shown in Table 4). After digestion of the AmOMT1 fragment (with restriction sites), it was constructed into the expression vector pMAL-c2X (purchased from New England Biolabs (Beijing) LTD.NEB) using T4-DNA ligase (purchased from Toyobo (Shanghai) Biotechnology Co., Ltd.) (the total volume of the ligation system was 7 μL: 3.5 μL Mix Buffer, 2.8 μL AmOMT1 fragment and 0.7 μL pMAL-c2X; reaction overnight at 4℃). Second, after the ligation system was transformed into TransT1, positive clones were selected. After plasmid extraction, it was transformed into the expression strain Novablue (purchased from Beijing Huayueyang Biotechnology Co., Ltd.) to obtain a prokaryotic expression bacterium carrying pMAL-c2X-AmOMT1.

[0050]

[0051] Note: Lowercase letters in the sequence are protective bases and restriction enzyme sites.

[0052] Example 3: The induction, purification, enzyme activity analysis, and product identification of the recombinant protein are as follows:

[0053] 1) Induction of recombinant proteins

[0054] Single colonies of pMAL-c2X-OMT and pMAL-c2X prepared in Example 2 were separately picked and cultured overnight at 37°C with shaking (200 rpm) in 2 mL of LB broth (containing 100 mg / L Amp, 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, all reagents purchased from Beijing Solarbio Science & Technology Co., Ltd.). 1 mL of the overnight culture was added to 100 mL of fresh LB broth (containing 100 mg / L Amp and 0.2% membrane-sterilized glucose), and cultured at 37°C with shaking until the OD600nm value reached 0.5-0.6. 1 mL of the culture was then collected as a control. 30 μL of IPTG (1 M, isopropyl-β-D-thiogalactopyranoside) was added to 100 mL of the culture, bringing the final concentration to 0.3 mM, and the culture was incubated at 16°C for 24 h. Centrifuge at 8,000×g for 3 min at 4℃ and collect the bacterial cells.

[0055] 2) Purification of recombinant proteins

[0056] The recombinant AmOMT1 protein was purified according to the pMAL fusion protein and purification system (New England BioLab Inc.) manual. In short, the collected bacterial pellet was resuspended in column buffer (2L system: 40mL 1.0M Tris-HCl (pH 7.5), 23.4g sodium chloride, 4mLEDTA (0.5M), 308mg DTT) and incubated overnight at -20°C. The next day, after thawing, the cells were sonicated to release the protein. The sample was centrifuged at 9,000×g for 30 min and then loaded. The affinity column packing was activated with column buffer (8 column volumes) at a flow rate of 1 mL / min. The sample was diluted 5-fold and loaded. After the sample had passed through the affinity column packing, it was washed with 12 column volumes of column buffer to remove contaminating proteins. Finally, the target protein was eluted with 5 column volumes of column buffer (10mM maltose). The solution was concentrated using a Millipore (30kDa) buffer and replaced with enzyme activation buffer. Following SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining was performed to confirm the recombinant protein. The electrophoresis and staining results are as follows: Figure 3 As shown.

[0057] 3) Determination of enzyme activity

[0058] The enzyme activity of the purified recombinant protein obtained above was determined. The enzyme activity reaction system was 50 μL, as shown in Table 5. After reacting at 37℃ for 2 h, the reaction was terminated with three volumes of methanol, centrifuged at 13,000 rpm for 10 min, and 2 μL of the sample was loaded.

[0059]

[0060] 4) Analysis and identification of enzyme active products

[0061] Recombinant AmOMT1 protein was successfully expressed using a prokaryotic expression system, and its function was further identified by enzyme activity analysis. The donor for the enzyme activity reaction was SAM, and the acceptor was 3',4',7-trihydroxyisoflavone. UPLC analysis of the obtained enzyme products yielded the following results: Figure 4 As shown.

[0062] The UPLC measurement parameters were as follows: UPLC model: Nexera UHPLC LC-30A system (SHIMADZU, Japan); column: Agilent Poroshell 120 SB-C18 (1.9μm, 2.1mm×50mm); mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile; flow rate: 0.3mL / min; elution gradient: 0-7min, 5%-100% B; 7-9min, 100% B; 9-10.5min, 100%-5% B; 10.5-11.5min, 5% B; DAD detection wavelength: 260nm.

[0063] Analysis of the UPLC spectrum of the enzyme active product revealed that only AmOMT1 was active against 3',4',7-trihydroxyisoflavone.

[0064] Simultaneously, mass spectrometry was used to identify the enzyme activity products, and the results were as follows: Figure 5 As shown. The mass spectrometry detection conditions were as follows: sample preparation before mass spectrometry was the same as before UPLC. Samples were separated using UPLC-MS / MS with an Agilent Poroshell 120 SB-C18 column (1.9 μm, 2.1 mm × 50 mm), the same mobile phase as UPLC, and an elution gradient of 0 min, 95% A; 5 min, 5% A, and finally equilibration at 95% A (5-7 mins). The flow rate was 0.20 mL / min, and the detection wavelength was the same as above. UPLC-MS / MS mass spectrometry conditions were: electrospray ionization, full ion scan, positive-ion (PI) mass spectrometry analysis. Nebulizer gas temperature: 300℃; nebulizer gas flow rate: 5.0 L / min; capillary voltage: 3500 V; nozzle voltage: 500 V; electron multiplier voltage: 200 V. MSTOF (Expt): Fragmentation voltage, 120V; cutoff voltage, 65V; acquisition mass spectrometry range m / z: 100-1000. The characteristic ion of 3',4',7-trihydroxyisoflavone in positive ion mode is 271.06.

[0065] Depend on Figure 5 It is known that AmOMT1 exhibits two product peaks (peak I and peak II) for the enzyme activity reaction with 3',4',7-trihydroxyisoflavone as the acceptor and SAM as the donor. The mass-to-charge ratios of both peaks I and II are 14.01 higher than the substrate mass-to-charge ratio (the increase in molecular weight after the removal of one molecule of water from a methyl group and a substrate hydroxyl group), indicating that both products are monomethylated products of 3',4',7-trihydroxyisoflavone. The parent ion in the primary and secondary mass spectra of peak I is consistent with that of peak II, but the characteristic ion fragments differ, suggesting that both peaks I and II are monomethylated products of 3',4',7-trihydroxyisoflavone. Based on the compound structural formula (e.g., ... Figure 1 As shown in the figure, 3',4',7-trihydroxyisoflavone has hydroxyl groups at the 7-position of ring A and the 3' and 4' positions of ring B, which can be O-methylated. Based on the retention times of vernix isoflavone and 3'-methyl daidzein standards, peak I was determined to be vernix isoflavone, and peak II was determined to be 3',4'-dihydroxy-7-methoxyisoflavone.

[0066] In summary, in vitro enzyme activity evidence shows that AmOMT1 encodes a methyltransferase that catalyzes the methylation of 3',4',7-trihydroxyisoflavone to produce vernix isoflavone and 3',4'-dihydroxy-7-methoxyisoflavone.

[0067] As can be seen from the above embodiments, the present invention provides an Astragalus membranaceus isoflavone methyltransferase protein AmOMT1, its encoding gene, primer set and its application. The Astragalus membranaceus isoflavone methyltransferase protein AmOMT1 can catalyze the methylation of 3',4',7-trihydroxyisoflavone to generate vernix isoflavone and 3',4'-dihydroxy-7-methoxyisoflavone.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of Astragalus membranaceus isoflavone methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to generate vernix isoflavone and 3',4'-dihydroxy-7-methoxyisoflavone, characterized in that, The amino acid sequence of the Astragalus membranaceus isoflavone methyltransferase protein AmOMT1 is shown in SEQ ID No.

1.

2. The application of a gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in catalyzing the methylation of 3',4',7-trihydroxyisoflavone to verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone, characterized in that, The nucleotide sequence of the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1 is shown in SEQ ID No.

2.

3. The application of a gene encoding the astragalus verbenaflavonoid methyltransferase protein AmOMT1 in the preparation of products that catalyze the methylation of 3',4',7-trihydroxyisoflavone to produce verbenaflavonoid and 3',4'-dihydroxy-7-methoxyisoflavone, characterized in that, The nucleotide sequence of the gene encoding the astragalus isoflavone methyltransferase protein AmOMT1 is shown in SEQ ID No. 2.

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