Scopoletin glycosyltransferase protein in traditional chinese medicine artemisia annua and coding gene and application thereof

Abstract

The present application discloses a Chinese medicine artemisia argyi scopolin glycosyltransferase protein and its coding gene and application. The protein is the protein in a) or b) as follows: a) the protein consisting of the amino acid sequence shown in sequence 2 in the sequence listing; b) the protein derived from a) by substitution and / or deletion and / or addition of one or several amino acid residues of the amino acid sequence shown in sequence 2 in the sequence listing and having scopolin glycosyltransferase activity. The present application is based on the fact that Chinese medicine artemisia argyi specifically enriches scopolin (up to 2.24 mg / g dry weight), and it is speculated that there is a highly active glycosyltransferase in it to catalyze the generation of scopolin. Through the transcriptome and proteome screening of two ecotypes of Chinese medicine artemisia argyi, a specific glycosyltransferase AaUGT88A54 catalyzing scopolin in Chinese medicine artemisia argyi is found and identified, and the scopolin glycosyltransferase protein and its coding sequence for biosynthesis of scopolin are provided.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a scopoletin glycosyltransferase protein in traditional Chinese medicine Artemisia annua, an encoding gene thereof and application. BACKGROUND

[0002] Scopoletin (7-hydroxy-6-methoxycoumarin) is a typical representative of the coumarin family, and its 7-OH glucosylation product is Scopolin. The structural formula is shown in Figure 1 Scopoletin is derived from a carbon skeleton C6-C3, containing a crystallographic skeleton core in a 1,2-benzopyrone structure, and having methoxylation and hydroxylation modification on the benzene ring (C6). Scopoletin is isolated from phenolic resin in Arabidopsis thaliana and other plants with phenylpropanoid pathway, and its medicinal value has attracted great attention all over the world. The in vitro pharmacological activities of scopoletin include antibacterial, antifungal, antituberculosis and antihypertensive properties. The proven in vivo pharmacological activities include anti-inflammatory, nerve-relieving, anti-diabetic and anti-hyperuricemia properties.

[0003] The antioxidant capacity of scopoletin and Scopolin in cells can inhibit the differentiation of pre-osteoclast RAW 264.7. In addition to having similar functions as its ligand, Scopolin has important effects on angiogenesis, liver protection, weight loss, anti-hepatoma, and osteoporosis relief. Scopolin can reduce the clinical symptoms of AIA in rats by inhibiting inflammation and angiogenesis, and this compound can be an effective drug for angiogenesis-related diseases and can be used as a structural basis for screening more effective synthetic analogs. Scopolin can reduce liver steatosis by activating SIRT1-mediated signal cascade, thereby enhancing liver SIRT1 activity and protein expression. In vivo and in vitro experiments show that Scopolin can prevent adipocyte differentiation and weight gain, indicating that Scopolin may be a potential bioactive compound for the treatment and prevention of human obesity. Scopolin extracted from Smilax china plays a role in anti-hepatocellular carcinoma by regulating the expression of glycolytic proteins GPI, GPD2 and PGK2. Scopolin may affect the interaction between Hsp90α and GPD2, which may provide a new potential treatment direction for hepatocellular carcinoma. Scopolin treatment can prevent OVX-induced loss of bone mineral density in osteoporotic mice, indicating that Scopolin may be a bioactive component for the treatment and prevention of osteoporosis.

[0004] Scopoletin has been biosynthesized in E. coli, and the glycosyltransferase catalyzing the glycosylation of scopoletin to generate scopolin has been cloned in Arabidopsis, tobacco and tomato. However, due to the weak catalytic activity of the enzyme, there is no report on the biosynthesis of scopolin. Therefore, there is an urgent need for an enzyme with high specificity to specifically synthesize scopolin, thereby providing a new reference for the design of drug synthesis. SUMMARY

[0005] Therefore, the present application is based on the enrichment of scopolin in traditional Chinese medicine Artemisia annua, and AaUGT88A54 protein is screened by transcriptome and proteome, and the production of scopolin catalyzed by the protein is identified by enzyme activity, thereby providing a glycosyltransferase sequence for the biosynthesis of scopolin.

[0006] The technical scheme of the present application is as follows:

[0007] A protein is a protein in a) or b) as follows:

[0008] a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

[0009] b) a protein derived from a) by substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having scopoletin glycosyltransferase activity. That is, one or several of the three substitutions and / or deletions and / or additions are processed.

[0010] The scopoletin glycosyltransferase activity is the activity of catalyzing the glycosylation of the A ring 7 position of scopoletin to generate scopolin.

[0011] The coding gene of the protein also belongs to the protection scope of the present application.

[0012] The coding gene is as shown in 1) or 2) or 3) as follows:

[0013] 1) the DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing;

[0014] 2) a DNA molecule hybridizing to the DNA molecule defined in 1) under stringent conditions;

[0015] 3) a DNA molecule having more than 90% homology to the DNA molecule defined in 1) or 2).

[0016] The coding gene contains 1392 nucleotides, as shown in SEQ ID NO: 1 in the sequence listing; and the protein encoded thereby contains 463 amino acids, as shown in SEQ ID NO: 2 in the sequence listing, which is named AaUGT88A54, and the protein encoded thereby is named AaUGT88A54.

[0017] Expression cassettes, recombinant expression vectors, transgenic cell lines or recombinant microorganisms containing the encoding gene also fall within the scope of protection of the present invention.

[0018] The use of the protein as scopoletin glycosyltransferase also falls within the protection scope of the present invention.

[0019] The scopoletin glycosyltransferase is an enzyme that catalyzes the glycosylation of position 7 of scopoletin A ring to generate scopoletin.

[0020] The use of the protein in catalyzing the conversion of scopoletin to scopoletin also falls within the protection scope of the present invention.

[0021] The use of the encoding gene in catalyzing the conversion of scopoletin to scopoletin also falls within the scope of protection of the present invention.

[0022] Based on the specific enrichment of scopoletin in the traditional Chinese medicinal herb Artemisia annua (up to 2.24 mg / g dry weight), this study hypothesizes the presence of a highly active glycosyltransferase that catalyzes the production of scopoletin. Through transcriptome and proteome screening of two ecotypes of the traditional Chinese medicinal herb Artemisia annua, the specific glycosyltransferase AaUGT88A54 that catalyzes scopoletin production in Artemisia annua was identified and characterized. This provides the scopoletin glycosyltransferase protein and its coding sequence for the biosynthesis of scopoletin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] For purposes of illustration and not limitation, the present invention will now be described with reference to preferred embodiments thereof, particularly with reference to the accompanying drawings, in which:

[0024] Figure 1 is the structural formula of scopoletin and scopolamine;

[0025] Figure 2 This is the verification result of cloning the gene into the pEASY blunt cloning vector;

[0026] Figure 3 This is the verification result of subcloning AaUGT88A54 into the expression vector and expression bacteria.

[0027] Figure 4 This is an SDS-Page gel image of the AaUGT88A54 recombinant protein.

[0028] Figure 5 This is the UPLC chart for the identification of the catalytic activity of AaUGT88A54 on scopoletin.

[0029] Figure 6 This is the MS identification diagram of the scopoletin catalytic product by AaUGT88A54. DETAILED DESCRIPTION

[0030] The application will be further described in conjunction with specific examples, but the application is not limited in any way by the examples.

[0031] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0032] Example 1, Traditional Chinese Medicine Artemisia annua Scopoletin Glycosyltransferase Protein and Its Encoding Gene and Application

[0033] Based on the transcriptome and proteome of traditional Chinese medicine Artemisia annua, combined with the conserved domain PSPG box of UGTs, 177 AaUGTs sequences greater than 300 aa were obtained, and finally AaUGT88A54 was locked.

[0034] Seeds of Chinese medicine Artemisia annua (non-patent literature describing Artemisia annua is: B. Liao, X. Shen, L. Xiang, S. Guo, S. Chen, Y. Meng, Y. Liang, D. Ding, J. Bai, D. Zhang, et al., Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield. Mol Plant. 15 (2022) 1310-1328. https: / / doi.org / 10.1016 / j.molp.2022.05.013.) collected in Hainan and Gansu were planted in a greenhouse for 3 months, RNA was extracted, reverse transcribed into cDNA, primer sequences were designed (as shown in Table 1), and mixed cDNA of roots and leaves was used as a template to clone an AaUGT gene fragment using KOD high-fidelity enzyme (KOD high-fidelity enzyme PCR system total volume was 50 μL: 5 μL 10X Buffer, 3 μL MgSO4, 5 μL dNTP (2 mM), 3 μL primer (10 mM), 1 μL template and 32 μL water, and the reaction program was as shown in Table 2. With the help of pEASY-Blunt vector (purchased from Beijing Zison Biotechnology Co., Ltd., product catalog number CB111-01 (20 rxns)), the AaUGT gene fragment was successfully connected to the vector (the connection system total volume was 2.5 μL: 0.5 μL Mix Buffer with enzyme and 2 μL template, 25 °C reaction for 2 h). The connection system was directly transformed into TransT1 competent cells, and positive clones were selected for sequencing (colony PCR system total volume was 20 μL: 13 μL Mix Buffer, 1 μL template, 1 μL primer and 5 μL water, program as shown in Table 3, Figure 2 ), compared with the transcript sequence, the nucleotide sequence had 100% similarity with the original data, and the actual sequencing result was used as the standard.

[0035] Table 1 Primer sequences used for cloning genes

[0036]

[0037] Note: Only the primer sequences of one gene successfully expressing recombinant proteins are listed.

[0038] Table 2 KOD high-fidelity enzyme PCR reaction program

[0039]

[0040]

[0041] Table 3 Colony PCR reaction program

[0042]

[0043] II. Obtaining the gene sequence and the protein sequence encoded thereby

[0044] The sequencing result shows that the gene amplified by using the primer in Table 1 contains 1392 nucleotides, as shown in Sequence 1 in the sequence listing; it encodes a protein containing 463 amino acids, as shown in Sequence 2 in the sequence listing, and the gene is named AaUGT88A54 and the protein encoded thereby is named AaUGT88A54.

[0045] III. Verification of the function of the gene

[0046] The function of the gene was verified by means of a prokaryotic system, a pMAL-c2X-AaUGT88A54 vector was constructed, and after the sequence was confirmed to be correct by sequencing, the vector was successfully transformed into a prokaryotic expression strain Novablue for in vitro verification.

[0047] First, an enzyme cutting site adapter was added to the AaUGT88A54 fragment (the system and procedure are consistent with the above gene cloning method, and the primer information is shown in Table 4); after the AaUGT88A54 fragment (with an enzyme cutting site) was cut, it was constructed into an expression vector pMAL-c2X (purchased from New England Biolabs., product catalog number E8200S) by using T4-DNA ligase (the total volume of the ligation system was 7 μL: 3.5 μL Mix Buffer, 2.8 μL AaUGT88A54 fragment and 0.7 μL pMAL-c2X; 4°C reaction overnight); secondly, after the ligation system was transformed into TransT1 (purchased when pEASY-Blunt vector was purchased), positive clones were selected; after the plasmid was extracted, it was transformed into an expression strain Novablue (purchased from Merck Millipore, Germany Darmstadt Merck KGaA Life Science Business Company, product catalog number 69284-3). The positive clones were identified by using a colony PCR program (Table 3). Figure 3 ).

[0048] Table 4 Primer sequences used for constructing a prokaryotic expression

[0049]

[0050] Note: The lower case letters in the sequence are protection bases, and the underlined bases are enzyme cutting site bases.

[0051] The induction, purification, enzyme activity analysis and product identification of the recombinant protein are as follows:

[0052] 1) Induction of recombinant protein

[0053] Single colonies of pMAL-c2X-AaUGT88A54 and pMAL-c2X were picked and inoculated into 3 mL LB liquid medium (containing Amp 100 mg / L) and incubated at 37°C overnight with shaking (200 rpm).

[0054] 1 mL of the overnight culture was inoculated into 100 mL fresh LB medium (containing Amp 100 mg / L, 0.2% membrane-sterilized glucose) and incubated at 37°C until the OD600 value reached 0.5-0.8. Then, 1 mL of the culture was collected as a control.

[0055] 30 μL of IPTG (1 M stock solution) was added to the 100 mL culture to a final concentration of 0.3 mM, and the culture was incubated at 16°C for 48 h.

[0056] The culture was centrifuged at 8,000 x g for 3 min at 4°C, and the supernatant was discarded.

[0057] 2) Purification of recombinant protein

[0058] The recombinant AaUGT88A54 protein was purified according to the manual of the pMAL fusion protein and purification system (New England BioLab Inc.). Briefly, the bacterial pellet collected above was resuspended in column buffer and incubated at -20°C overnight. The next day, the sample was thawed, and the cells were disrupted by sonication to release the protein. After centrifugation at 9,000 x g for 30 min, the sample was loaded onto the column. The affinity column was activated with 12 column volumes of column buffer at a flow rate of 1 mL / min. The sample was diluted 5-fold and loaded onto the column. After the sample passed through the column, the column was washed with 12 column volumes of column buffer to remove the impurities. Finally, the target protein was eluted with 5 column volumes of column buffer (containing 10 mM maltose). The eluate was concentrated by low-speed centrifugation (3,000 x g, 4°C) using a Millpore (30 KDa) filter and then replaced into the enzyme activity assay buffer. After SDS-PAGE and Coomassie blue staining, the recombinant protein was found to have a size of approximately 100 kDa (the tag protein, MBP or maltose-binding protein, is 42.5 kDa), which was consistent with the predicted size of AaUGT88A54 (51 kDa). Figure 4 ).

[0059] 3) Determination of enzyme activity

[0060] Enzyme activity reaction system was 100 μL, as shown in Table 5. After 30 min of reaction at 30°C, the reaction was terminated with an equal volume of methanol, and centrifuged at 13,000 rpm for 10 min. 2 μL was taken for sampling after membrane filtration (0.22 μm).

[0061] Table 5 Recombinant protein enzyme activity reaction system

[0062]

[0063] 4) Enzyme activity product analysis and identification

[0064] Analysis of enzyme activity product UPLC spectrum Figure 5 ), it was found that there was a new product peak in the reaction system of AaUGT88A54 and scopolin.

[0065] UPLC conditions:

[0066] UPLC model: Nexera UHPLC LC-30A system (SHIMADZU, Japan).

[0067] Mobile phase: A phase: 0.1% formic acid aqueous solution; B phase: acetonitrile.

[0068] Elution gradient: 0-7 mins, 5%-100% B; 7-9 mins, 100% B; 9-10.5 mins, 100%-5% B; 10.5-11.5 mins, 5% B.

[0069] DAD detection wavelength: 335 nm.

[0070] Further identification by mass spectrometry found that the mass-to-charge ratio of the enzyme activity product peak P1 (acidification + COOH) of AaUGT88A54 for scopolin was 206 more than that of the substrate (one molecule of water was removed from the substrate hydroxyl and the molecular weight was increased after acidification), indicating that the product was a monoglucoside of scopolin. According to the compound structural formula Figure 1 ), scopolin only has a hydroxyl group at position 7 of ring A that can be O-glycosylated, so it is speculated that the product is scopolin. At the same time, we found that the enzyme activity product peak had the same retention time and mass spectrometry fragment information as the scopolin standard Figure 6 ). In summary, in vitro enzyme activity evidence shows that AaUGT88A54 gene encodes a glycosyltransferase that catalyzes the glycosylation of scopolin to produce scopolin.

[0071] Mass spectrometry conditions:

[0072] The sample was separated by UPLC MS / MS, the column was Eclipse Plus C18 RRHD (1.8 μm, 2.1 x 50 mm i.d.; Agilent), the mobile phase was the same as UPLC, the elution gradient was 0 min, 95% A; 7 mins, 5% A, finally 95% A balance (1 min), the flow rate was 0.30 mL / min, and the detection wavelength was the same as above.

[0073] UPLC MS / MS mass spectrum conditions: electrospray ionization, full ion scan, negative ion mode negative-ion (EI) mass spectrum analysis. Heat gas temperature: 300°C; gas flow, 5.0 L / min, capillary voltage, 3500V; nozzle voltage, 500V; delta EMV, 200V. MS TOF (Expt): collision voltage (Fragmentor), 120V; skimmer, 65V; acquisition mass spectrum range m / z: 100-1000.

[0074] 5) Catalytic activity of AaUGT88A54

[0075] To further understand the catalytic characteristics of the AaUGT88A54 recombinant protein, in the embodiments of the present application, the catalytic activity of the AaUGT88A54 recombinant protein on scopolin was detected. The total volume of the enzyme activity reaction system was 50 μL, and the system included 5 μg of AaUGT88A54 recombinant protein, 100 μM of UDP-glucose, and 0-400 μM of scopolin (10, 50, 100, 200, and 400 μM, respectively). After 30 min of reaction at 30°C, the reaction was terminated by equal volume of methanol, and after high-speed centrifugation at 14000 rpm, 2 μL was taken for UPLC detection after passing through a 0.22 μm membrane. It was found that the K M mM M was 0.54 min -1 mM -1 , indicating that the AaUGT88A54 recombinant protein had high catalytic activity on scopolin (as shown in Table 6).

[0076] Table 6 Catalytic activity of AaUGT88A54 recombinant protein enzyme on scopolin

[0077]

[0078] The foregoing detailed description has set forth various embodiments of the devices and / or processes via the use of specific terminology. However, embodiments thereof can be practiced with the exact description not being presented in detail. The term "device" should be understood to encompass devices operating in various modes, such as active mode, sleep mode, hibernate mode, and the like. The terms "coupled" and "connected," along with derivatives thereof, can be used. It should be understood that these terms are not intended as synonyms for each other. Rather, particular circuitry that can be said to be coupled to, or connected with, other circuitry can be some of the other circuitry that can communicate in some way, while other circuitry can be some of the other circuitry that can not be in communication with that particular circuitry.

Claims

1. The use of a protein with an amino acid sequence as shown in SEQ ID NO: 2 in catalyzing the glycosylation of anamidine A at the 7th position of the ring to transform into scopolin.

2. The use of a coding gene with a nucleotide sequence as shown in SEQ ID NO: 1 in catalyzing the glycosylation of anamidine A at the 7th position of the ring to transform into scopolin.

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