Polygonatum cyrtonema PcOMT1 gene and application thereof in promoting synthesis and methylation of high isoflavone
By cloning and expressing the polysaccharide PcOMT1 gene and overexpressing genes in tobacco, the problem of insufficient research on the biosynthesis pathway of high isoflavones was solved, the content of high isoflavones was significantly improved, and its catalytic methylation was verified. Function.
Patent Information
- Application Number
- CN202411307397.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-19
- Publication Date
- 2025-02-14
Smart Images
Figure CN119432884A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant biotechnology and relates to a Polygonatum cyrtonema PcOMT1 gene and an application thereof in promoting the synthesis and methylation of isoflavones. Background Art
[0002] Polygonatum cyrtonema is a perennial herb of the genus Polygonatum in the family Asparagaceae. It is named for "the essence of Kun soil" and was first recorded in the first pharmaceutical monograph in my country, "Shennong Bencao Jing" (25-220 AD). It is a traditional classic medicinal and edible plant. Polygonatum cyrtonema is one of the three base source species of medicinal Polygonatum in the 2020 edition of the "Chinese Pharmacopoeia". Its medicinal part, the rhizome, contains the specific effective ingredient isoflavones. Isoflavones have a unique carbon skeleton and are a special class of flavonoid compounds. Its parent structure (C6-C3-C1-C6) has one more carbon atom than isoflavones (C6-C3-C6), and the carbon skeleton has a 9-specificity. Modern pharmacological studies have shown that isoflavones have strong biological activity in anti-neuroinflammation and blood sugar regulation.
[0003] Isoflavones are relatively rare in nature and are mainly distributed in Asparagaceae and Leguminosae plants. Polygonatum cyrtonema is a representative species of the genus Polygonatum in the Asparagaceae family. About 15 isoflavones and their derivatives have been isolated from Polygonatum cyrtonema. At present, there have been many studies on the biological activity of isoflavones, but few studies on their biosynthetic pathways. The carbon skeleton (C6-C3-C6) synthesis pathway of common flavonoid compounds is relatively clear, but the biosynthetic mechanism of the carbon skeleton (C6-C3-C1-C6) of isoflavones in plants has not been reported. The upstream phenylpropanoid pathway in the plant flavonoid synthesis pathway is relatively conservative in plants, and the possibility of adding carbon atoms is relatively low. The inference of the biogenic pathway shows that the increase in carbon atoms of isoflavones is likely to be the addition of a carbon atom to the 2'-OH position of 2',4',6',4-tetrahydroxychalcone, thereby forming a unique carbon skeleton of C6-C3-C1-C6. Based on the fact that Polygonatum cyrtonema contains high levels of isoflavones, it is possible that there is a specific oxygen methyltransferase in Polygonatum cyrtonema that can catalyze the methylation of 2'-OH of chalcone.
[0004] The prokaryotic expression system of the FOMT gene has been widely used in the in vitro functional verification of flavonoid O-methyltransferase. Studies have shown that FOMT can catalyze the methylation of the hydroxyl sites of most flavonoid skeletons. The most common catalytic sites are the 3'-OH and 5'-OH sites of the B ring, and the 7'-OH site of the A ring and the 4'-OH site of the B ring are also common. FOMTs with methylation sites at the 5'-OH, 6'-OH and 8'-OH of the A ring have also been identified in plants: CdFOMT5' in Citrus depressa can specifically catalyze 5'-OH methylation, PaF6OMT in Plagiochasma appendiculatum can specifically catalyze 6'-OH methylation of flavonoids, and ObF8OMT-1 in Ocimum basilicum can specifically catalyze methylation of the 8'-OH site of flavonoids. There are few reports on FOMT catalyzing the 3'-OH site of the C ring of flavonoids. Only ShMOMT3 in tomatoes specifically catalyzes the 3'-OH site of myricetin. Plant FOMT methylation modification occurs on both the A and B rings, and mostly occurs at the 3'-OH and 4'-OH sites of the B ring and the 6'-OH, 7'-OH and 8-OH sites of the A ring. Among them, F7OMTs methylated at the 7'-OH site of the A ring are widely isolated. The methylation modification sites of FOMT are different in different species. Among them, O-methylation modification of flavonoids in dicotyledonous plants such as soybeans and alfalfa mostly occurs at 3'-OH and 4'-OH sites, while monocotyledonous plants such as rice mostly occur at 3'-OH, 5'-OH and 7'-OH sites. However, there are currently no relevant reports on the functions of the FOMT gene in Polygonatum cyrtonema. Summary of the invention
[0005] In order to solve the above problems, the present invention provides a Polygonatum cyrtonema PcOMT1 gene and its application in promoting the synthesis and methylation of isoflavones.
[0006] The present invention provides a Polygonatum cyrtonema PcOMT1 gene, which has a nucleotide sequence as shown in SEQ ID No: 1, or a sequence in which one or more nucleotides are deleted, added and / or substituted but the function remains unchanged. Preferably, the nucleotide sequence of the Polygonatum cyrtonema PcOMT1 gene is SEQ ID No: 1.
[0007] The invention also provides a protein encoded by the Polygonatum cyrtonema PcOMT1 gene, which is composed of the amino acids shown in SEQ ID No.2.
[0008] The present invention also provides a method for promoting high isoflavone synthesis, which comprises preparing a modified plant containing the Polygonatum cyrtonema PcOMT1 gene, wherein the expression of the Polygonatum cyrtonema PcOMT1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID No: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the product obtained by transcription and the product obtained by translation can be increased.
[0009] The present invention also provides the use of the Polygonatum cyrtonema PcOMT1 gene in promoting the synthesis of isoflavones, and prepares a modified plant containing the Polygonatum cyrtonema PcOMT1 gene, wherein the expression of the Polygonatum cyrtonema PcOMT1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID No: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the increase in the product obtained by transcription and the product obtained by translation can be an increase.
[0010] In one embodiment, the plant is Polygonatum sibiricum or Nicotiana tabacum. Preferably, the plant is Nicotiana tabacum.
[0011] In one embodiment, the Polygonatum cyrtonema PcOMT1 gene is connected to a vector, transformed into tobacco through Agrobacterium-mediated transformation, and then screened, cultured, and transgenic strains are obtained.
[0012] The present application obtains the Polygonatum cyrtonema PcOMT1 gene by gene cloning, constructs a plant expression vector, and transiently transforms the Polygonatum cyrtonema PcOMT1 gene into Nicotiana benthamiana. The results show that the overexpressed Polygonatum cyrtonema PcOMT1 gene significantly increases the content of high isoflavones in tobacco leaves ((3R)-3-[(4-methoxyphenyl)methyl]-2,3-dihydro-5,7-dihydroxy-8-methoxy-6-methyl-4H-1-benzopyran-4-one (HJkn0236), (3R)-5,7-dihydroxy-8-methyl-3-(2',4'-dihydroxybenzyl)-chromogen-4-one (HJkn0239)). The results of the Polygonatum cyrtonema PcOMT1 gene cloning and in vivo functional verification of tobacco prove that the Polygonatum cyrtonema PcOMT1 gene has the function of promoting the synthesis of high isoflavones.
[0013] The present invention also provides the use of the Polygonatum cyrtonema PcOMT1 gene in catalyzing 2'-OH, 4'-OH methylation, and prepares a modified plant containing the Polygonatum cyrtonema PcOMT1 gene, wherein the expression of the Polygonatum cyrtonema PcOMT1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID No: 1. It should be noted that in the concept of "increased expression", "expression" refers to the process in which a gene is transcribed and translated to produce a biologically active protein; an increase in the intensity of the above process is an increase in expression, and specifically, the increase in the product obtained by transcription and the product obtained by translation can be an increase.
[0014] In one embodiment, the plant is Polygonatum cyrtonema or Nicotiana tabacum.
[0015] The present application clones the PcOMT1 gene of Polygonatum cyrtonema and constructs a prokaryotic expression vector of the target gene, obtains the protein of the PcOMT1 gene of Polygonatum cyrtonema through protein induction and purification, and performs in vitro enzyme activity functional verification. Using isoliquiritigenin as a methyl acceptor and SAM as a methyl donor, UHPLC-Q-TOF / MS analysis is used to preliminarily verify that PcOMT1 can catalyze isoliquiritigenin to produce 2'-methoxy-4',4-hydroxychalcone and 2',4'-dimethoxy-4-hydroxychalcone. The in vitro functional verification of the PcOMT1 gene of Polygonatum cyrtonema proves that the PcOMT1 gene has the function of catalyzing 2'-OH, 4'-OH methylation. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:
[0017] Figure 1 The total RNA electrophoresis result of the stem and leaf of Polygonatum cyrtonema of the present invention is shown in the figure, wherein M: DL5000 DNA Marker; 1: Polygonatum cyrtonema leaf.
[0018] Figure 2 The PCR amplification result of the PcOMT1 gene of Polygonatum cyrtonema of the present invention, wherein M: DL 2000 DNA Marker; 3: PcOMT1 gene amplification result.
[0019] Figure 3 The PCR bacterial test result of the PcOMT1 gene of Polygonatum cyrtonema of the present invention is shown in the figure. In the figure, M: DL 2000 DNA Marker; 11-15: different single colonies of the PcOMT1 gene.
[0020] Figure 4The results of double restriction enzyme digestion of the pCAMBIA1380-35S-GFP vector of the present invention and amplification of the PcOMT1 gene with homology arms, in which M: DL 5000 DNA Marker; 2: PcOMT1; 3: pCAMBIA1380-35S-GFP linear vector after restriction enzyme digestion.
[0021] Figure 5 The bacterial test results after GV3101 was transformed with pCAMBIA1380-35S-GFP-PcOMT1 of the present invention, in which M: DL 2000 DNA Marker; 6-10: single colonies with different PcOMT1 genes.
[0022] Figure 6 This is a construction diagram of the pCAMBIA1380-35S-GFP-PcOMT1 vector of the present invention.
[0023] Figure 7 It is the relative expression level of the PcOMT1 gene of the Polygonatum cyrtonema of the present invention.
[0024] Figure 8 The present invention's broad targeted metabolome identified changes in the content of high isoflavones in tobacco overexpressing the Polygonatum cyrtonema PcOMT1 (PC05G03386) gene.
[0025] Fig. 9 This is a construction diagram of the pGEX-4T-1-PcOMT1 vector of the present invention.
[0026] Fig.10 This is the SDS-PAGE analysis of the PcOMT1 protein of the present invention, wherein CL: protein supernatant resuspension; FT: protein flow-through; W1-3: protein washing solution; E1-5: protein elution solution.
[0027] Fig.11 The PcOMT1 of the present invention catalyzes UPLC analysis with isoliquiritigenin as substrate (1 hour).
[0028] Fig.12 The spectra of the product of isoliquiritigenin catalyzed by PcOMT1 of the present invention and the theoretical product 2',4'-dimethoxy-4-hydroxychalcone, in which A: the primary mass spectrum of the product of isoliquiritigenin catalyzed by PcOMT1; B: the secondary mass spectrum of the product of isoliquiritigenin catalyzed by PcOMT1 protein; C: the secondary mass spectrum of 2',4'-dimethoxy-4-hydroxychalcone.
[0029] Fig.13 This is the liquid phase diagram of the product (half an hour) of isoliquiritigenin catalyzed by PcOMT1 of the present invention. DETAILED DESCRIPTION
[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0032] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0033] 1. Experimental Materials
[0034] The Polygonatum cyrtonema used in the experiment was obtained from the Wildlife Conservation and Utilization Team of the National Key Laboratory of Zhejiang Agriculture and Forestry University (the culture conditions were natural conditions). The Nicotiana benthamiana used for metabolite content determination was cultured in the plant growth room of this laboratory (culture temperature was 25°C, light time was 16h·d -1 ).
[0035] 2. Experimental Methods
[0036] 2.1 Extraction of total RNA from Polygonatum cymbidium
[0037] 2.1.1 Extraction steps of total RNA from Polygonatum cyrtonema (kit)
[0038] The total RNA of Polygonatum cyrtonema was extracted using the MiniBEST Plant RNA Extraction Kit from TaKaRa. The instructions can be found on the official website of TaKaRa (https: / / www.takarabiomed.com.cn / ).
[0039] 2.1.2 Detection of total RNA from Polygonatum cymbidium
[0040] Take an appropriate amount of Polygonatum cyrtonema total RNA sample and spot it for about 15 minutes at 110V to detect RNA integrity. The electrophoresis solution needs to be replaced and the operation should be as quick as possible to avoid RNA degradation to the greatest extent. RNA concentration and A230 / A260 and A260 / A280 are parameters for judging the quality of the extracted RNA, which are obtained by measuring 1μL of total RNA sample with a spectrophotometer.
[0041] 2.2 Acquisition of the PcOMT1 gene from Polygonatum cyrtonema
[0042] 2.2.1 Design of full-length primers for the PcOMT1 gene in Polygonatum cyrtonema
[0043] The full-length primers of the CDS region of the PcOMT1 gene of Polygonatum cyrtonema were designed using the primer design software Primer5.0, which was synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.1 below:
[0044] Table 2.1 Primer sequences used for cloning PcOMT1
[0045]
[0046] 2.2.2 Synthesis of the first strand of cDNA
[0047] RNA was collected from leaves and flowers of Polygonatum cyrtonema and analyzed according to PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit instructions to obtain cDNA. Remove genomic DNA reaction, the system is shown in Table 2.2, gently mix, react at 42℃ for 2 minutes, and place on ice after the reaction. Then perform reverse transcription reaction, the system is shown in Table 2.3, gently mix, react at 37℃ for 15 minutes, and then place at 85℃ for 5 seconds. After the reaction, store at -20℃.
[0048] Table 2.2 Reaction system for removing genomic DNA
[0049]
[0050] Table 2.3 Reverse transcription reaction system
[0051]
[0052] 2.2.3 Amplification of the PcOMT1 gene fragment of Polygonatum cyrtonema
[0053] The cDNA of Polygonatum cyrtonema obtained by the above reverse transcription was diluted 5 times and then PCR amplified. The 25 μL reaction system is as shown in Table 2.4.
[0054] Table 2.4 PCR amplification reaction system
[0055]
[0056] The PCR reaction procedure was: pre-denaturation: 98°C for 2 min; 30 cycles (denaturation: 98°C for 10 s; annealing at 50°C for 10 s; extension at 72°C for 1 min); final extension: 72°C for 3 min; storage: 12°C. PcOMT1 is expressed at a low level in leaves, and its sequence similarity is high, making PCR difficult and the product specificity low. It was synthesized by GenScript Biotech Co., Ltd.
[0057] 2.2.4 Agarose gel electrophoresis
[0058] (1) Weigh 1.5 g agarose powder and dissolve it in 150 mL 1×TAE buffer. Heat in a microwave oven for about 3 minutes until the liquid becomes transparent. Add EB to the gelatin plate, pour the dissolved agarose liquid into the gelatin plate, and mix well.
[0059] (2) Place the electrophoresis plate, insert the comb, and slowly pour in the agarose solution prepared in (1). Let it stand for 40 minutes until the gel turns milky white.
[0060] (3) After the gel solidifies, remove the comb and place the electrophoresis plate with the gel in the electrophoresis tank.
[0061] (4) Pour 1×TAE electrophoresis buffer into the electrophoresis tank so that the liquid level is 1-2 mm above the surface of the agarose gel.
[0062] (5) DNA maker and PCR products were spotted into the wells as controls.
[0063] (6) After running at 120 V for 15 min, remove the gel and place it in a gel imager for observation and photography.
[0064] 2.2.5 PCR product gel recovery
[0065] A DNA gel recovery kit (Jinsha Biotechnology) was used to recover the PCR product. The instructions for the kit can be found on the official website of Jinsha Biotechnology (https: / / genesand.com / ).
[0066] 2.2.6 Connection
[0067] The ligation system is shown in Table 2.5. Mix gently and react at room temperature for 5 minutes. After the reaction is completed, place on ice.
[0068] Table 2.5 Connection system
[0069]
[0070] 2.2.7 E. coli transformation
[0071] The E. coli used for transformation was Treliefe 5a Chemically Competent Cell from Tsingke Biotechnology. For instructions, please refer to the official website of Tsingke Biotechnology (http: / / www.tsingke.net / ).
[0072] 2.2.8 Screening of positive clones
[0073] Pick a single colony from the long bacterial plate and put it into LB liquid containing the corresponding antibiotic, put it into a 37℃ shaker at 200r, and take out the turbid bacterial liquid after overnight for PCR verification. The system is as shown in Table 2.6. The PCR reaction program is: pre-denaturation: 98℃2min; 30 cycles (denaturation: 98℃10s; annealing 50℃10s; extension 72℃1min); final extension: 72℃3min; storage: 12℃.
[0074] Take 300 μL of the positive bacterial solution and send it to Youkang Biotechnology Co., Ltd. for sequencing verification.
[0075] Table 2.6 PCR verification reaction system
[0076]
[0077] 2.2.9 Extraction of positive clone plasmids was performed using a high-purity plasmid DNA minipreparation kit (Jinsha Biotech). The kit instructions are available on the official website of Jinsha Biotech ( https: / / genesand.com / ).
[0078] 2.3 Construction of pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector
[0079] The homologous recombination method was used to construct the pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector. The restriction sites on the pCAMBIA1380-35S-GFP vector are SalI and Xhol. The primers required for constructing the pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector were designed using SnapGene and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.7.
[0080] Table 2.7 Primer sequences used in pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector
[0081]
[0082] 2.3.1 Double restriction digestion of vector
[0083] In order to obtain the linearized vector, the plant expression vector pCAMBIA1380-35S-GFP was digested with restriction endonucleases SalI and XholⅠ. The digestion reaction system is shown in Table 2.8, and the reaction was carried out at 37℃ for 1h and placed on ice.
[0084] Table 2.8 Enzyme digestion reaction system
[0085]
[0086] 2.3.2 Agarose gel electrophoresis
[0087] Same as 2.2.4.
[0088] 2.3.3 Rubber recycling
[0089] Same as 2.2.5.
[0090] 2.3.4 Connection
[0091] Use the full-form gold biological -Basic Seamless Cloning and Assembly Kit was used to connect the target gene with the linear vector after double enzyme digestion by homologous recombination. The system was as shown in Table 2.9, and the reaction was performed at 50℃ for 20min.
[0092] Table 2.9pCAMBIA1380-35S-GFP-PcOMT1 vector connection system
[0093]
[0094] 2.3.5 E. coli transformation
[0095] Same as 2.2.7.
[0096] 2.3.6 Screening of positive clones
[0097] Same as 2.2.8.
[0098] 2.3.7 Extraction of positive clone plasmid
[0099] Same as 2.2.9.
[0100] 2.3.8 Agrobacterium transformation
[0101] The Agrobacterium used for transformation was GV3101 Chemically Competent Cell from Tsingke Biotechnology. For instructions, please refer to the official website of Tsingke Biotechnology (http: / / www.tsingke.net / ).
[0102] 2.3.9 Screening and saving positive clones
[0103] (1) Pick a single colony from the plate and place it in LB liquid containing Rif (100 μg / mL) and Kan (100 μg / mL) and culture at 28°C with shaking for 2 days.
[0104] (2) PCR verification of Agrobacterium culture.
[0105] (3) Take 500 μL of positive bacterial solution and add 500 μL of 50% glycerol, and store it in a -80°C ultra-low temperature refrigerator for later use.
[0106] 2.4 Tobacco transient transformation
[0107] (1) Take the bacterial solution stored at -80°C and spread it on Kan and Rif double-antibody plates and grow it at 28°C for two days. When Agrobacterium colonies grow, select a single colony and transfer it to 1 mL of double-antibody LB liquid medium and culture it at 28°C and 200 rpm. Continue to transfer the bacterial solution to 50 mL of double-antibody LB liquid medium and culture it overnight until the OD600 is about 0.6-0.8.
[0108] (2) Prepare injection buffer for tobacco transient expression.
[0109] (3) Centrifuge the bacterial solution at 3000 rpm for 10 min, discard the supernatant, add injection buffer to resuspend the precipitate, and continue centrifuging at 3000 rpm for 10 min. Discard the supernatant, and repeat twice.
[0110] (4) Resuspend the precipitate again with injection buffer, and the final OD600 of the Agrobacterium culture is between 0.3-0.5.
[0111] (5) Use a disposable 1 mL syringe to inject the Agrobacterium bacterial solution into the mesophyll from the back of the tobacco leaf and wipe off the residual bacterial solution on the leaf.
[0112] (6) The injected tobacco is cultured in the dark for 2-3 days.
[0113] 2.7 Metabolome of transiently transformed and genetically transformed tobacco
[0114] 2.7.1 Sample extraction
[0115] (1) Cut tobacco leaves that showed significant gene expression according to qRT-PCR results, wrap them in tin foil, and quickly freeze them in liquid nitrogen and store them at -80°C.
[0116] (2) The sample was freeze-dried in a vacuum oven and ground into powder using a grinder.
[0117] (3) Dissolve 50 mg of sample powder in 1 mL of 70% methanol internal standard extract.
[0118] (4) Add 500 μL petroleum ether, vortex for 5 min, let stand to separate layers, and centrifuge at 12,000 rpm for 10 min at 4 °C.
[0119] (5) After centrifugation, all the supernatant was filtered through a 0.22 μm PTFE filter and stored at -20°C until LC-MS / MS analysis.
[0120] 2.7.2 Chromatographic mass spectrometry acquisition conditions
[0121] The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (Applied Biosystems 6500QTRAP, https: / / sciex.com.cn / ).
[0122] The liquid phase conditions mainly include:
[0123] 1) Chromatographic column: Agilent SB-C18 1.8 μm, 2.1 mm × 100 mm;
[0124] 2) Mobile phase: Phase A is ultrapure water (with 0.1% formic acid added), phase B is acetonitrile (with 0.1% formic acid added);
[0125] 3) Elution gradient:
[0126] 0.00-9.00min: the proportion of phase B increases linearly from 5% to 95%;
[0127] 9.00-10.00min: The proportion of phase B is 95%;
[0128] 10.00-11.00min: the proportion of phase B drops to 5%;
[0129] 11.00-14.00min: the proportion of phase B is 5%;
[0130] 4) Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.
[0131] Mass spectrometry conditions mainly include:
[0132] The temperature of the electrospray ionization (ESI) source was 500°C; the ion spray voltage (IS) was 5500V (positive ion mode) / -4500V (negative ion mode); the ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set to 50, 60 and 25 psi, respectively, and the collision-induced ionization parameter was set to high. The QQQ scan used the MRM mode, and the collision gas (nitrogen) was set to medium. The DP and CE of each MRM ion pair were completed by further optimization of the declustering potential (DP) and collision energy (CE). A specific set of MRM ion pairs was monitored in each period according to the metabolites eluted in each period.
[0133] 2.8 In vitro functional verification of the PcOMT1 gene in Polygonatum cyrtonema
[0134] 2.8.1 Construction of expression vector
[0135] The pGEX-4T-1-PcOMT1 overexpression vector was constructed by homologous recombination. The restriction sites on the pGEX-4T-1 vector are BamHⅠ and XholⅠ. The primers required for constructing the pGEX-4T-1-PcOMT1 overexpression vector were designed using SnapGene and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. The primers are shown in Table 2.13.
[0136] Table 2.13 Primer sequences used in pGEX-4T-1-PcOMT1 overexpression vector
[0137]
[0138] 2.8.2 Double digestion of vector
[0139] In order to obtain the linearized vector, the prokaryotic expression vector pGEX-4T-1 was digested with restriction endonucleases BamHI and XholⅠ. The digestion reaction system is shown in Table 2.14, and the reaction was carried out at 37℃ for 1 hour and placed on ice.
[0140] Table 2.14 Enzyme digestion reaction system
[0141]
[0142] 2.8.3 Agarose gel electrophoresis
[0143] Same as 2.2.4.
[0144] 2.8.4 Rubber Recycling
[0145] Same as 2.2.5.
[0146] 2.8.5 Connection
[0147] Use the full-form gold biological -Basic Seamless Cloning and Assembly Kit was used to connect the target gene with the linear vector after double enzyme digestion by homologous recombination. The system was as shown in Table 2.15, and the reaction was performed at 50°C for 20 min.
[0148] Table 2.15 Vector ligation reaction system
[0149]
[0150] 2.8.6 E. coli transformation
[0151] Same as 2.2.7
[0152] 2.8.7 Screening of positive clones
[0153] Same as 2.2.8.
[0154] 2.8.8 Extraction of positive clone plasmid
[0155] Same as 2.2.9.
[0156] 2.9 Prokaryotic expression and purification of pGEX-4T-1-PcOMT1
[0157] 2.9.1 Transformation of target gene
[0158] Take the pGEX-4T-1-PcOMT1 plasmid and transform the Transetta (DE3) expression strain. The transformation method is the same as above. After transformation, pick a single colony and add it to 1 mL LB liquid culture medium containing 100 mg / mL Amp and shake culture at 37°C and 200 rpm until the bacterial solution becomes turbid. Add 50% glycerol to the strains that test positive and store them at -80°C.
[0159] 2.9.2 Induced expression of target protein
[0160] (1) Using empty Transetta (DE3) as a negative control, 500 μL of both the experimental group and the control group were added to 10 mL of LB liquid medium containing Amp and cultured at 37°C with shaking at 200 rpm until turbidity;
[0161] (2) Take 10 mL of activated bacterial solution, add 500 mL of LB liquid medium containing Amp, and culture at 37°C with shaking at 200 rpm until OD600 is between 0.5 and 0.6;
[0162] (3) Add IPTG inducer, and the final concentration of IPTG in the bacterial solution is 0.5 mM;
[0163] (4) Incubate at 16°C with shaking at 200 rpm for 12 h.
[0164] 2.9.3 Extraction of target protein
[0165] (1) Centrifuge the bacterial solution after IPTG induction at 5000 rpm for 10 min at 4°C, discard the supernatant, and collect the bacterial cells;
[0166] (2) Resuspend in 40 mL PBS solution, centrifuge at 5000 rpm for 10 min at 4°C, discard the supernatant, and resuspend in 40 mL PBS solution;
[0167] (3) Use an ultrasonic cell disruptor, No. 2 amplitude, 33% power, disrupt for 2 seconds, pause for 5 seconds, and continue for 30 minutes.
[0168] (4) Centrifuge the disrupted bacterial solution at 14,000 rpm at 4°C for 10 min, transfer the supernatant to a new centrifuge tube, add PBS solution to the precipitate and resuspend it, and keep both the supernatant and the resuspended solution for gel running.
[0169] (5) Take 1 mL of GST-tag and add 1 mL of PBS solution. Add it to the gravity column and discard the effluent. Repeat 4 times. For the last time, keep 1 mL of the mixture and add it to the supernatant. Rotate slowly on a shaker at 4°C for 1 h.
[0170] (6) The supernatant containing the GST-tag is passed through a gravity column, and the flowthrough is the effluent. The target protein binds to the GST-tag in the gravity column, and the protein is eluted with 5 mL of Elution Buffer to obtain the purified protein.
[0171] 2.9.4 SDS-PAGE electrophoresis analysis
[0172] (1) Using Omni-Easy TM One-step PAGE rapid preparation kit (10%) was used to prepare electrophoresis gel. For instructions, please refer to http: / / www.epizyme.cn / index.php?c=article&id=1225.
[0173] (2) Take 40 μL of supernatant, precipitate resuspension, flowthrough and purified protein into 1.5 mL centrifuge tubes, add 10 μL of 5× protein loading buffer respectively, boil at 100°C for 10 min, and then centrifuge at 12000 rpm for 2 min.
[0174] (3) Take 20 μL of protein sample and load it on 10% PAGE gel at 120 V for 70 min.
[0175] (4) Remove the colloid, cut off the concentrated gel, place the separation gel in Coomassie Brilliant Blue rapid staining solution, stain for 30 min on a shaker at 20 rpm, and then decolorize with clean water overnight.
[0176] (5) The next day, observe the decolorization of the gel.
[0177] 2.9.5 Target protein concentration
[0178] (1) Add the purified protein to the upper layer of the ultrafiltration tube, centrifuge at 4000 rpm for 20 min at 4°C, and discard the effluent.
[0179] (2) Add 10 mL of PBS buffer to the upper layer of the ultrafiltration tube, centrifuge at 4000 rpm for 30 min, discard the effluent, and repeat once.
[0180] (3) Collect the remaining liquid on the upper layer of the ultrafiltration tube as the concentrated protein solution and store it at -80°C for subsequent experiments.
[0181] 2.9.6 Protein concentration determination
[0182] The protein concentration was determined using the BCA Protein Assay Kit. The instructions can be found at www.epizyme.cn.
[0183] 2.10 Verification of the catalytic function of the PcOMT1 gene in Polygonatum cyrtonema
[0184] The in vitro enzymatic reaction system is shown in Table 2.16. The experimental group is a PBS solution of pGEX-4T-1-PcOMT1 protein after induction expression and purification; the positive control group is a PBS solution of CHOMT protein after induction expression and purification, and the negative control is boiled protein. The substrates are isoliquiritigenin and 2'-O-methylisoliquiritigenin (concentration 1mM). The reaction is carried out at 37°C and 200rpm for 30 minutes or 1 hour.
[0185] Table 2.16 In vitro enzymatic reaction system
[0186]
[0187] (2) After the reaction was completed, 1 mL of methanol solution was added to terminate the reaction.
[0188] (3) The reaction solution was centrifuged at high speed for 20 min, and an appropriate amount of the upper layer liquid was added to an Agilent liquid phase vial, and the enzyme activity product was analyzed by ultra-performance liquid chromatography (UPLC).
[0189] 2.11 Detection of in vitro enzyme activity products
[0190] The samples were qualitatively determined by UHPLC-Q-TOF / MS. Chromatographic conditions: The samples were separated by Agilent 1290 InfinityLC ultra-high performance liquid chromatography system (UHPLC) C-18 column; column temperature 40°C; flow rate 0.4 mL / min; injection volume 2 μL; mobile phase composition A: water + 25 mM ammonium acetate + 0.5% formic acid, B: methanol; gradient elution program is as follows: 0---0.5 min, 5% B; 0.5---10 min, B linearly changes from 5% to 100%; 10.0---12.0 min, B is maintained at 100%; 12.0---12.1 min, B linearly changes from 100% to 5%; 12.1---16 min, B is maintained at 5%; the samples were placed in an automatic sampler at 4°C during the entire analysis process. In order to avoid the influence of fluctuations in instrument detection signals, the samples were analyzed continuously in a random order. Mass spectrometry conditions: AB Triple TOF 6600 mass spectrometer was used to collect the primary and secondary spectra of the samples. The ESI source conditions after chromatographic separation were as follows: ion source gas 1 (Gas1): 60, ion source gas 12 (Gas2): 60, gas curtain (CUR): 30, source temperature: 600℃, ion voltage floating (ISVF) ±5500V (positive and negative modes); TOF MS scan m / z range: 60-1000Da, integrated ion scan m / z range: 25-1000Da, TOFMS scan accumulation time 0.20 seconds / spectrum, integrated ion scan accumulation time 0.05 seconds / spectrum; secondary mass spectra were obtained using information-dependent analysis (IDA), and the high-sensitivity mode was used, declustering voltage (DP): ±60V (positive and negative modes), collision energy: 35±15eV, IDA settings were as follows to exclude isotopes within 4Da, and candidate ions to be monitored in each cycle: 10. The mass spectrometry data were analyzed using MSDIAL (ver.4.9.221218 Windows x64).
[0191] 3 Experimental results
[0192] 3.1 Extraction of total RNA from Polygonatum cymbidium
[0193] The concentration of extracted RNA was determined, and the results are shown in Table 3.1. The 260 / 280 value of RNA from Polygonatum cyrtonema leaves was in the range of 1.8-2.2. The results of RNA agarose gel electrophoresis are shown in Table 3.1. Figure 1 , 28S and 18S bands were bright and clear, and the RNA integrity was good. Therefore, the RNA quality of Polygonatum cyrtonema leaves was good and could be used in subsequent reverse transcription cDNA experiments.
[0194] Table 3.1 RNA concentration determination of Polygonatum cyrtonema root, stem and leaf
[0195]
[0196] 3.2 Acquisition of the PcOMT1 gene from Polygonatum cyrtonema
[0197] Agarose gel electrophoresis was used to detect the target gene product amplified from cDNA and the company's synthetic gene. The results were as follows Figure 2 The target fragment size of the PcOMT1 gene of Polygonatum cyrtonema is 1098bp. It can be seen from the electrophoresis result that the fragment size of the PCR amplification product is basically consistent with the theoretical gene length. Therefore, it is preliminarily determined that the product fragment obtained by PCR amplification is the PcOMT1 gene of Polygonatum cyrtonema. The bacterial test results after ligation and transformation are shown in Figure 3 As shown, the sequencing results returned after the bacterial solution with positive bacterial test was sent to the company for sequencing showed that it was the same as the sequence of the Polygonatum cyrtonema genome resequencing, indicating that the PcOMT1 gene of Polygonatum cyrtonema was successfully cloned.
[0198] 3.3 Construction of pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector
[0199] 3.3.1 Obtaining linear vector and target gene
[0200] The circular pCAMBIA1380-35S-GFP vector was double-digested with SalI and XholI restriction endonucleases to obtain a linear pCAMBIA1380-35S-GFP vector. The PcOMT1 gene fragment of Polygonatum cyrtonema was amplified by PCR to obtain a PcOMT1 gene fragment with homology arms. The linear vector pCAMBIA1380-35S-GFP and the PcOMT1 gene fragment with homology arms were gel-recovered ( Figure 4 ) and then used to construct the vector.
[0201] 3.3.2 Vector construction and bacterial testing
[0202] After pCAMBIA1380-35S-GFP-PcOMT1 was transformed into GV3101 Agrobacterium, single clone plaques were picked from the plate. The bacterial test results were as follows: Figure 5 .
[0203] The position of the band obtained from the bacterial test was consistent with the size of the PcOMT1 gene, and the positive bacterial solution was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing verification and comparison. The sequencing results showed that the pCAMBIA1380-35S-GFP-PcOMT1 overexpression vector was successfully constructed. The vector schematic diagram is shown in Figure 6 shown.
[0204] 3.4 Metabolome analysis and expression analysis of tobacco plants transiently transformed with the PcOMT1 gene of Polygonatum cyrtonema
[0205] Figure 7The results showed that the PcOMT1 gene of Polygonatum cyrtonema was significantly expressed in tobacco lines. Based on the UPLC-MS / MS detection platform, extensive targeted metabolomics analysis was performed on the leaves of tobacco overexpressing the PcOMT1 gene of Polygonatum cyrtonema. Each sample group had 3 biological replicates, and the metabolites of the samples were qualitatively and quantitatively analyzed by mass spectrometry. Compared with the empty sample, the following isoflavones in the leaves of PcOMT1 overexpressing tobacco were significantly increased: (3R)-3-[(4-methoxyphenyl)methyl]-2,3-dihydro-5,7-dihydroxy-8-methoxy-6-methyl-4H-1-benzopyran-4-one (HJkn0236), (3R)-5,7-dihydroxy-8-methyl-3-(2',4'-dihydroxybenzyl)-chromogen-4-one (HJkn0239) ( Figure 8 The results of in vivo functional verification of the PcOMT1 gene in tobacco from Polygonatum cyrtonema proved that the PcOMT1 gene in Polygonatum cyrtonema has the function of promoting the synthesis of high isoflavones.
[0206] 3.5 Construction of prokaryotic expression vector pGEX-4T-1-PcOMT1
[0207] 3.5.1 Obtaining linear vector and target gene
[0208] The circular pGEX-4T-1 vector was double-digested with BamHI and XholⅠ restriction endonucleases to obtain a linear pGEX-4T-1 vector, which was recovered by gel and used to construct a vector. The PcOMT1 gene of Polygonatum cyrtonema was amplified by PCR to obtain a PcOMT1 gene fragment with homology arms, which was also recovered by gel and used to construct a vector.
[0209] 3.5.2 Vector construction and bacterial testing
[0210] After pGEX-4T-1-PcOMT1 was transformed into dh5α, a single clone plaque was picked from the plate. The position of the band obtained by bacterial test was consistent with the size of the PcOMT1 gene fragment of Polygonatum cyrtonema. The positive bacterial solution was sent to Hangzhou Youkang Biotechnology Co., Ltd. for sequencing verification and comparison.
[0211] The sequencing results showed that the pGEX-4T-1-PcOMT1 prokaryotic expression vector was successfully constructed, and the PcOMT1 vector was then transformed into the Transetta expression strain. Fig. 9 shown.
[0212] 3.6PcOMT1 protein expression and purification
[0213] 3.6.1 pGEX-4T-1-PcOMT1 SDS-PAGE results
[0214] After prokaryotic expression and crushing and centrifugation, the protein supernatant resuspension, protein flow-through, protein washing solution, and protein elution solution of PcOMT1 protein were subjected to SDS-PAGE electrophoresis. After electrophoresis, Coomassie Brilliant Blue fast staining solution was used for staining and decolorization overnight. The results are as follows: Fig.10 As shown in the figure, the size of PcOMT1 protein is 39.89 kDa, and the size of GST tag on pGEX-4T-1 vector is 26 kDa, so the size of recombinant protein after fusion of PcOMT1 protein and GST tag is 65.89 kDa.
[0215] 3.6.2 Determination of target protein concentration after concentration
[0216] Taking a single time as an example, the ELISA reader measures the absorbance value of each well of the ELISA plate at a wavelength of 562nm as shown in Table 3.2.
[0217] Table 3.2 Absorbance values of each well at wavelength A562
[0218]
[0219] With the concentration of the protein standard solution as the x-axis value and the absorbance of the protein standard solution as the y-axis value, a standard curve was drawn to calculate the PcOMT1 protein concentration.
[0220] The linear regression equation was derived based on the absorbance, and we obtained y = 0.0002x + 0.0102, R 2 =0.9986, where x is the concentration of the protein standard solution (ug / mL), and y is the absorbance of the measured protein standard solution at wavelength A562 (nm). The concentration of the PcOMT1 protein concentrate sample was calculated based on this equation for the subsequent catalytic characteristics study.
[0221] 3.7 In vitro functional verification of the PcOMT1 gene in Polygonatum cyrtonema
[0222] Comparison of the liquid chromatograms of the in vitro enzyme activity product of Polygonatum cyrtonema PcOMT1 and the theoretical product 2'-O-methylisoliquiritigenin standard, the results are as follows Fig.11 and Fig.13 As shown in the figure, it was found that the product obtained after 1 hour of PcOMT1 in vitro enzymatic activity was not the theoretical product 2'-O-methyl isoliquiritigenin (4,4'-dihydroxy-2'-methoxychalcone), while the product obtained after half an hour of PcOMT1 in vitro enzymatic activity was the theoretical product 2'-O-methyl isoliquiritigenin. The peak of the product obtained by 1 hour of catalysis was compared with the primary mass spectrum, and it was found that the m / z of the product obtained by PcOMT1 in vitro enzymatic activity was 283 [MH] - The suspected product ( Fig.12A), namely the dimethyl substituted product. The secondary mass spectrum analysis of the product revealed the presence of [MH-18] - Fragment ions ( Fig.12 B) By comparing with the online website pmhub (https: / / pmhub.org.cn), it was found that the substance 2',4'-dimethoxy-4-hydroxychalcone has a similar mass-to-nuclear ratio and the same fragment ion [MH-18] as the suspected product. - ( Fig.12 C).
[0223] To confirm that the product of PcOMT1 in vitro enzyme activity is a chalcone with one more methyl group in the 2'-O-methyl isoliquiritigenin skeleton, 2'-O-methyl isoliquiritigenin was used as substrate and PcOMT1 protein catalyzed the reaction to obtain a small amount of product with a peak time similar to that of the product with isoliquiritigenin as substrate, see Fig.13 Therefore, it was confirmed that the product was 2',4'-dimethoxy-4-hydroxychalcone, proving that PcOMT1 can catalyze the methylation of 2'-OH, 4'-OH.
[0224] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A Polygonatum cyrtonema PcOMT1 gene, characterized in that: The Polygonatum cyrtonema PcOMT1 gene has the nucleotide sequence shown in SEQ ID No: 1, or a sequence in which one or several nucleotides are deleted, added and / or substituted but the function remains unchanged.
2. The gene according to claim 1, characterized in that The nucleotide sequence of the gene is SEQ ID No:
1.
3. The protein encoded by the gene according to claim 1 or 2, characterized in that The protein consists of the amino acids shown in SEQ ID No.
2.
4. A method for promoting high isoflavone synthesis, the method comprising preparing a modified plant containing the Polygonatum cyrtonema PcOMT1 gene, characterized in that: Compared with the wild-type plant, in the modified plant, the expression of the Polygonatum cyrtonema PcOMT1 gene is increased; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID NO:
1.
5. The application of the Polygonatum cyrtonema PcOMT1 gene in promoting the synthesis of isoflavones, characterized in that: A modified plant containing the Polygonatum cyrtonema PcOMT1 gene is prepared, wherein the expression of the Polygonatum cyrtonema PcOMT1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID NO:
1.
6. The use according to claim 5, characterized in that: The plant is polygonatum or tobacco.
7. The use according to claim 6, characterized in that: The plant is tobacco.
8. The use according to claim 7, characterized in that: The Polygonatum cyrtonema PcOMT1 gene is connected to a vector, transformed into tobacco through Agrobacterium-mediated transformation, and then screened, cultured and transgenic strains are obtained.
9. The use of the Polygonatum cyrtonema PcOMT1 gene in catalyzing 2'-OH, 4'-OH methylation, characterized in that: A modified plant containing the Polygonatum cyrtonema PcOMT1 gene is prepared, wherein the expression of the Polygonatum cyrtonema PcOMT1 gene is increased in the modified plant relative to the wild-type plant; wherein the base sequence of the Polygonatum cyrtonema PcOMT1 gene is shown in SEQ ID NO:
1.
10. The use according to claim 9, characterized in that: The plant is polygonatum or tobacco.