CN114807082B - Diqian flavonoid glucuronyl glycosyltransferase and encoding gene and application thereof - Google Patents

Abstract

The invention provides a dittany flavonoid glucuronyl glycosyltransferase and a coding gene and application thereof, belonging to the technical fields of genetic engineering and enzyme engineering. The invention clones and obtains the glycosyltransferase which can efficiently catalyze the glycosylation of the dihydroflavone (naringenin, hesperetin, pinocembrin and eriodictyol) and the glucuronidation of the flavone, the dihydroflavone and the flavonol from liverwort for the first time. In vitro enzyme activity functional identification proves that MpUGT742A1 has higher catalytic activity on the dihydroflavone, can catalyze and generate dihydroflavone-7-O-glucose/glucuronide, and can also convert various flavone and flavonol compounds to generate mono-or di-glucuronide, thereby being applicable to biosynthesis and other medicinal glycoside compounds such as scutellarin, and having higher economic value and wide application prospect.

Description

Diqian flavonoid glucuronyl glycosyltransferase and encoding gene and application thereof

Technical Field

The invention belongs to the technical fields of genetic engineering and enzyme engineering, and in particular relates to a dittany flavonoid glucuronic acid glycosyltransferase and a coding gene and application thereof.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Flavonoids are important secondary metabolites in nature, are widely present in plants, and they exist mainly in the form of glycosides in plants. Glycosylation of flavonoids in plants is catalyzed by Glycosyltransferases (GT), which transfer activated sugar molecules to acceptor substrates to produce structurally diverse glycoside products. Glycosylation modifications can improve the solubility of a compound and affect its bioavailability and bioactivity in humans.

Flavone glycosides have a variety of pharmacological activities, such as antioxidant, anti-inflammatory and neuroprotective functions. Flavonoid glucuronides are compounds having one or more glucuronic acid substitutions on a flavonoid, and have great potential for pharmaceutical use due to their structural and metabolic specificity in humans. Some glucuronide medicinal activities have been mined, such as scutellarin, and have been developed as clinical medicines for treating cardiovascular and cerebrovascular diseases. Luteolin-3' -O-glucuronide can inhibit bladder cancer cell proliferation. However, the content of the compounds in plants is low, the extraction and separation processes are complex, and the manual acquisition is difficult, so that the identification of flavonoid glucuronyl transferase can be used for biosynthesis of the compounds by a high-efficiency and simple method.

The flavonoid glycosyltransferases identified at present are mostly characterized by taking UDP-glucose as a sugar donor, and few glucuronic acid glycosyltransferases (UGAT) are reported and are mainly identified from Labiatae plants. Mosses are an important plant group for the transition from aquatic to terrestrial, and are in vivo rich in structurally diverse secondary metabolites, whereas glycosyltransferases in mosses are currently only a few identified. Liverwort (Marchantia polymorpha) is a model plant of the moss species and has been identified in vivo as also comprising some mono-or disaccharide flavonoid glucuronides, such as luteolin-7, 3 '-O-glucuronide, luteolin-7-O-glucuronide, luteolin-4' -O-glucuronide and apigenin-7-O-glucuronide. However, the inventors found that the studies of Glycosyltransferases (GTs) catalyzing the production of flavonoid glucuronides in liverwort have not been reported yet.

Disclosure of Invention

Based on the defects of the prior art, the invention provides the dittany flavonoid glucuronosyltransferase and the encoding gene and application thereof. The glucuronyl transferase from liverwort of the invention is found by research, and the enzyme can catalyze the glucuronidation of flavone, flavanone and flavonol to generate various monoglycoside and disaccharide glycoside products, and the glucuronyl transferase also comprises some medicinal compounds such as scutellarin, and the like, and can catalyze the glycosylation of the flavanone (naringenin, hesperetin and pinocembrin), thus having higher practical application value and economic value.

In order to achieve the technical purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a protein designated as MpUGT742A1, said protein designated as MpUGT742A1 being derived from Dichium (Marchantia polymorpha). Experiments prove that the compound can efficiently catalyze the glucuronidation of flavone, flavanone and flavonol to generate various monoglycoside and disaccharide glycoside products, wherein the compound also comprises some medicinal compounds such as scutellarin and the like, and in addition, the compound can catalyze the glycosylation of flavanone (naringenin, hesperetin and pinocembrin).

Wherein the MpUGT742A1 has an amino acid sequence shown in SEQ ID NO. 1.

SEQ ID NO.1 consists of 513 amino acid residues.

In a second aspect of the present invention, there is provided a gene encoding the MpUGT742A 1.

Wherein the gene has the nucleotide sequence as set forth in any one of (a 1) or (a 2):

(a1) A nucleotide sequence shown as SEQ ID NO. 2;

(a2) A nucleotide sequence complementary to (a 1).

SEQ ID NO.2 consists of 1542 nucleotides, of which nucleotides 1 to 1539 are coding sequences and nucleotides 1530 to 1542 are transcribed to terminate the codon termination peptide chain synthesis.

In a third aspect of the present invention, a recombinant expression vector containing the above-mentioned coding gene, a recombinant cell or transformant containing the above-mentioned coding gene are also within the scope of the present invention.

The recombinant expression vector is obtained by effectively connecting the genes to an expression vector, and the expression vector can be a plasmid; the plasmid further comprises pET32a and pGWB5;

the recombinant cell may be any one or more of a bacterial cell, a fungal cell or a plant cell;

the bacterial cells include E.coli (e.g., BL21 (DE 3)), agrobacterium tumefaciens (e.g., GV 3101), agrobacterium rhizogenes, and the like.

The plant cells include tobacco epidermal cells.

In the fourth aspect of the present invention, the use of the protein MpUGT742A1 as a glycosyltransferase is also within the scope of the present invention.

In a fifth aspect of the invention, the coding gene, recombinant expression vector, transformed cell or transgenic plant is used in the preparation of MpUGT742A 1.

In a sixth aspect of the present invention, there is provided the use of the protein MpUGT742A1 as follows (b 1) - (b 2):

(b1) Glucuronidation or glucosylation of catalytic flavones and flavonoids;

(b2) Preparing glycoside or uronic acid glycoside compounds;

in the (b 1), the flavonoid and flavonoid compounds include but are not limited to flavonoid compounds, flavonol compounds and dihydroflavonoid compounds;

wherein the flavonoid compounds include, but are not limited to, apigenin, luteolin, baicalein and scutellarin;

the flavonols include, but are not limited to, quercetin, kaempferol and isorhamnetin;

the dihydroflavonoids include, but are not limited to naringenin, hesperetin, eriodictyol, glycyrrhizin and pinocembrin.

More specifically, the protein MpUGT742A1 is capable of catalyzing the glucosylation of dihydroflavonoids and the glucuronidation of flavonoids, flavanoids and flavonols;

experiments prove that the MpUGT742A1 has higher catalytic activity on naringenin, hesperetin and pinocembrin, and catalyzes the generation of flavanone-7-O-glucoside. In fact, it has high catalytic activity on flavonoid substrates of various structural types (flavones, flavanones, flavonols) and catalyzes the production of various mono-and disaccharide glycoside products.

In the (b 2), the glycoside compound includes a dihydroflavone glycoside compound;

the uronic acid glycoside compounds comprise flavonoid glucuronide, dihydroflavonoid glucuronide and flavonol glucuronide compounds;

the beneficial effects of one or more of the technical schemes are as follows:

the MpUGT742A1 from liverwort provided by the technical proposal is glucuronyl transferase which is first discovered in moss plants and can catalyze glucuronidation of flavonoid, flavanone and flavonols to generate various monoglycoside and bisglycoside; it also catalyzes the glucosylation of the dihydroflavonoids. The full-length sequence of the gene was obtained from the cDNA using PCR techniques. And (3) transforming escherichia coli BL21 (DE 3) by constructing a pET32a protein expression vector to obtain the target protein. In vitro enzyme activity functional identification proves that MpUGT742A1 can efficiently catalyze dihydroflavone to generate corresponding glucoside or glucuronide, and can catalyze flavone and flavonol to generate corresponding glucuronide, and the enzyme can be used for biosynthesis of various flavonoid glycosylation products, so that the enzyme has high economic value and wide application prospect.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

Fig. 1: electrophoretogram of ORF amplification product of the objective gene MpUGT742A 1.

Fig. 2: SDS-PAGE electrophoresis of the MpUGT742A1 protein. Wherein: m is the molecular mass standard of protein; lane 1: supernatant of MpUGT742A 1; lane 2: purified protein of MpUGT742A 1.

Fig. 3: the MpUGT742A1 takes UDP-glucose as a main enzyme activity catalytic reaction HPLC spectrum of a sugar donor, and LC-MS spectrum and a reaction formula of a product. The sugar receptors are naringenin, pinocembrin and hesperetin respectively. Each enzyme activity catalytic reaction is used as a control by the catalytic reaction of the empty carrier.

Fig. 4: the MpUGT742A1 takes UDP-glucuronic acid as a sugar donor, and flavone (scutellarin, baicalein, apigenin and luteolin) as an HPLC (high performance liquid chromatography) spectrum of a main enzyme activity catalytic reaction of a sugar acceptor and an LC-MS spectrum of a product. Each enzyme activity catalytic reaction is used as a control by the catalytic reaction of the empty carrier.

Fig. 5: mpUGT742A1 takes UDP-glucuronic acid as a sugar donor, and dihydroflavone (naringenin, glycyrrhizin, hesperetin and pinocembrin) as an HPLC spectrum of a main enzyme activity catalytic reaction of a sugar acceptor and an LC-MS spectrum of a product. Each enzyme activity catalytic reaction is used as a control by the catalytic reaction of the empty carrier.

Fig. 6: mpUGT742A1 takes UDP-glucuronic acid as a sugar donor, takes flavonol (kaempferol, quercetin and isorhamnetin) as an HPLC (high performance liquid chromatography) spectrum of a main enzyme activity catalytic reaction of a sugar acceptor and takes LC-MS spectrum of a product. Each enzyme activity catalytic reaction is used as a control by the catalytic reaction of the empty carrier.

Fig. 7: conversion of a range of flavonoids by MpUGT742A1 with UDP-glucuronic acid as sugar donor.

Fig. 8: subcellular localization map of mtungt 742 A1.

Wherein: a: green fluorescent signal under excitation light; b: fluorescence signal of chloroplast under excitation light

C: tobacco epidermal cells under natural light; d: A. b, C.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

Example 1 cloning of the expression Gene MpUGT742A1

1.1 CTAB-PVP method for extracting total RNA of liverwort

(1) Taking fresh liverwort plant material, cleaning, sucking water with filter paper, placing in a precooled mortar, adding liquid nitrogen, and grinding into powder.

(2) Taking a proper amount of powder into a 2mL inlet centrifuge tube pre-cooled in advance, adding 600-800 mu l of CTAB-PVP extracting solution preheated at 65 ℃, and mixing uniformly upside down.

The preparation method of the CTAB-PVP extraction buffer solution comprises the following steps:

100mM Tris-HCl (pH 8.0), 2% CTAB (w/v), 2% PVP (w/v), 25mM EDTA,2M NaCl, mercaptoethanol added to 0.2% after autoclaving; solution configuration ddH treated with DEPC ₂ O, after autoclaving, the mixture is ready for use.

(3) Water bath at 65 ℃ for 30min, and reverse mixing once every 10min.

(4) After cooling to room temperature, 600-800. Mu.l of chloroform was added, and after mixing was reversed, the mixture was centrifuged at 13,000rpm at 4℃for 10min.

(5) Transferring the supernatant to a new centrifuge tube with 2mL inlet, adding 600-800 μl chloroform, shaking and mixing well, and centrifuging at 4deg.C at 13,000rpm for 10min.

(6) The above procedure was repeated (i.e., three extractions with chloroform).

(7) The supernatant was carefully aspirated and transferred to a fresh 1.5mL centrifuge tube, 1/3 of the 8M LiCl was added, and the mixture was allowed to stand at-20℃for 3 hours or more.

(8) Centrifuge at 13,000rpm for 10min at 4℃and discard the supernatant.

(9) The precipitate was washed 2-3 times with 700. Mu.l of 75% ethanol (DEPC water formulation). Centrifuging, discarding the supernatant, and volatilizing residual ethanol.

(10) Total RNA was prepared by dissolving RNA in 30. Mu.l of sterilized water after Proteinase K treatment. The concentration and quality of the extracted RNA were measured using a BioPhotometer plus nucleic acid protein meter.

1.2 Full-length amplification of MpUGT742A1 Gene

1.2.1 primer design

The open reading frame of MpUGT742A1 was found using software Bioxm 2.6 (Open Reading Frame). Full-length primers MpUGT742A1-F/R were designed on both sides of the ORF, and the gene was amplified.

1.2.2 cDNA Synthesis

The cDNA template strand was obtained by PCR technique using total RNA of the extracted liverwort as a template and using PrimerScript RT Master Mix reverse transcription system.

The reverse transcription system and reverse transcription procedure were as follows:

(1) Removal of genomic DNA

The components are added into an import PCR tube, gently mixed and then put in a water bath at 42 ℃ for 5min.

(2) Reverse transcription PCR

The reverse transcription procedure in the PCR instrument was: 37 ℃ for 15min; denaturation at 85℃for 15s, incubation at 4 ℃.

The reverse transcription product was stored at-20℃and diluted 10-fold before use.

1.2.3 amplification of the Gene of interest

The amplification was performed using the diluted reverse transcribed cDNA as a template and MpUGT742A1-F/R as a primer.

The amplification system and the amplification procedure were as follows:

the components are added into a 200 mu L PCR tube to be uniformly mixed, and the mixture is put into a PCR instrument for amplification after low-speed centrifugation according to the following procedures: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 52℃for 15s, elongation at 72℃for 45s,33 cycles; extending at 72℃for 10min.

And (3) detecting the PCR reaction product by agarose gel electrophoresis, and cutting and recycling the target size strip by the following method.

The PCR products were subjected to agarose gel electrophoresis (1.4%, W/V, g/100 ml) and recovered using TIANGEN gel recovery kit. The method comprises the following steps:

(1) After agarose gel electrophoresis of the PCR product, ethidium Bromide (EB) is used for dying for 5min, a gel block containing the band with the target size is cut off rapidly under an ultraviolet lamp, and the gel block is placed into a 1.5mL centrifuge tube.

(2) 200. Mu.L of solution PC was added and the gel was dissolved in a 55℃water bath for 5-6 min. The tubes were inverted every 2min to allow complete dissolution.

(3) The adsorption column CB2 was placed in a 2mL collection tube, and the above sol was transferred to the adsorption column CB2 and centrifuged at 12,000rpm for 1min, and the filtrate in the collection tube was discarded.

(4) 600. Mu.L of the rinse PW was added to the column CB 2. Centrifuge at 12,000rpm for 1min, discard the filtrate.

(5) Repeating the operation step (4).

(6) The filtrate was discarded, and the adsorption column CB2 was centrifuged at 12,000rpm for 2 minutes at room temperature to remove the rinse solution as much as possible.

(7) The adsorption column CB2 is placed in a new 1.5mL centrifuge tube, and the centrifuge tube is opened until the ethanol volatilizes. Add 30. Mu.L ddH to the center of the column membrane ₂ O, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min, and collecting DNA solution for immediate use or preservation at-20deg.C.

1.3 destination fragment blunt end vector ligation

The above gel recovery product fragment was ligated to the blunt-ended vector pTOPO according to the following reaction system:

and uniformly mixing the reaction systems, putting the mixture into a PCR instrument to react for 5 minutes at 25 ℃, and then converting the final product into the escherichia coli DH5 alpha.

1.4 conversion

E.coli DH5 alpha competent cells (50. Mu.L) preserved at-80℃were removed and thawed on ice, 5. Mu.L of ligation product was added, gently mixed by blowing, and left on ice for 30min; after 45s of heat shock in a water bath at 42 ℃, the mixture is rapidly placed on ice for 2min, 500 mu L of antibiotic-free LB culture medium is added, then the mixture is subjected to shaking culture for 1h in a culture box at 37 ℃,200 mu L of conversion liquid is coated on LB solid culture medium (containing 100 mu g/mL of ampicillin resistance), and the mixture is subjected to static culture at 37 ℃ for 12h to 16h.

LB Medium component (1L): 5g of yeast extract, 10g of tryptone and 10g of NaCl, and then adding water for dissolution to a volume of 1L. After the solid medium was added to agar (12 g/L), the mixture was autoclaved.

1.5 identification of recombinant Positive clones

5 single clones were randomly selected and inoculated into 200. Mu.L of LB medium, and cultured with shaking at 37℃for 4 hours. Colony PCR was performed using the bacterial solution as a template. The system is as follows:

amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 52℃for 30s, elongation at 72℃for 60s,32 cycles; extending at 72 ℃ for 10min;

after colony PCR, agarose gel electrophoresis can amplify positive monoclonal with bright and single target size band, and the positive clone with proper band size is sent to sequence. Positive clone with successful sequencing was stored: 930 mu L of bacterial liquid is added with 70 mu LDMSO, and the mixture is frozen at the temperature of minus 80 ℃ after being evenly mixed.

Example 2 Gene protein expression and enzymatic functional analysis

2.1 extraction of MpUGT742A1-pTOPO plasmid

Plasmid was extracted using plasmid miniprep kit (TIANGEN):

(1) The strain MpUGT742A1-pTOPO-DH 5. Alpha. Was streaked on LB plate (containing 100. Mu.g/mL Amp), after 12h at 37℃a single clone was grown, and the single clone was picked up in 4mL of medium containing Amp resistance and cultured at 37℃for 10h at 110 rpm.

(2) The bacterial liquid was centrifuged at 12,000rpm for 1min at room temperature, and the supernatant was discarded, and the bacterial cells were collected and the supernatant was discarded as much as possible.

(3) 150. Mu.L of the solution P1 was added to the centrifuge tube in which the bacterial cells were precipitated, and the solution was vortexed until the bacterial cells were completely suspended.

(4) 150. Mu.L of the solution P2 was added to the centrifuge tube, and the tube was gently turned upside down for 6-8 times to allow the cells to be sufficiently lysed.

(5) 350. Mu.L of solution P5 was added to the centrifuge tube and immediately mixed up and down quickly, at which point flocculent precipitate would appear. After standing for 2min, the mixture was centrifuged at 12,000rpm for 5min.

(6) The supernatant collected in the previous step was transferred to an adsorption column CP3 (the adsorption column was put into a collection tube). Centrifuge at 12,000rpm for 1min, and discard the waste liquid in the collection tube.

(7) To the adsorption column CP3, 300. Mu.L of the rinse solution PWT was added, and the mixture was centrifuged at 12,000rpm for 1min, and the waste liquid in the collection tube was discarded.

(8) The adsorption column CP3 was placed in a collection tube, centrifuged at 12,000rpm for 2min, and the residual rinse solution in the adsorption column was removed.

(9) Placing the adsorption column CP3 into a clean centrifuge tube, volatilizing ethanol, suspending and dripping 30-50 μl of distilled water into the middle part of the adsorption film, centrifuging at 12,000rpm for 2min, and collecting plasmid solution into the centrifuge tube.

2.2 amplification of the MpUGT742A1 ORF

The constructed positive monoclonal plasmid is used as a template, and the primer pair MpUGT742A1-pET32a-F/R and PrimerSTAR Max DNA polymerase with restriction enzyme cutting sites are used for amplifying the ORF of the target gene MpUGT742A 1.

The ORF was amplified using the corresponding primer using the MpUGT742A1-pTOPO plasmid as template, with the following amplification procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 10s, annealing at 52℃for 15s, elongation at 72℃for 45s,33 cycles; extending at 72℃for 10min.

And (3) after the PCR products are separated by gel electrophoresis, carrying out gel recovery on the fragments according to a gel recovery kit. (results see FIG. 1)

2.3 enzyme digestion

The PCR product gel recovery fragments of vector pET32a and MpUGT742A1 were digested with BamHI and HindIII, respectively, in the following manner:

the enzyme digestion is carried out in a water bath at 37 ℃ for 3 hours. And adding 10×loading buffer into the enzyme digestion product to terminate the reaction, performing agarose gel electrophoresis, and selecting a proper strip for gel recovery, wherein the gel recovery method is the same as that described above.

2.4 ligation, transformation and Positive validation

The target fragment after cleavage was ligated with the vector pET32a (purchased from Novagen) after cleavage with T4 DNA Ligase (purchased from Takara) as follows:

after the above components were thoroughly mixed, they were connected overnight at 16 ℃. The ligation product was competent to transform E.coli DH 5. Alpha. The transformation method is the same as above. And (3) selecting a monoclonal to verify positive, sequencing, selecting a monoclonal bacterial cell with correct sequencing, and extracting the MpUGT742A1-pET32a plasmid. The constructed prokaryotic expression vector plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) by a thermal shock method, and the transformation, screening and identification methods are the same as those described above.

2.5 Prokaryotic expression of MpUGT742A1 recombinant protein

2.5.1 recombinant protein Induction purification

(1) The strain MpUGT742A1-pET32a-BL21 positive clone was picked up and inoculated into 4mL LB medium containing Amp resistance, and shake-cultured overnight at 37℃and 110 rpm.

(2) The cultured bacterial liquid is inoculated into 200mL culture medium containing Amp resistance according to the proportion of 1:100, and is cultured until the OD600 is approximately equal to 0.5 under the same condition. Adding 0.5mM IPTG into the bacterial liquid, and culturing in a shaking table at 16 ℃ and 110rpm for 16-18 hours to induce the expression of the target protein.

(3) And (3) bacterial collection: the bacterial solution was centrifuged at 5000rpm and the supernatant was discarded after 5 minutes.

(4) Washing: adding a proper amount of Binding buffer washing liquid into a centrifuge tube according to the bacterial amount, re-suspending the bacterial, centrifuging at 5,000rpm for 5min, collecting bacterial cells, washing twice, and adding 15-20 mL Binding buffer to re-suspend the bacterial cells.

(5) Cracking: placing the bacterial liquid into an ice-water mixture, performing ultrasonic bacterial cell lysis, centrifuging at 12,000rpm at 4 ℃ for 20min, collecting the supernatant, purifying by a column, and preparing a part of supernatant for SDS-PAGE to observe the protein expression.

(6) Separating: the collected supernatant was fed to an equilibrated Ni-NTA column, and after the supernatant was completed, a column volume of eluent (containing 20mM imidazole) was added to wash out the foreign proteins, and then the recombinant protein of interest was collected by 5mL Elution buffer (containing 250mM imidazole concentration).

(7) Ultrafiltration: placing the eluted protein solution into a ultrafiltration tube with a protein molecule of 30,000Da, centrifuging for 10min at 4,000rcf, adding Binding buffer for 2-3 times, and concentrating target protein.

(8) The concentrate was aspirated into a 2mL collection tube, the protein concentration was determined, and the sample was left.

(9) After adding 10% glycerol into the protein, quick freezing the protein with liquid nitrogen and storing the protein in a refrigerator at the temperature of minus 80 ℃ for standby.

Binding buffer: 2.42g Tris-HCl and 29.22g NaCl are respectively weighed, dissolved in water, pH is regulated to 8.0, volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is preserved at 4 ℃.

An execution buffer: 2.42g Tris-HCl, 29.22g NaCl and 34g imidozole are respectively weighed, dissolved in water, pH is regulated to 8.0, the volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is stored at 4 ℃.

2.5.2 concentration determination of protein

Protein concentration was determined using Bradford protein concentration determination kit.

(1) Total dissolved protein standard BSA, 10. Mu.L was diluted to 100. Mu.L with 0.9% NaCl to a final concentration of 0.5mg/mL as standard.

(2) Standards were added to 96-well plates at 0,1,2,4,8,12,16,20 μl, with 0.9% nacl made up to 20 μl. Three are made in parallel.

(3) The protein samples left behind were diluted appropriately with 0.9% NaCl, and 20. Mu.L was added as well. Each 3 were made in parallel.

(4) 200 mu L G of 250 staining solution is added to each well and left at room temperature for 3-5min.

(5) And measuring the absorbance value (A595) at 595nm by using an enzyme-labeled instrument, drawing a standard curve according to the protein concentration of the standard substance and the corresponding absorbance, and calculating the protein concentration in the sample according to the standard curve.

2.5.3 SDS-PAGE electrophoresis

Expression, isolation and purification of the target protein were detected by denaturing polyacrylamide gel electrophoresis (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE).

(1) And fixing the glass plate on the glue frame by adopting vertical plate electrophoresis.

(2) Preparing 12% separating gel, adding into electrophoresis apparatus, sealing with water, and standing until the separating gel is solidified.

(3) Preparing 5% concentrated gel, and pouring out the upper water layer. And (3) uniformly mixing the prepared 5% concentrated glue, immediately pouring the mixture, inserting the hole comb between glass plates (avoiding the generation of bubbles), and pulling out the hole comb after the gel is solidified.

(4) And respectively adding 2×loading buffer and metal bath at 100deg.C for 5min to combine protein with loading buffer.

Centrifuging at 13,000rpm for 5min, taking 10 μl of supernatant, and spotting while sucking 3 μl of protein Marker.

(5) And adding a proper amount of electrophoresis buffer solution into the electrophoresis tank, performing electrophoresis at a constant voltage of 90V, changing to 120V constant voltage electrophoresis when the sample is electrophoresed to the separation gel, and stopping electrophoresis until bromophenol blue reaches the lower edge of the gel.

(6) Taking off the albumin glue, putting the albumin glue into coomassie brilliant blue R-250 staining solution for soaking and staining, and lightly shaking and staining for 4 hours at room temperature.

(7) Washing the dyeing liquid on the surface of the protein gel with distilled water for 2-3 times, placing the washed protein gel in the decolorizing liquid for decolorizing for 2 hours, and replacing the decolorizing liquid for several times in the decolorizing process until the background of the protein gel is washed clean.

(8) The results of the protein electrophoresis are shown in FIG. 2.

The preparation solution and the proportion of the SDS-PAGE separating gel and the concentrated gel are as follows:

2.6 in vitro enzyme Activity function identification of proteins

2.6.1 in vitro enzyme Activity assay

The MpUGT742A1 is used for in vitro enzyme activity functional identification, and a reaction system added with pET32a protein is used as a control group. The sugar donor is UDP-glucose or UDP-glucuronic acid, and the substrate is flavone, flavonol, dihydroflavone, dihydrochalcone, dibenzyl and phenylpropion. The enzyme activity reaction system is as follows:

mixing the above components, reacting at 30deg.C for 30min, reacting with UDP-glucose as sugar donor, adding equal volume of ethyl acetate, extracting twice with equal volume of ethyl acetate, mixing organic phases, and volatilizing solvent. Redissolving with 100 μl of methanol, and performing enzyme activity reaction analysis by HPLC; enzyme activity reaction using UDP-glucuronic acid as sugar donor, adding methanol to stop reaction, centrifuging at 12,000rpm for 30min, collecting supernatant, and performing enzyme activity reaction analysis by HPLC.

2.6.2 analysis of enzyme Activity products

To verify the in vitro enzymatic function of the MpUGT742A1, HPLC was used to detect the products of the enzymatic reactions described above (FIGS. 3, 4, 5, 6). The analysis used ZORBAX SB-B18,5 μm, 4.6X1150 mm (Agilent) column, detection wavelength 254nm,280nm and 346nm, flow rate 1.0mL/min, sample injection amount 20. Mu.L. The liquid phase analysis conditions were as follows:

when UDP-glucose is used as a sugar donor, the HPLC analysis conditions are as follows:

when UDP-glucuronic acid is used as a sugar donor, the HPLC analysis conditions are as follows:

LC-MS was used for the identification of the enzyme activity product. The analysis used Hypersil Gold,1.9 μm,100×2.1mm column, detection wavelengths 254nm,280nm and 350nm, flow rate 0.3mL/min, and sample injection amount 2. Mu.L. The analysis conditions were:

the results of the enzymatic reaction analysis using UDP-glucose as the sugar donor are shown in Table 1.

TABLE 1 conversion of partial substrate by MpUGT742A1

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^a The enzyme activity reaction glycosyl donor is UDP-glucose;

^b detecting trace products;

^c failure to detect the product;

^d catalytic activity is expressed as conversion (%) + -STDEV.

The result of the enzymatic reaction of MpUGT742A1 with UDP-glucose as a sugar donor and dihydroflavone as a sugar acceptor is shown in figure 3, which has high catalytic activity on naringenin, hesperetin and pinocembrin and catalyzes the generation of dihydroflavone-7-O-glucoside. The results of the enzymatic reaction of MpUGT742A1 with UDP-glucuronic acid as the sugar donor are shown in FIGS. 4, 5, 6 and 7. It has high activity on flavonoid substrates (flavone, flavanone, flavonol) of various structural types, and catalyzes the production of various monoglycoside and disaccharide glycoside products.

Example 3 Gene subcellular localization

3.1 construction of the Gene GFP positioning vector

Gateway primers were designed based on the gene of interest MpUGT742A 1:

amplifying by using the MpUGT742A1-pET32a plasmid as a template, wherein the amplification system and the amplification conditions are the same as above, and performing gateway reaction on the amplified and purified product:

(1) The BP reaction system is as follows:

(a) Removal of BP Clonase ^TM mix reagent is placed on ice for 2min, according to the followingThe components are added into an EP pipe in turn by the upper reaction system, and are blown and evenly mixed by a gun tip.

(b) The mixture was incubated at 25℃for 4-6h.

(c) After the reaction was completed, 0.5. Mu. L Proteinase K solution was added thereto, and the mixture was gently mixed and placed in a water bath at 37℃for 10 minutes to terminate the reaction.

(d) The final reaction product was transformed into E.coli DH 5. Alpha. And plated on LB plates containing gene resistance and incubated at 37 ℃. The method for identifying ligation transformation and positive monoclonal is the same as above.

(2) The plasmids sequenced successfully (MpUGT 742A1-pDONR 207) were subjected to the LR reaction according to the following protocol:

(a) Placing the mixed solution at 25 ℃ for reaction for about 6 hours, then adding 0.5 mu L Proteinase Ksolution, gently mixing, and reacting at 37 ℃ for 10 minutes to terminate the reaction;

(b) After the reaction, the final reaction product was transformed into E.coli DH 5. Alpha. And spread on LB plates containing Kan resistance, and cultured at 37 ℃. The method for identifying ligation transformation and positive monoclonal is the same as above. The final positive plasmid MpUGT742A1-pGWB5 was obtained after successful sequencing.

3.2 transformation of Agrobacterium by Freeze thawing

(1) Taking out the agrobacteria competent cells GV3101 at-80 ℃, melting on ice, taking 1 mug of MpUGT742A1-pGWB5 plasmid and pGWB5 empty vector plasmid to be respectively added into the GV3101 competent cells, lightly blowing and mixing by a pipetting gun, and carrying out ice water bath for 5min;

(2) Quick-freezing with liquid nitrogen for 5min, placing in water bath at 37deg.C for 5min, and then placing in ice for 5min;

(3) Adding 400 mu L of non-resistance YEP liquid culture medium, and culturing at 30 ℃ for 2-3h in a shaking way;

(4) 200. Mu.L of the bacterial liquid was applied to YEP solid medium (containing 50. Mu.g/mL Kan, 100. Mu.g/mL Rif). Standing at 30deg.C for 2-3d;

(5) And (3) selecting a monoclonal, inoculating the monoclonal into a culture medium, performing shake culture, identifying the positive colony by colony PCR, and taking the positive clone for bacterial storage for later use.

YEP medium component (1L): yeast extract 10g, tryptone 10g, naCl 5g, and water to dissolve and fix volume. After the solid medium was added to agar (12 g/L), the mixture was autoclaved.

3.3 transient transformation of tobacco epidermal cells by Agrobacterium

(1) MpUGT742A1-pGWB5-GV3101, pGWB5-GV3101 and inhibitor protein silenced p19 were streaked, incubated at 30℃for 36h, and then picked and inoculated into 3mL of YEP liquid culture medium (containing Kan 50. Mu.g/mL, gent 50. Mu.g/mL, rif 100. Mu.g/mL) with shaking at 30℃for about 36h with shaking at 200 rpm.

(2) The bacterial liquid is prepared according to the following proportion of 1:50 were inoculated into 5mL of YEP liquid medium (containing Kan 50. Mu.g/mL, gent 50. Mu.g/mL, and Rif 100. Mu.g/mL) and shake-cultured for about 10 hours.

(3) Activating the bacterial liquid again according to the following steps of 1:50 were inoculated into 20mL of YEP liquid medium (containing Kan 50. Mu.g/mL, gent 50. Mu.g/mL, rif 100. Mu.g/mL) and shaken up to OD600 of 0.4-0.6.

(4) Collecting bacteria, centrifuging at 4000rpm for 20min, and discarding supernatant; the tobacco conversion solution is washed for 1 time, centrifuged and the supernatant is discarded.

(5) The thalli are resuspended with a small amount of transformation liquid, and OD600 is regulated to be approximately equal to 1.0.

(6) MpUGT742A1-pGWB5-GV3101 and pGWB5-GV3101 were mixed with p19 at a ratio of 1:1, and left to stand in the dark for 3-5 hours.

(6) Agrobacteria were infiltrated into the lower epidermal cells of tobacco lamina using a 1mL syringe.

(7) After 36h, the fluorescence signal of the lower epidermal cell (setting 488nm argon excitation light, GFP signal emission wavelength 495-570nm, chloroplast emitted signal wavelength 650-760 nm) was detected on the laser confocal microscope by using the agrobacterium infiltrated leaf, and the result is shown in fig. 8.

Tobacco conversion liquid: MES-KOH (pH 5.6), na3PO 42 mM, glucose 0.5% (v/v), acetosyringone 100 μl.

While the foregoing description of the embodiments of the present invention has been presented in conjunction with the drawings, it should be understood that it is not intended to limit the scope of the invention, but rather, it is intended to cover all modifications or variations within the scope of the invention as defined by the claims of the present invention.

SEQUENCE LISTING

<110> university of Shandong

<120> Diqian flavonoid glucuronosyltransferase and encoding gene and application thereof

<130>

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 513

<212> PRT

<213> MpUGT742A1 amino acid sequence

<400> 1

Met Thr Gly Asp Thr Leu Ser Ser Pro Thr Ser Val Glu Glu Val Asn

1 5 10 15

Gly Lys Ser His Ser Lys Thr Ser Gly Asn Gly His Leu Leu Val Leu

20 25 30

Gly Phe Gly Ser Gln Ala His Val Val Asn Ser Phe Lys Val Gly Met

35 40 45

Tyr Leu Ala Glu Arg Gly Val Thr Ile Thr Tyr Val Ser Arg Gln Lys

50 55 60

Tyr Ile Asn Gln Leu Lys Gln Ser Tyr Ser Pro Glu Lys Leu Lys Ala

65 70 75 80

Leu Gly Ile Arg Thr Val Gly Leu Ala Asp Gly Tyr Glu Gly Thr His

85 90 95

Ile Phe Asp Arg Ala Thr Arg Phe Glu Gln Val Phe Gln Pro Tyr Leu

100 105 110

Glu Glu Leu Ile Ala Asp Arg Lys Ala Gly Leu Ala Ile Pro Thr Ala

115 120 125

Ile Leu Ala Asp Arg Phe Leu Gln Phe Ala Lys Asp Val Ala Gln Lys

130 135 140

Leu Glu Ile Lys Arg Tyr Val Phe Phe Ser Ala Ser Val Ser Glu Pro

145 150 155 160

Met Met Tyr Met Ala Val Gln Glu Leu His Arg Lys Gly Thr Val Arg

165 170 175

Lys Leu Glu Asn Gly Glu Phe Val Gly Leu Glu Asn Val Pro Asn Val

180 185 190

Pro Gly Cys Asp Trp Met Arg Gln Gln Asp Leu Pro Trp Val Leu Trp

195 200 205

Ala Asp Thr Lys Ala Met Leu Asp Ile Ser His Thr Met Thr Asp Ala

210 215 220

Asp Gly Leu Val Leu Asn Tyr Phe Asp Asp Leu Gly Pro Arg Cys Leu

225 230 235 240

Glu Thr Leu Gly Asn Ser Leu Ser Ala Gln Ser Gly Thr Thr Gly Lys

245 250 255

Ala Pro Lys Leu Phe Thr Ile Gly Pro Leu Ser Asn Ala Ala Thr Ser

260 265 270

Val Asn Val Ala Cys Asn Gly Gly Glu Leu Lys Lys Thr Glu Met Gln

275 280 285

Cys Phe Asp Trp Leu Asp Gln Gln Pro Thr Ser Ser Val Leu Tyr Val

290 295 300

Cys Phe Gly Ser Ile Phe Arg Pro Asp Ala Pro Gln Leu Tyr Glu Leu

305 310 315 320

Ala Leu Gly Leu Glu Ala Ser Asn Gln Arg Phe Leu Leu Val Leu Pro

325 330 335

Ala Thr Glu Arg Gln Gly Leu Arg Lys Asp Gly Ala Val Thr Leu Glu

340 345 350

Asp Glu Leu Pro Glu Asn Phe Ala Ser Arg Val Ser Asp Arg Gly Leu

355 360 365

Ile Val Tyr Gly Trp Val Pro Gln Ile Gln Met Leu Ala His Thr Ser

370 375 380

Val Gly Gly Phe Met Ser His Cys Gly Trp Ser Ser Cys Leu Glu Ser

385 390 395 400

Phe Gly Ser Gly Val Pro Met Ile Ala Trp Pro Met Ala Ala Asp Gln

405 410 415

Asn Pro Asn Cys Arg Tyr Val Val Asn Glu Leu Lys Val Gly Ile Glu

420 425 430

Leu Thr Gly Lys Lys Gly Asp Met Val Phe Ala Ser Phe Ser Met Arg

435 440 445

Val Thr Gly Lys Lys Tyr Asp Thr Phe Val Glu Lys Asp Glu Ile Ala

450 455 460

Arg Ala Val Glu Val Leu Met Glu Gly Glu Glu Gly Lys Ala Thr Arg

465 470 475 480

Ala Arg Ala Gln Ala Leu Arg Val Lys Leu Gly Ala Ala Leu Ala Val

485 490 495

Gly Gly Ser Ser Tyr Arg His Leu Gln Glu Leu Ala Asp His Ile Asn

500 505 510

Glu

<210> 2

<211> 1542

<212> DNA

<213> MpUGT742A1 nucleotide sequence

<400> 2

atgacggggg atactctgtc ctcacccact tcggtcgaag aggtcaatgg aaaatctcat 60

tcgaagacta gcgggaacgg gcatttgctc gtgctgggat tcggcagcca agctcatgtc 120

gtcaatagct tcaaggtagg aatgtacctt gcggaaagag gcgtgaccat cacgtacgtc 180

agtcggcaga agtacatcaa ccagctcaag cagagctact cgcccgagaa gctcaaggca 240

ttgggcatcc ggacggtagg tttggccgat ggatacgaag gaacgcacat cttcgatcga 300

gcgactcgct tcgaacaagt ctttcaaccg tatctggagg agctgatcgc agacagaaag 360

gcaggactcg ccatccccac tgccatcttg gccgacaggt ttcttcaatt cgccaaggat 420

gtcgcccaga agctggaaat caaaaggtac gtcttcttca gcgcttctgt ctcagagccg 480

atgatgtaca tggccgtgca agagctccac agaaaaggga cggtaagaaa actcgagaat 540

ggggaatttg taggcctcga gaatgttccg aatgttccag ggtgcgattg gatgcgacag 600

caggacttgc cctgggtttt atgggctgac actaaggcaa tgttggacat aagccacacc 660

atgacggacg cggatggtct tgtcctcaac tacttcgacg acttgggccc cagatgcctc 720

gaaactctcg gaaattcact ctctgcccaa agtggcacga caggaaaggc accgaagctg 780

ttcacgatcg ggccgttgtc gaacgcggcc acgtctgtga acgtggcatg caacggcggc 840

gaactgaaga agacggaaat gcaatgcttc gactggctcg accagcagcc gacgagctcc 900

gtcctgtacg tctgcttcgg ctcgatattc cggcccgacg cgccgcagct ctacgagctg 960

gctctgggcc tggaagccag caaccaacgg tttcttctgg tcctgcccgc caccgagcgg 1020

caaggacttc ggaaggatgg agctgtcact ttggaggacg agctgcccga aaacttcgcc 1080

tcgcgcgtca gcgaccgggg actgattgtt tatggctggg tgccgcaaat ccagatgctg 1140

gcgcatacgt ccgtcggagg tttcatgtcc cactgcggct ggagctcttg cctcgagagc 1200

ttcggcagcg gcgttcccat gattgcctgg cccatggccg ccgaccagaa tcccaattgc 1260

aggtacgtgg tgaacgagct gaaggtgggc atcgagctga cgggcaagaa aggggacatg 1320

gtctttgctt cgttttcaat gcgagtgacc gggaagaagt acgacacgtt cgtggagaaa 1380

gacgagatcg cgcgggcggt cgaagtgttg atggagggag aagaagggaa ggccacgagg 1440

gcgagagctc aggccttgag ggtgaagctc ggggcagcgt tagccgttgg cgggtcgtcg 1500

taccggcatc tgcaggagct cgccgatcac atcaacgagt ga 1542

Claims (5)

1. Use of a protein msugt 742A1 in any one of the following (b 1) - (b 2):

(b1) Glucuronidation or glucosylation of catalytic flavones and flavonoids;

(b2) Preparing glycoside or uronic acid glycoside compounds;

in the (b 1), the flavonoid and flavonoid compounds include flavonoid compounds, flavonol compounds and dihydroflavonoid compounds;

in the (b 2), the glycoside compound includes a dihydroflavone glycoside compound;

the uronic acid glycoside compounds comprise flavonoid glucuronide, dihydroflavonoid glucuronide and flavonol glucuronide compounds;

the amino acid sequence of the protein MpUGT742A1 is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein the flavonoids comprise apigenin, luteolin, baicalein and scutellarin.

3. The use according to claim 1, wherein the flavonols comprise quercetin and kaempferol.

4. The use according to claim 1, wherein the dihydroflavonoids comprise naringenin, hesperetin, eriodictyol, glycyrrhizin and pinocembrin.

5. Use according to claim 1, wherein the protein mtugt 742A1 catalyzes the glucosylation of dihydroflavonoids and glucuronidation of flavonoids, flavanoids and flavonols.

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