CN110218711B

CN110218711B - Enzyme protein and coding gene and application thereof

Info

Publication number: CN110218711B
Application number: CN201910500134.2A
Authority: CN
Inventors: 黄璐琦; 高伟; 李媛
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2019-06-11
Filing date: 2019-06-11
Publication date: 2021-09-14
Anticipated expiration: 2039-06-11
Also published as: CN110218711A

Abstract

The invention relates to the technical field of biology, in particular to an enzyme protein, and a coding gene and application thereof. The invention provides a protein which is (a1) or (a 2): (a1) a protein consisting of an amino acid sequence shown in sequence 1; (a2) and (b) a protein derived from (a1) by substitution and/or deletion and/or addition of one or several amino acid residues and having a glycosyltransferase function. The protein of the invention can be used for glycosylating flavonoid compounds with different structures and glycosylating different hydroxyl groups of the flavonoid compounds through transcriptome sequencing identification and separation.

Description

Enzyme protein and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an enzyme protein, and a coding gene and application thereof.

Background

Herba Andrographitis, also known as herba Andrographitis, herba Swertiae Dilutae, Indian grass, radix Scutellariae, and herba Siphonostegiae. Herba Salviae Plebeiae, herba Swertiae Dilutae, Indian grass, radix Scutellariae, and herba Siraitiae chinensis. Contains diterpene lactone compounds, and contains abundant flavonoid compounds in the rhizome. Various andrographolidumes and various injections have antipyretic effect; also has the functions of resisting pathogenic microorganism, resisting inflammation and enhancing immunity; the total flavonoids can inhibit platelet aggregation induced by ADP in vitro, and have pharmacological effects of resisting platelet and proliferation. Glycosylation is a key modification step that occurs in various biological processes, particularly in secondary metabolic pathways, which alters the stability, polarity, solubility, biological activity, toxicity, etc. of the substrate molecule. In recent years, chemical and enzymatic synthesis of glycosylation has been greatly advanced. But the chemical glycosylation reaction has the defects of more side reactions and intermediates, poor regioselectivity and stereoselectivity, low yield, poor solvent compatibility, complex extraction and separation, complicated protection and deprotection steps and the like. The use of glycosyltransferases to glycosylate natural and non-natural products is an emerging area in synthetic biology and has developed rapidly in recent years. The newly discovered glycosyltransferase with specific substrate recognition can help to clarify the glucoside biosynthesis pathway and has important theoretical and practical values. At present, researches on the andrographis paniculata ketotransferase are less at home and abroad. Therefore, the novel andrographis paniculata flavone transferase which has a catalytic effect on flavonoids with different structures has important significance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel protein with glycosyltransferase, which meets the requirement of glycosylation on flavonoid compounds with different structures.

In order to solve the above technical problem, the present invention provides a protein named ApUFGT 2.

The protein is derived from Andrographis paniculata (Andrographis paniculata).

A protein which is (a1) or (a2) as follows:

(a1) a protein consisting of an amino acid sequence shown in sequence 1;

(a2) and (b) a protein derived from (a1) by substitution and/or deletion and/or addition of one or several amino acid residues and having a glycosyltransferase function.

Wherein the sequence 1 consists of 479 amino acid residues

The invention also provides a gene encoding the protein.

Wherein the gene is a DNA molecule described in the following (b1) or (b 2):

(b1) the nucleotide sequence is the DNA sequence shown in the sequence 2;

(b2) a DNA sequence obtained by substituting and/or deleting and/or adding (b1) by one or more nucleotides.

Wherein, the sequence 2 consists of 1440 nucleotides, wherein, the 1 st to 1440 th are coding sequences.

Recombinant expression vectors, expression cassettes or recombinant bacteria containing said genes are also intended to be within the scope of the present invention.

The use of said proteins as glycosyltransferases is also intended to be within the scope of the present invention.

The above applications may be for non-disease diagnostic and/or non-disease therapeutic purposes.

The use of said proteins in the biosynthesis of or in the preparation of flavonoid glycosides is also intended to be within the scope of the present invention.

In the application, the flavone glycoside is apigenin glycoside, wogonin glycoside or luteolin glycoside, namely, the application of the protein in the biosynthesis of apigenin glycoside or the preparation of apigenin glycoside is also within the protection scope of the invention. The application of the protein in the biosynthesis of wogonin glycoside or the preparation of wogonin glycoside is also within the protection scope of the present invention. The application of the protein in the biosynthesis of luteolin glucoside or the preparation of dehydrated luteolin glucoside is also within the protection scope of the invention.

The application of the gene in preparing the glycosyl transferase is also within the protection scope of the invention.

The application of the recombinant expression vector, the expression cassette or the recombinant bacterium in the preparation of the protein of claim 1 is also within the protection scope of the invention.

The application of the recombinant expression vector, the expression cassette or the recombinant bacterium in the preparation of the glycosyl transferase also belongs to the protection scope of the invention.

The invention discloses a protein with a function of polysaccharide transferase in andrographis paniculata, which can be used for glycosylating flavonoid compounds with different structures through transcriptome sequencing identification and separation, and the protein also has multiple functions and can be used for glycosylating different hydroxyl groups of the flavonoid compounds. The protein is an effective enzymatic tool for synthesizing small bioactive glucosides with different structures, and has important significance for flavonoid compound resource development, drug design and discovery of new active lead compounds.

Drawings

FIG. 1 SDS-PAGE of affinity chromatography purification of recombinant proteins. M, standard protein marker (Thermo Scientific);

FIG. 2 is a graph showing the results when apigenin is used as a substrate, wherein FIG. 2A is a chromatogram and FIG. 2B is a mass spectrum;

FIG. 3 is a graph showing the results when the substrate is wogonin, wherein FIG. 3A is a chromatogram, and FIG. 3B is a mass spectrum, wherein 2 and 2a in FIG. 3A represent the substrate and the product, respectively. FIG. 3B shows the mass spectrum of product 2 a;

FIG. 4 is a graph showing the results when luteolin is used as a substrate, wherein FIG. 4A is a chromatogram and FIG. 4B is a mass spectrum.

Detailed Description

Escherichia coli Transetta (DE 3): beijing Quanjin Biotechnology Ltd. pEASY-Uni Seamless Cloning and Assembly Kit: beijing Quanjin Biotechnology (TransGen Biotech) Inc. Ni-NTA agarose affinity chromatography column: qiagen, Wisconsin, USA. Apigenin (CAS number: 520-36-5), wogonin (CAS number: 632-85-9), luteolin (CAS number: 491-70-3), chrysin (CAS number: 480-40-0), daidzein (CAS number: 486-66-8), kaempferol (CAS number: 520-18-3), naringenin (CAS number: 480-41-1), phloretin (CAS number: 60-82-2) and genistein (CAS number: 446-72-0) were all purchased from Yunnan West Living Biotechnology GmbH. apigenin-7-O-glucoside (CAS number: 578-74-5), kaempferol-7-O-glucoside (CAS number: 16290-07-6), daidzin (CAS number: 552-66-9), naringenin-7-O-glucoside (CAS number: 529-55-5), and genistin (CAS number: 529-59-9) were all purchased from Dougu-Prov scientific and technological development, Inc. The vector HIS-MBP-pET28a is described in the following references: combining the Single Key Amino acid response for the Novel catalytic function of ent-Kaurene oxidase supported by NADPH-cytochromeP450 products in tripterygium Wilfordii; frontiers in Plant Science, www.frontiersin.org; october 2017, Volume 8 and Article 1756, which can be obtained from the Chinese medicine resource center of Chinese academy of sciences of traditional Chinese medicine, so that the application experiment can be repeated and the product can not be used for other purposes.

Example 1

Discovery of flavone glycosyltransferase protein ApUGT2 in andrographis paniculata and coding gene thereof

Transcriptome sequencing was performed by MEJA (methyl jasmonate) induction, and 9 candidate glycosyltransferases were found in leaf transcriptome, the expression of which was induced by methyl jasmonate. Further carrying out prokaryotic expression and functional verification on the nine genes, and finding that the protein ApUGT2 is glycosyltransferase which can exclusively participate in biosynthesis of flavone glycosides and cannot catalyze the biosynthesis of diterpenoids in the andrographis paniculata.

The amino acid of the glycosyltransferase protein ApUGT2 is shown as a sequence 1 in a sequence table, and a coding region in a cDNA corresponding to the glycosyltransferase protein ApUGT2 is shown as a sequence 2 in the sequence table.

Example 2

Functional verification

Construction of recombinant plasmid

1. Synthesizing a double-stranded DNA molecule shown in a sequence 2 of the sequence table, namely ApUFGT2 gene.

2. And (3) taking the DNA molecules obtained in the step (1) as a template, carrying out PCR amplification by adopting a primer pair consisting of F1 and R1, and recovering PCR amplification products.

F2：5’-TCCAGGGGCCCGAATTCGGAATGTCGGCCGCCACCGCC-3’；

R2：5’-AGTGCGGCCGCAAGCTTGTTATTGTAACGATACAGCTC-3’。

3. The vector HIS-MBP-pET28a is taken and double digestion is carried out by restriction enzymes BamH I and Sal I, and the linearized vector skeleton is recovered. (described in the following documents: combining the Single Key Amino acid responsive function for the Novel catalyst function of ent-Kaurene oxidase Supported by NADPH-Cytochrome P450 derivatives in tripterygium Wilfordii; Frondiers in Plant Science, www.frontiersin.org; October 2017, Volume 8, Article 1756.)

4. Taking the PCR amplification product obtained in the step 2, adopting pEASY-Uni Seamless Cloning and Assembly Kit and operating according to the instruction, Cloning the PCR amplification product to a vector HIS-MBP-pET28a, and obtaining a recombinant plasmid.

The ApUFGT2 gene shown in SEQ ID No.2 is used for replacing a fragment between BamHI and SalI recognition sites of HIS-MBP-pET28a, other sequences of HIS-MBP-pET28a are kept unchanged, and a recombinant expression vector is obtained and named as pET28a-ApUFGT 2. pET28a-ApUFGT2 can express the protein ApUFGT2 shown in sequence 1 in the sequence table.

Secondly, preparing crude enzyme solution

1. And (2) taking the recombinant plasmid pET28a-ApUFGT2 constructed in the step one, introducing into escherichia coli Transetta (DE3) (purchased from Beijing all-style gold biotechnology, Inc.) to obtain a recombinant bacterium.

2. Inoculating the recombinant strain obtained in step 1 into liquid LB culture medium containing 0.1mg/100ml ampicillin, and performing shake culture at 37 deg.C and 250rpm to OD_600nm＝0.6-1.0。

3. After completion of step 2, IPTG was added to the system so that the concentration thereof in the system was 1mM, and the system was cultured with shaking at 180rpm at 16 ℃ for 12 hours.

4. And 3, centrifuging at 4 ℃ and 10000g for 20min, collecting thalli, re-suspending with precooled PB buffer (containing 1mM EDTA, 10% glycerol and 1mM PMSF, wherein the solvent is PBS buffer with pH7.0 and 50 mM), then performing ultrasonic bacteria breaking (30% power, 5s of ultrasound and 5s interval for 10min) in an ice bath, centrifuging at 4 ℃ and 15000g for 15min, collecting supernatant, namely crude enzyme liquid, and naming the supernatant as supernatant A.

The above procedure was carried out using the vector HIS-MBP-pET28a in place of the recombinant plasmid, and the resulting supernatant was designated supernatant B.

Thirdly, preparing the protein

1. And (3) taking the supernatant A obtained in the third step, and purifying by using a Ni-NTA agarose affinity chromatography column (purchased from Qiagen, Wisconsin, USA) to obtain ApUFGT 2.

The specific method comprises the following steps: filtering the supernatant with 0.45 μm membrane to obtain protein, loading to Ni-NTA agarose affinity chromatography column, and incubating at 4 deg.C for 2 hr; the samples were eluted with different concentrations of imidazole/Pb buffer [0.02M Na2HPO4-NaH2PO4(pH7.4) and 0.5M NaCl, the imidazole concentration being 50, 100, 200, 300 or 500 mM. The protein was then concentrated and the buffer was changed to desalting buffer (50mM Tris-HCl, pH7.4) by means of Amicon Ultra-30K filter (Millipore, USA). Protein concentration was determined using a modified Bradford protein detection kit (shanghai bio-technology corporation, china). The purified protein was verified by SDS-PAGE and detected. The results are shown in FIG. 1.

Fourth, verification test that protein has glycosyltransferase function

The following flavone substrates were set up separately: comprises flavones of herba Andrographitis, apigenin (CAS number: 520-36-5), wogonin (CAS number: 632-85-9), luteolin (CAS number: 491-70-3), flavones of non-herba Andrographitis, chrysin (CAS number: 480-40-0), daidzein (CAS number: 486-66-8), kaempferol (CAS number: 520-18-3), naringenin (CAS number: 480-41-1), phloretin (CAS number: 60-82-2), and genistein (CAS number: 446-72-0). The reaction system consisted of 50mM Tris-HCl (ph 8.0), 8. mu.g purified protein, 320. mu.M substrate and 3200. mu.M UDP glucose in a total volume of 100. mu.l. The reaction was carried out at 30 ℃ for 12 hours, quenched by the addition of twice the volume of methanol, the mixture was thoroughly shaken, centrifuged at 12000g for 10 minutes and the supernatant was finally filtered through a 0.22 μm filter and then subjected to liquid phase and mass spectrometry.

The LC-MS analysis was a Waters acquisition I-Class UPLC tandem Waters Zevo G2-S Q-TOF MS (Waters corporation, USA) column, a Waters acquisition UPLC BEH C18 column (2.1X 50mm,1.7 μm) at 40 ℃. The sample size was 1. mu.L. The flow rate of the mobile phase was 0.4 mL/min. The mobile phase consists of liquid A and liquid B. The solution A is 0.1 percent (volume percentage content) formic acid aqueous solution. The liquid B is acetonitrile. And (3) an elution process: the volume fraction of the liquid A in the mobile phase is linearly reduced from 95% to 83% in 0-3 min; the volume fraction of the liquid A in the mobile phase is linearly reduced from 83% to 65% in 3-12 min; and the volume fraction of the liquid A in the mobile phase is linearly reduced from 65% to 40% in 12-14.5 min. The ionization mode is an electrospray positive ion mode; scanning range, 50-1500 Da; scanning time, 0.2 seconds; cone voltage, 40V; source temperature, 100 ℃; the dissolved gas temperature, 450 ℃; cone gas flow, 50 liters/hour; the flow rate of the desolventizing agent is 900L/h; collision energy, 20-50V. The data were analyzed using Masslynxtm software (version 4.1, Waters Co., Milford, MA, USA) using French (200 pg/. mu.l, 10. mu.l/min) as a reference (m/z 556.2766 ESI. (+). exact mass number Leucine enkephalin as calibrator).

Wherein, the result chart of the substrate of apigenin is shown in figure 2. In FIG. 2, the upper panel A is a chromatogram and the lower panel B is a mass spectrum. In FIG. 2, (1c) corresponds to the reaction product, (1 c') corresponds to the product standard, and (3) corresponds to the substrate standard. The peak of the reaction product is 1 peak and is 1c, and the mass spectrum of the product 1c is 1 more than that of the substrate by 162 mass-to-charge ratio (after one molecule of water is removed by one glucose and substrate light group, the molecular weight of the product is increased), which indicates that the product is the corresponding glycoside compound. And the peak of the product standard 1 c' corresponds to the peak of the reaction product 1c, it can be judged that the same substance, that is, apigenin-7-O-glucoside is contained in the reaction product of the substrate apigenin and the protein of the present invention (ApUFGT 2). It can be seen that the protein of the present invention (ApUFGT2) has a glycosyltransferase function for apigenin.

The results obtained when the substrate was wogonin are shown in FIG. 3. In FIG. 3, the upper panel A is a chromatogram and the lower panel B is a mass spectrum. The mass spectrum of the reaction product with 1 peak, 2a shown on the graph, is shown in the graph B in the graph 3, the mass charge ratio of the product 2a is 1 to 162 (the molecular weight of the product is increased after one molecule of water is removed by one glucose and substrate light group) more than that of the substrate, and the product is the corresponding glycoside compound. It can be seen that the protein of the present invention (ApUFGT2) has a glycosyltransferase function with wogonin.

Wherein the result of the substrate is luteolin is shown in FIG. 4. In FIG. 4, the upper panel A is a chromatogram and the lower panel is a mass spectrum. The reaction product peaks had 5 peaks, 3a, 3B, 3c, 3d and 3e, and the mass spectra of the

products

3a, 3B, 3c, 3d and 3e corresponded to B5, B4, B3, B2 and B1 in fig. 4, respectively. From the mass spectrum, the mass-to-charge ratio of the product is 1 or 2 162 more than that of the substrate (the molecular weight of the product is increased after one molecule of water is removed from one glucose and substrate light group), which indicates that the product is the corresponding glycoside compound. Indicating that the product is the corresponding glycoside compound. It can be seen that the protein of the present invention (ApUFGT2) has a glycosyltransferase function for luteolin.

The results of the above-described validation test of the glycosyltransferase function using each of the compounds in table 1 as a substrate were shown in table 1, and the conversion, which is the mass of the product after the reaction/(total mass of the product after the reaction + mass of the remaining substrate after the reaction), was calculated from the results of the chromatography. Wherein, the elution process of the substrate 4-11 is from '0-3 min', the volume fraction of the A liquid in the mobile phase is linearly reduced from 95% to 83%; the volume fraction of the liquid A in the mobile phase is linearly reduced from 83% to 65% in 3-12 min; changing the volume fraction of the liquid A in the mobile phase from 65% to 40% in 12-14.5min to 0-6min, and linearly reducing the volume fraction of the liquid A in the mobile phase from 95% to 75%; and the volume fraction of the liquid A in the mobile phase is linearly reduced from 75% to 60% in 6-15 min.

TABLE 1 conversion of the protein of the invention (ApUFGT2) when reacted with different substrates

The conversion rates of the protein of the invention (ApUFGT2) when reacted with different substrates are shown in Table 1. As can be seen from the results in Table 1, the protein of the invention (ApUFGT2) has good glycosylation conversion rate on flavonoid substrates.

Sequence listing

<110> institute of traditional Chinese medicine of Chinese academy of traditional Chinese medicine

<120> enzyme protein, coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 479

<212> PRT

<213> Andrographis paniculata (Andrographis paniculata)

<400> 1

Met Ser Ala Ala Thr Ala Thr Ala Ala Ala Val His Val Leu Val Phe

1 5 10 15

Pro Tyr Pro Ala Gln Gly His Met Ile Pro Leu Leu Asp Phe Thr His

20 25 30

His Leu Ala Ala Arg Cys Gly Val Ala Val Thr Val Val Val Thr Pro

35 40 45

Gln Asn Leu Arg Gln Leu Asn Pro Leu Leu Ala Ala His Pro Arg Ser

50 55 60

Val Thr Ala Leu Val Leu Pro Phe Pro Ala His Pro Ala Ile Pro Ala

65 70 75 80

Gly Val Glu Asn Thr Val Asp Leu Pro Ala Gly Gly Phe Arg His Met

85 90 95

Met Val Ala Leu Glu Gly Leu Arg His Pro Ile Ala Asp Trp Phe Arg

100 105 110

Thr His Pro Ser Pro Pro Ala Ala Ile Ile Ser Asp Met Phe Leu Gly

115 120 125

Trp Thr Asn His Leu Ala Ala Glu Leu Gly Val Pro Gly Tyr Ala Phe

130 135 140

Phe Pro Ser Gly Phe Phe Ala Ile Ser Phe Ile Arg Ser Leu Trp Cys

145 150 155 160

Gln Ser Pro Glu Leu Lys Asn Gly Asp Asp Arg Asn Thr Ala Val Gly

165 170 175

Phe Pro Glu Ile Pro Asn Ser Pro Ile Tyr Pro Trp Trp Gln Leu Ser

180 185 190

Pro Ile Phe Arg Ser Tyr Val Arg Gly Asp Pro Lys Ser Glu Phe Ile

195 200 205

Lys Asp Ser Phe Leu Ala Asn Phe Val Lys Ser His Gly Leu Val Gly

210 215 220

Asn Thr Phe Tyr Ala Leu Glu Gly Val Tyr Ser Gln Tyr Leu Lys Lys

225 230 235 240

Val Leu Gly His Asn Arg Val Trp Ala Ile Gly Pro Val Leu Pro Arg

245 250 255

Ser Asp Pro Ile His Arg Gly Gly Pro Ser Ser Ile Ser Pro Asp Glu

260 265 270

Ile Leu Ser Trp Leu Asp Thr Cys Gln Asp Arg Ser Val Val Tyr Ile

275 280 285

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

290 295 300

Ala Ala Gly Leu Glu Lys Ser Thr Val Lys Phe Ile Leu Ser Val Lys

305 310 315 320

Ala Ala Thr Gln Gly His Ala Ala Gly Met Tyr Gly Ala Leu Pro Pro

325 330 335

Gly Phe Asp Gly Arg Val Ala Gly Arg Gly Leu Val Ile Arg Gly Trp

340 345 350

Ala Pro Gln Val Leu Ile Leu Arg Asn Thr Ala Val Ser Ala Phe Leu

355 360 365

Thr His Cys Gly Trp Asn Ser Val Leu Glu Ser Ile Ala Ala Gly Val

370 375 380

Pro Met Leu Ala Trp Pro Met Glu Ala Asp Gln Phe Leu Asn Ala Thr

385 390 395 400

Leu Leu Val Asp Gln Leu Gly Val Ala Val Arg Val Cys Glu Gly Arg

405 410 415

Glu Thr Val Leu Pro Ala Glu Asp Leu Val Arg Phe Leu Glu Gly Thr

420 425 430

Val Gly Asp Glu Trp Ser Glu Lys Thr Ala Arg Ala Ala Ala Leu Arg

435 440 445

Lys Ala Ala Ala Asp Ala Val Ser Asp Gly Gly Ser Ser Val Asn Asp

450 455 460

Leu Glu Asp Phe Ala Thr Glu Leu Cys Gly Ala Val Ser Leu Gln

465 470 475

<210> 2

<211> 1440

<212> DNA

<213> Andrographis paniculata (Andrographis paniculata)

<400> 2

atgtcggccg ccaccgccac cgccgccgcc gtccatgtcc ttgtcttccc ataccctgcc 60

cagggccaca tgattcccct cctcgatttc acccaccacc tcgccgcccg ctgcggcgtg 120

gccgtcaccg tcgtcgtcac cccccagaac ctccggcaac tcaaccccct cctcgccgcc 180

catccccgtt ccgtcaccgc cctcgtcctc cccttcccgg cgcaccccgc cattcccgcc 240

ggcgtcgaga acaccgtcga cctccccgcc ggcggattcc gccatatgat ggtcgccctc 300

gagggcctcc gccaccccat cgccgactgg ttccgtaccc acccctcgcc gccggcggcc 360

atcatctccg acatgttcct cggctggacc aaccacctcg ccgccgagct cggcgtcccc 420

ggctacgctt tcttcccctc cggcttcttc gctatctcct tcatccgctc gctgtggtgt 480

cagtcgccgg agctcaaaaa cggcgacgac agaaacacgg cggtcggatt cccagaaatt 540

cccaattccc cgatttaccc ctggtggcag ttatccccaa ttttccgtag ctacgtaaga 600

ggagacccaa aatcggaatt tatcaaagat tctttcctgg ccaatttcgt aaaaagccat 660

gggttggtag gcaacacgtt ctacgcattg gaaggtgtgt attcacagta cctcaaaaaa 720

gtgctgggtc acaaccgggt ctgggcaatc ggacccgtat tgccccggtc cgacccgatc 780

catcggggcg gacccagctc catttcgccc gacgagatcc tctcctggct cgacacgtgt 840

caagatcgca gcgtggtgta catctgcttc gggagccagg ccgtgttgac gaacaagcag 900

atggcggagc tggcggcggg gctggagaag tccaccgtga aatttatttt atctgtgaag 960

gcggcgacgc aggggcacgc cgccgggatg tacggcgccc tgccgccggg gttcgatggt 1020

cgggtcgccg ggaggggttt ggtgattcgg gggtgggcgc cgcaggtgct gatactgcgg 1080

aacacggccg tgtcggcgtt tctgacgcac tgtgggtgga attcagtgct ggagagtatc 1140

gccgccggcg ttccgatgct ggcgtggccg atggaggccg accagtttct gaatgcgacg 1200

ctgctggtgg atcagttggg agtggcggtt agggtttgcg aggggcggga gacggtgctt 1260

ccggcggagg atctggtgag gtttctggaa ggaactgtcg gggatgagtg gtcggagaag 1320

acggcccgtg cggcggcgct gaggaaggcg gcggcggacg ccgttagcga cggcggaagt 1380

tcggtcaatg atttggagga ttttgcgact gaactttgcg gagctgtatc gttacaataa 1440

Claims

1. An application of protein in biosynthesis of participating in flavonoid glycoside or preparation of flavonoid glycoside is provided, wherein the flavonoid glycoside is apigenin glycoside, wogonin glycoside or luteolin glycoside;

the protein is composed of an amino acid sequence shown in a sequence 1.