CN110184250B

CN110184250B - Flavone glycosyltransferase protein, and coding gene and application thereof

Info

Publication number: CN110184250B
Application number: CN201910500135.7A
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: 2021-01-15
Anticipated expiration: 2039-06-11
Also published as: CN110184250A

Abstract

The invention relates to the technical field of biology, in particular to a flavone glycosyltransferase protein, and a coding gene and application thereof, wherein the protein 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

Flavone glycosyltransferase protein, and coding gene and application thereof

Technical Field

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

Background

Flavonoid glycosides are natural organic compounds with a series of important physiological activities and widely exist in the nature. Flavonoid glycosides are present in plants in the form of glycosides. For example, one of the secondary metabolites in andrographis paniculata is flavonoids and their glycosides. In recent years, people pay more and more attention to the pharmacological actions of the andrographis paniculata flavonoid glycoside such as antiplatelet and antiproliferation, and provide opportunities for further development and clinical application of the andrographis paniculata flavonoid glycoside. 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. How to carry out glycosylation reaction on natural products and non-natural products by using glycosyltransferase is an emerging field in synthetic biology, and the development is rapid 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 flavone glycosyltransferase protein participating in the synthesis of flavone glycosides, and meet the requirement of glycosylation of flavone compounds with different structures.

In order to solve the above technical problem, the present invention provides a protein named ApUFGT 3.

The protein named ApUFGT3 is derived from Andrographis paniculata (Andrographis paniculata).

The invention provides a protein named ApUFGT3, wherein ApUFGT3 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.

Wherein, the sequence 1 consists of 363 amino acid residues.

A gene encoding the protein.

Wherein the gene has a DNA sequence shown as a sequence 2.

Recombinant expression vectors, expression cassettes or recombinant bacteria containing the genes.

The use of the protein ApUFGT3 as a glycosyltransferase is also within the scope of the present invention.

The sequence 2 consists of 1392 nucleotides, wherein the 1 st to 1392 nd are coding sequences.

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

The application of the ApUFGT3 gene in preparing 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 preparing the glycosyl transferase also belongs to the protection scope of the invention.

The application of the protein ApUFGT3 in the synthesis of or preparation of flavone glycosides is also within the protection scope of the present invention.

The flavone glycoside compound is apigenin glycoside, wogonin glycoside or luteolin glycoside. Namely, the application of ApUFGT3 in the biosynthesis of apigenin glycoside or the preparation of apigenin glycoside is also within the protection scope of the present invention. The application of ApUFGT3 in the biosynthesis of wogonin glycoside or in the preparation of wogonin glycoside is also within the scope of the present invention. The application of ApUFGT3 in the biosynthesis of luteolin glycoside or in the preparation of luteolin glycoside is also within the protection scope of the invention.

The application of the recombinant bacterium in the preparation of the protein ApUFGT3 is also within the protection scope of the invention.

The invention discloses novel andrographis paniculata polysaccharide transferase (ApUFGT3), which can be used for glycosylating flavonoid compounds with different structures through transcriptome sequencing identification and separation, and also shows multiple functions, and can be used for glycosylating different hydroxyl groups of the flavonoid compounds to form flavone glycoside. The glycosyltransferase (ApUFGT3) is an effective enzymatic tool for synthesizing small bioactive glucosides with different structures, and has important significance for flavonoid 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 ApUGT3 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 ApUGT3 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 ApUGT3 is shown as a sequence 1 in a sequence table, and a coding region in a cDNA corresponding to the glycosyltransferase protein ApUGT3 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 ApUFGT3 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.

F3：5’-TCCAGGGGCCCGAATTCGGAATGGATCCCAATGTCGAAG-3’；

R3：5’-AGTGCGGCCGCAAGCTTGTTACTTTGCTTCATTTTTCTC-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 ApUFGT3 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 3. pET28a-ApUFGT3 can express the protein ApUFGT3 shown in sequence 1 in the sequence table.

Secondly, preparing crude enzyme solution

1. And (2) taking the recombinant plasmid pET28a-ApUFGT3 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 3. 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 is a mass spectrum. In FIG. 2A, 1a, 1b and 1c correspond to the reaction product, (1 c') to the product standard and 1 to the substrate standard, respectively. The reaction product has three peaks, 1a, 1B and 1c, and the mass spectra correspond to the following B3, B2 and B1, respectively, i.e. the mass-to-charge ratio of the product is 1 or 2 more than that of the substrate (the product has increased molecular weight after one glucose and substrate are removed from one molecule of water), which indicates that the product is the corresponding glycoside compound. The peak of the product standard corresponds to the peak of the reaction product, and the same substance is judged.

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 charge ratio of the product 2a is 1 more than that of the substrate, namely the product 2a, which is 1 in number, as seen from the mass spectrum (after one molecule of water is removed from one glucose and substrate light group, the molecular weight of the product is increased), indicating that the product is the corresponding glycoside compound.

Wherein the result of the substrate is luteolin is shown in FIG. 4. In FIG. 4, the upper diagram is a chromatogram and the lower diagram 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 more than that of the substrate, and the mass-to-charge ratio of the product is 162 (after one molecule of water is removed from one glucose and substrate light group, the molecular weight of the product is increased), which indicates that the product is the corresponding glycoside compound.

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 (ApUFGT3) when reacted with different substrates

The conversion rates of the protein of the invention (ApUFGT3) 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 (ApUFGT3) has good glycosylation conversion rate on flavonoid substrates.

Sequence listing

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

<120> flavone glycosyltransferase protein, and coding gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 463

<212> PRT

<213> Andrographis paniculata (Andrographis paniculata)

<400> 1

Met Asp Pro Asn Val Glu Asp Arg Thr Pro His Cys Leu Leu Leu Pro

1 5 10 15

Tyr Pro Asn Gln Gly His Ile Asn Pro Ile Leu Gln Phe Ala Lys Arg

20 25 30

Leu Ser His Thr Arg Arg Arg Ile Gln Ile Thr Phe Ile Leu Thr Lys

35 40 45

Phe Leu Leu Lys Ser Thr Thr Ala Ala Ala Ala Ala Ala Glu Ala Asp

50 55 60

Ile Ser Phe Arg Ser Ile Ser Asp Gly Phe Asp Asp Gly Gly Arg Ala

65 70 75 80

His Ala Lys Ser Phe Glu Glu Tyr Thr Asp Arg Phe Glu Leu Val Gly

85 90 95

Arg Glu Thr Leu Thr Glu Leu Leu Arg Glu Leu Ser Asp Ser Gly Arg

100 105 110

Pro Val Asp Cys Val Val Tyr Asp Pro Phe Ile Pro Trp Val Leu Asp

115 120 125

Val Ala Lys Gly Phe Gly Leu Pro Ala Ala Ala Phe Phe Thr Gln Ser

130 135 140

Cys Ala Val Asn Ser Val Tyr His Gln Val Tyr Cys Gly Arg Leu Arg

145 150 155 160

Pro Pro Leu Arg Glu Asn Glu Val Ala Val Val Ala Ala Glu Leu Pro

165 170 175

Pro Leu Lys Ala Glu Glu Leu Pro Ser Phe Ile Glu Val His Gly Ser

180 185 190

Tyr Pro Val Val Phe Glu Met Ile Lys Ser Gln Phe Arg Asn Val Glu

195 200 205

Lys Ala Asp Trp Ile Phe Val Asn Thr Phe Tyr Lys Leu Glu Glu Lys

210 215 220

Ile Ile Asn Ser Leu Ser Glu Phe Trp Pro Ile Lys Ala Ile Gly Pro

225 230 235 240

Ser Ile Pro Ser Met Cys Leu Asp Lys Arg Leu Gln Asp Asp Glu Asp

245 250 255

Tyr Gly Leu Ser Leu Phe Glu Pro Ser Leu Ser Val Cys Leu Asn Trp

260 265 270

Leu Asp Lys His Glu Ser Lys Ser Val Ile Tyr Ile Ser Phe Gly Ser

275 280 285

Leu Val Gln Leu Thr Ile Glu Gln Thr Gln Glu Leu Ser Gln Ala Leu

290 295 300

Met Ile Leu Asp Lys Pro Phe Ile Trp Ile Val Arg Lys Ser Glu Glu

305 310 315 320

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

325 330 335

Trp Gly Pro Gln Leu Lys Val Leu Gly His Asp Ala Ile Gly Cys Phe

340 345 350

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

355 360 365

Val Pro Met Val Ala Met Pro Gln Trp Thr Asp Gln Asn Thr Asn Ala

370 375 380

Lys Phe Val Thr Asp Ile Trp Lys Val Gly Val Trp Ala Lys Lys Asp

385 390 395 400

Cys Lys Gly Ile Val Lys Ser Asn Val Ile Ile Asp Cys Val Glu His

405 410 415

Val Met Glu Asp Gly Glu Glu Ile Arg Lys Asn Ala Ile Met Trp Lys

420 425 430

Glu Phe Ala Arg Glu Ala Val Asp Lys Gly Gly Ser Ser Asp Thr Asn

435 440 445

Ile Glu Asp Phe Ile Ile Thr Leu Lys Glu Lys Asn Glu Ala Lys

450 455 460

<210> 2

<211> 1392

<212> DNA

<213> Andrographis paniculata (Andrographis paniculata)

<400> 2

atggatccca atgtcgaaga ccgcacgcct cactgcctac ttctgcccta cccaaaccaa 60

ggtcacatca atcctatcct ccaattcgcc aagcgattgt cccacacgcg ccgccgcatc 120

caaatcacct tcatcctcac aaaattcctc ctcaaatcca ccaccgccgc cgccgccgcc 180

gccgaagccg acatctcttt caggtcgatt tccgacggct tcgacgacgg cggtagagca 240

cacgccaaga gcttcgagga gtacaccgac cgattcgagc ttgtcggccg ggaaacccta 300

acggagctgc tccgcgagct ttcggattcg ggtcgacccg tggactgtgt ggtgtacgac 360

ccgtttatcc cgtgggtcct cgacgttgcc aagggcttcg gcctcccggc ggcggcgttc 420

ttcacgcagt cgtgcgccgt gaacagcgtc taccaccagg tctactgcgg gcggctgcgg 480

ccgccgctgc gggagaatga ggtggcggtg gtggcggcgg agctgccgcc gctgaaggcg 540

gaggagctgc cgtcgtttat tgaggtgcac gggtcgtacc cggtggtgtt cgaaatgata 600

aagagtcagt tccgcaacgt ggagaaagct gattggatat tcgtcaacac tttctacaaa 660

ctggaggaga agataattaa ttctttatct gaattttggc caataaaagc aattggacca 720

tcgataccat cgatgtgctt agacaaaagg ttgcaagatg atgaagatta tggtctaagc 780

ctctttgaac cctcattaag tgtttgccta aattggctcg ataaacatga atctaaatcg 840

gtcatctaca tttctttcgg aagtttagtt caattaacga ttgaacaaac tcaagaactc 900

tcgcaagcat tgatgatatt agacaaaccc tttatatgga ttgttcgaaa atcagaagaa 960

tctaaacttc caaataactt tccaccggaa aatggattga tcgtgtcatg gggcccacaa 1020

ctaaaagtat taggacacga tgcaatcgga tgtttcatta cacactgtgg atggaactcg 1080

acgcttgagg cattaagcct aggggtaccc atggtggcta tgccacaatg gaccgatcaa 1140

aacactaatg caaaatttgt tacggatatt tggaaagtcg gtgtgtgggc taagaaagat 1200

tgtaagggaa tagtaaagag caacgtgatt atcgattgtg tagaacacgt gatggaagat 1260

ggggaagaaa ttagaaagaa tgctattatg tggaaggaat ttgcgcggga agccgttgat 1320

aagggaggga gttcagatac aaatattgaa gactttataa ttacattgaa agagaaaaat 1380

gaagcaaagt aa 1392

Claims

1. The use of a protein designated ApUFGT3 as a flavoketotransferase, wherein ApUFGT3 is a protein consisting of the amino acid sequence depicted in SEQ ID NO. 1.

2. Use of a gene encoding the protein ApUFGT3 of claim 1 in the preparation of a xanthoxyltransferase, wherein the nucleotide sequence of the gene is shown in SEQ ID NO 2.

3. Use of a recombinant expression vector, expression cassette or recombinant bacterium for the production of a ketoxytransferase, wherein the recombinant expression vector, expression cassette or recombinant bacterium comprises a gene as claimed in claim 2.

4. The application of a protein named ApUFGT3 in participating in synthesis of flavone glycoside or preparing the flavone glycoside, wherein the protein named ApUFGT3 is a protein consisting of an amino acid sequence shown in a sequence 1, and the flavone glycoside is apigenin glycoside, wogonin glycoside or luteolin glycoside.