CN113322243B - Protein UGT236 and coding gene and application thereof - Google Patents

Abstract

The invention discloses a protein UGT236, and a coding gene and application thereof. The protein UGT236 is a protein with an amino acid sequence shown as SEQ ID NO. 2. Experiments prove that the protein UGT236 has activity of glycosyltransferase, and can efficiently catalyze phloretin to generate phlorizin, namely the protein UGT236 can be used for preparing the phlorizin by taking the phloretin as a substrate. The invention has important application value.

Description

Protein UGT236 and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein UGT236, and a coding gene and application thereof.

Background

Phlorizin is a 2' -glucoside of dihydrochalcone and widely exists in tissues such as apple roots, stems, leaves and fruits. Recently, the lithocarpus polystachyus rehd of the evergreen plant of lithocarpus of Fagaceae is found to be rich in phlorizin, and the content of young leaves is as high as 3.1%. In recent years, research shows that phlorizin has various pharmacological activities, such as antioxidant activity, hyperglycemia and complications resistance, lipid reduction activity, anti-inflammatory activity, antitumor activity, retina protection activity, liver protection activity, skin whitening activity, sterilization activity and the like, and indicates that phlorizin has potential development values in various fields such as medicine, beauty treatment and agriculture.

Phlorizin is mainly extracted from plants such as apple branches or lithocarpus polystachyus and is easily limited by resources and the like at present, similar compounds exist in the plants, the separation and purification process is long, and environmental pollution is easily caused. With the development of synthetic biology technology, it is a promising approach to excavate key enzyme genes of phlorizin biosynthesis pathway from plants and to reconstruct the biosynthesis pathway of phlorizin from heterologous microorganisms to directly produce phlorizin. The final key step of phlorizin biosynthesis is that phloretin is catalyzed by specific glycosyltransferase to add glucose on 2' -OH to finally generate phlorizin. A plurality of glycosyltransferases which can catalyze phloretin to generate phlorizin are identified from apples and other species at present, but have the defects of low activity and easy generation of byproducts, which also limits the application of the glycosyltransferases in the biosynthesis of the phlorizin. The identification of more active and specific glycosyltransferases from plants or obtained by enzymatic engineering is a hotspot in the current studies on phlorizin biosynthesis.

Isatis tinctoria (Isatis indigotica fort.) is a two-year-old herb of Isatis tinctoria of Brassicaceae. The isatis root is one of the isatis root and the isatis leaf is one of the indigowoad leaf sources, is a common traditional Chinese medicine, has the effects of clearing heat and removing toxicity, cooling blood and relieving sore throat, and is clinically used for treating diseases such as influenza, epidemic parotitis, epidemic encephalitis B, acute and chronic hepatitis, herpes zoster and the like.

Disclosure of Invention

The invention aims to provide a glycosyl transferase capable of catalyzing phloretin to generate phlorizin.

The glycosyltransferase provided by the invention is named as protein UGT236 and is derived from isatis tinctoria. The protein UGT236 may be a 1) or a 2) or a 3) as follows:

a1 2) the amino acid sequence is the protein shown in SEQ ID NO 2;

a2 2) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of the protein shown in SEQ ID NO. 2;

a3 A protein with glycosyltransferase activity catalyzing phloretin to generate phlorizin is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a 1) or a 2).

Wherein, SEQ ID NO 2 consists of 482 amino acid residues.

In order to facilitate the purification of the protein of a 1), a tag as shown in Table 1 may be attached to the amino-terminus or the carboxy-terminus of the protein shown in SEQ ID NO: 2.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (generally 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in a 3) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a 3) above can be obtained by deleting one or several amino acid residues of the codon in the DNA sequence shown in SEQ ID NO. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in Table 1 above to the 5 'end and/or 3' end thereof.

Nucleic acid molecules encoding the protein UGT236 also belong to the scope of protection of the invention.

The nucleic acid molecule encoding the protein UGT236 can be specifically a DNA molecule shown as b 1) or b 2) or b 3) or b 4) as follows:

b1 The coding region is a DNA molecule shown in SEQ ID NO. 1;

b2 A DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1;

b3 A DNA molecule which has 75% or more identity to the nucleotide sequence defined in b 1) or b 2), is derived from Isatis tinctoria and encodes the protein UGT 236;

b4 ) hybridizes with the nucleotide sequence limited by b 1) or b 2) under strict conditions, and is derived from isatis tinctoria and encodes the protein UGT236.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO. 1 consists of 1449 nucleotides, and the nucleotide of SEQ ID NO. 1 codes an amino acid sequence shown in SEQ ID NO. 2.

The nucleotide sequence encoding the protein UGT236 of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides, which are artificially modified to have 75% or more identity to the nucleotide sequence of the protein UGT236 isolated in the present invention, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as the protein UGT236 is encoded.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the present invention encoding the protein UGT236 consisting of the amino acid sequence shown in SEQ ID NO. 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

Expression cassettes, recombinant vectors or recombinant microorganisms containing any of the above-described nucleic acid molecules are also within the scope of the present invention.

The recombinant vector containing any one of the nucleic acid molecules can be a recombinant plasmid obtained by inserting a DNA molecule shown in SEQ ID NO. 1 into a multiple cloning site of an expression vector.

The recombinant vector containing any one of the nucleic acid molecules can be specifically the recombinant plasmid pET28a-HIS-MBP-UGT236 mentioned in the examples. The recombinant plasmid pET28a-HIS-MBP-UGT236 is obtained by replacing a small DNA fragment between a restriction enzyme BamHI recognition sequence and a restriction enzyme NotI recognition sequence of a pET28a-HIS-MBP prokaryotic expression vector with a DNA molecule with the nucleotide sequence shown in SEQ ID NO. 1. The nucleotide sequence of the pET28a-HIS-MBP prokaryotic expression vector (circular) can be shown as SEQ ID NO. 3.

The recombinant microorganism containing any of the above-described nucleic acid molecules may be a recombinant bacterium obtained by introducing a recombinant vector containing any of the above-described nucleic acid molecules into a starting microorganism.

The starting microorganism may be Escherichia coli. The escherichia coli may be e.coli Rossetta (DE 3).

The recombinant microorganism containing any one of the nucleic acid molecules can be specifically the recombinant bacterium A mentioned in the examples.

The invention also protects the application of the protein UGT236, or the nucleic acid molecule, or the expression cassette, the recombinant vector or the recombinant microorganism containing the nucleic acid molecule, which can be c 1) or c 2) as follows:

c1 Use as a glycosyltransferase;

c2 Use in the preparation of a product having a glycosyltransferase function;

the glycosyltransferase has an activity to catalyze the production of phlorizin from phloretin.

The invention also provides the application of any one of the protein UGT236, or any one of the nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the nucleic acid molecules, which can be c 3) or c 4) as follows:

c3 Use in the production of phlorizin);

c4 Use) for the preparation of a product for the production of phlorizin.

In the application, the phlorizin production takes phloretin as a substrate.

The invention also provides the application of any one of the protein UGT236, or any one of the nucleic acid molecules, or an expression cassette, a recombinant vector or a recombinant microorganism containing any one of the nucleic acid molecules, which can be c 5) or c 6) as follows:

c5 Use in catalyzing phloretin;

c6 Application in the preparation of products for catalyzing phloretin.

Experiments prove that the protein UGT236 has the activity of glycosyltransferase, and can efficiently catalyze phloretin to generate phlorizin, namely the protein UGT236 can be used for preparing the phlorizin by taking the phloretin as a substrate. The invention has important application value.

Drawings

FIG. 1 is a UGT236 domain prediction analysis.

FIG. 2 is a UGT phylogenetic tree (adjacency) analysis.

FIG. 3 is a schematic diagram of the construction of the recombinant plasmid pET28a-HIS-MBP-UGT236 and SDS-PAGE of the expression product.

Fig. 4 shows UPLC detection results in example 2.

FIG. 5 shows the results of Q-TOF-MS measurements in example 2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are all conventional ones unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Coli Rosseta (DE 3) competence, pEASY-Uni Seamless Cloning and Assembly Kit, DNA gel recovery Kit are all products of Beijing Quanjin Biotechnology, inc. PrimeScript 1st Strand cDNA Synthesis Kit is a product of TaKaRa, takara, inc., baoyuan (Dalian) engineering Co., ltd. Restriction enzymes and Prestained Protein Ladder are products of Beijing GmbH, N.England (NEB) Biotechnology. KOD-Plus-Neo high fidelity PCR enzyme is a product of Toyo textile (Shanghai) Biotechnology Ltd. IPTG (Isopropyl-. Beta. -D-thiogalactoside) and PMSF (Phenylmethylenesulfonyl fluoride) are products of Sigma company, USA.

The primers were synthesized by Shanghai Bioengineering Co., ltd. Sequencing was performed by Beijing Rui Bo Biotechnology Inc.

The primers in the following examples were each synthesized by Biotechnology engineering (Shanghai) GmbH.

The sequencing in the following examples was performed by the Kyorkstoke Biotechnology Ltd.

The nucleotide sequence of the pET28a-HIS-MBP prokaryotic expression vector (circular) is shown as SEQ ID NO. 3.

Example 1 preparation of recombinant glycosyltransferase

1. Acquisition of Gene encoding glycosyltransferase derived from Isatis tinctoria (UGT 236 Gene)

The present inventors have found a gene encoding glycosyltransferase (UGT 236 gene) in Isatis tinctoria through a large number of experiments. The nucleotide sequence of UGT236 gene is shown in SEQ ID NO. 1. The UGT236 gene codes protein UGT236, and the amino acid sequence of the protein UGT236 is shown as SEQ ID NO. 2.

Nucleotide homology searches were performed on the UGT236 gene and protein UGT236 sequences in the Non-redundant GenBank CDS translation + PDB + Swissprot database using the BLAST program in the NCBI database.

The domain prediction analysis of UGT236 is shown in fig. 1, where a is the conserved domain prediction, B is the PSPG position, and C is the PSPGbox sequence.

The phylogenetic tree analysis (adjacency) of UGT is shown in FIG. 2, wherein C2 is phrophetin 2-O-glycosyltransferase, and UGT236 is grouped with the identified C2 UGT.

The results indicate that the protein UGT236 has a high homology at the amino acid level with UGT in other species, while having a typical active site domain.

2. Construction of recombinant plasmid pET28a-HIS-MBP-UGT236

The structure of the recombinant plasmid pET28a-HIS-MBP-UGT236 is shown in A in FIG. 3.

1. Extracting the total RNA of the hairy root of isatis tinctoria, and then carrying out reverse transcription by using PrimeScript 1st Strand cDNA Synthesis Kit to obtain the cDNA of the hairy root of isatis tinctoria.

2. Taking cDNA of the woad hairy root as a template, and adopting a primer F:5' -AGGGGCCCGAATTCGGATCCATGAAGATCGAGCTCGTG-3' (underlined is the restriction site for the restriction enzyme BamHI) and primer R:5' -GTGGTGCTCGAGTGC GGCCGCAnd (3) carrying out PCR amplification on a primer pair consisting of CTAGACCACAACTTCGACT-3' (the restriction enzyme cutting site of the restriction enzyme NotI is underlined) to obtain a PCR amplification product.

The reaction system was 20. Mu.L, and consisted of 5. Mu.L of KOD Buffer (10X) (a component of the KOD-Plus-Neo high fidelity PCR enzyme itself), 5. Mu.L of dNTP (concentration of 2.0 mmol/L), 2. Mu.L of an aqueous solution of primer F (concentration of 10. Mu. Mol/L), 2. Mu.L of an aqueous solution of primer R (concentration of 10. Mu. Mol/L), 1. Mu.L of the KOD-Plus-Neo high fidelity PCR enzyme, 2. Mu.L of an aqueous cDNA solution of Isatis tinctoria hairy roots (cDNA containing 20ng of Isatis tinctoria hairy roots), and 3. Mu.L of sterile double distilled water.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 2min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 1min, and 34 cycles; extension at 68 ℃ for 7min.

3. And (3) taking the PCR amplification product obtained in the step (2), carrying out agarose gel electrophoresis, and then recovering a DNA fragment of about 1500bp by using a DNA gel recovery kit.

4. Taking pET28a-HIS-MBP prokaryotic expression vector, using restriction enzyme BamHI and NotI to cut enzyme, and using DNA gel recovery kit to recover about 6.5kb vector skeleton.

5. And (3) connecting the DNA fragment obtained in the step (3) with the vector framework obtained in the step (4) by using pEASY-Uni Seamless Cloning and Assembly kit to obtain a recombinant plasmid pET28a-HIS-MBP-UGT236.

The recombinant plasmid pET28a-HIS-MBP-UGT236 was sequenced. According to the sequencing result, the structure of the recombinant plasmid pET28a-HIS-MBP-UGT236 is described as follows: the small DNA fragment between the recognition sequence of the restriction enzyme BamHI and the recognition sequence of the restriction enzyme NotI of the pET28a-HIS-MBP prokaryotic expression vector is replaced by a DNA molecule with the nucleotide sequence shown in SEQ ID NO. 1.

3. Expression of recombinant glycosyltransferases

1. E.coli Rosseta (DE 3) competent cells were transformed with the recombinant plasmid pET28a-HIS-MBP-UGT236 to obtain recombinant bacteriacide A.

2. A single clone of the recombinant bacterium A was inoculated into 5mL of LB liquid medium (containing 50. Mu.g/mL kanamycin), and cultured overnight at 37 ℃ with shaking at 250rpm to obtain a culture broth.

3. After completion of step 2, the culture broth was inoculated (inoculation ratio 1: 100) into 500mL of LB liquid medium (containing 50. Mu.g/mL kanamycin) and cultured at 37 ℃ with shaking at 250rpm until OD is reached _600nm The concentration of IPTG was adjusted to about 0.6 and 1mM in the system, followed by shaking culture at 16 ℃ and 250rpm for 169h, centrifugation at 4 ℃ and 3000g for 10min, and collection of the cells.

4. And (3) taking the thallus obtained in the step (3), washing the thallus by using precooled pure water for 2 times, and then adding 3mL PB Buffer into the thallus obtained by centrifuging every 50mL of bacterial liquid for resuspension to obtain a resuspension solution.

PB Buffer: 50mM Tris-Cl buffer, pH7.4, containing 1mM EDTA, 1mM PMSF, 10% (v/v) glycerol.

5. And (4) after the step 4 is finished, taking the heavy suspension, carrying out ultrasonic disruption by using a cell disruptor (the interval is 5s under the frequency of 10KG, the disruption is 5s, and the total disruption is 5 min), then carrying out centrifugation at 4 ℃ and 13000rpm for 15min, and collecting supernatant, wherein the supernatant is the crude enzyme solution (induced by IPTG) of the recombinant bacterium A.

According to the method, the recombinant plasmid pET28a-HIS-MBP-UGT236 is replaced by pET28a-HIS-MBP prokaryotic expression vector to obtain recombinant bacterium B (used as a reference bacterium), and then crude enzyme liquid of the recombinant bacterium B is further obtained.

Replacing the step 3 with the step 3X according to the method, and obtaining a crude enzyme solution (without IPTG induction) of the recombinant bacterium A without changing other steps. Step 3X is: the culture broth was inoculated (inoculation ratio 1: 100) to 500mL of LB liquid medium (containing 50. Mu.g/mL kanamycin), and cultured at 37 ℃ and 250rpm with shaking to OD _600nm The value was about 0.6, followed by shaking culture at 16 ℃ and 250rpm for 169h, centrifugation at 4 ℃ and 5000g for 10min, and cell collection.

And (3) carrying out SDS-PAGE on the crude enzyme solution of the recombinant bacterium A (induced by IPTG), the crude enzyme solution of the recombinant bacterium B and the crude enzyme solution of the recombinant bacterium A (not induced by IPTG).

The results are shown in B in FIG. 3 (M is a Prestained Protein Ladder, V + is the crude enzyme solution of recombinant bacterium B, 236-is the crude enzyme solution of recombinant bacterium A (without IPTG induction), and 236+ is the crude enzyme solution of recombinant bacterium A (with IPTG induction)). The result shows that the crude enzyme solution (induced by IPTG) of the recombinant bacterium A has an obvious specific protein expression band, the corresponding molecular weight is 100kD, and the size is consistent with the expected size; the crude enzyme solution of the recombinant bacterium A (without IPTG induction) and the crude enzyme solution of the recombinant bacterium B have no band with the molecular weight of 100 kDa.

The crude enzyme solution (induced by IPTG) of the recombinant bacterium A contains recombinant glycosyl transferase.

Example 2 recombinant glycosyl transferase catalyzing phloretin to phlorizin

The solution to be tested is the crude enzyme solution of the recombinant bacterium A (induced by IPTG) or the crude enzyme solution of the recombinant bacterium B prepared in example 1.

1. Preparing a reaction system. The reaction system consisted of 300. Mu.L of the test solution, 1.5. Mu.L of phloretin solution (solvent DMSO, concentration 40 mM) and 3. Mu.L of uridine diphosphate-beta-O-D-glucose solution (solvent ultrapure water, concentration 40 mM).

2. Taking the reaction system prepared in the step 1, and reacting for 8 hours at 30 ℃ and 400 rpm.

3. And (3) taking the system which finishes the step 2, adding 2 times of pure methanol (aiming at terminating the reaction), and oscillating and extracting.

4. After completion of step 3, the cells were centrifuged at 13000rpm for 15min and the supernatant was collected.

5. After completion of step 4, the supernatant was taken, filtered through a PTFE filter with a pore size of 0.22. Mu.M, and the filtrate was collected.

6. The filtrate and control were subjected to UPLC analysis.

The reference substance is a mixture of phloretin, trilobatin and phlorizin.

The UPLC conditions are as follows: and (3) chromatographic column: ACQUITY UPLC HSST3 (2.1 mm. Times.100mm, 1.7 μm); mobile phase: acetonitrile (a) with 0.1% formic acid: water with 0.1% formic acid (B) =12:88; flow rate: 0.5mL/min; the detection wavelength was 285nm, the column temperature was 40 ℃ and the sample size was 2. Mu.L.

The experimental results are shown in figure 4 (empty vector is crude enzyme solution of recombinant bacterium B, UGT236 is crude enzyme solution of recombinant bacterium A (induced by IPTG), substrate is phloretin, P1 is phloretin, P2 is trilobatin, and P3 is phloridzin). The result shows that the retention time of phlorizin in the control in UPLC is 3.41min, and the retention time of phloretin in UPLC is 6.53min; a characteristic peak exists in a filtrate collected after the crude enzyme solution (induced by IPTG) of the recombinant bacterium A reacts at the retention time of 3.41 min; and no characteristic peak is detected in the filtrate collected after the crude enzyme solution of the recombinant bacterium B reacts.

7. And (3) carrying out qualitative analysis on the filtrate and a reference substance collected after the crude enzyme solution (induced by IPTG) of the recombinant bacterium A reacts by using Q-TOF-MS.

The mass spectrum conditions are as follows: waters Xevo G2-S QTOF-MS mass spectrum adopts an electrospray ionization source (ESI) and a negative ion detection mode; the scanning range m/z is 50-1500, the scanning time is 0.2s, the capillary tube voltage is 2000V, the taper hole voltage is 40V, the desolvation gas nitrogen is 900L/h, the desolvation temperature is 450 ℃, and the ion source temperature is 100 ℃.

The analysis result of the filtrate collected after the crude enzyme solution (induced by IPTG) reaction of the recombinant bacterium A is shown in A in FIG. 5.

The results of the analysis in the control are shown in FIG. 5B.

The results show that in the filtrate collected after the crude enzyme solution (induced by IPTG) reaction of the recombinant bacterium A, the product molecular ion peak is M/z435.1382[ M-H2 ]] ^- And (2) a substrate phloretin m/z 273.0810[ 2 ] M-H] ^- The difference of molecular weight is 162, which indicates that the product is in root barkAdding a molecule of glucose on the basis of the glucose; at the same time, the peak time, molecular weight and fragment ion of phlorizin are consistent with those of a reference substance, which shows that the product M/z435.1382[ M-H ] is] ^- Is phlorizin.

The above results indicate that the crude enzyme solution of recombinant bacteriabe (induced with IPTG) prepared in example 1 has glycosyltransferase activity, which catalyzes the production of phlorizin from phloretin.

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

<120> protein UGT236 and coding gene and application thereof

<160>3

<170> PatentIn version 3.5

<210> 1

<211> 1449

<212>DNA

<213> Isatis tinctoria (Isatis indigotica fort.)

<400> 1

atgaagatcg agctcgtgtt cataccgtca ccgggaattg gtcatctcag gtcaaccgtc 60

gagctagcga agcaactagt cgacagagat gatcgtcttt ccatcaccgt tatcatcatt 120

cctcgctctt ccggtggtga cgccactgac tccgcccaaa tctcgtccct cttcgcggcg 180

tctcaggttc gtctccgtta cgaaaccatc tccgtcgcgg atgaaccaac cgcggatcgt 240

ctgccgactc aactctacat caagaaccaa aagccgcaag tgagagatgc agtcgcgaaa 300

atcctcgatc cagcacgagt tgactcgcct ccgcggctcg ccggattcgt cgtcgacatg 360

ttctgtacct cgatgataga tctagccgac gagttcggag ttccgactta catggtgtac 420

acgtcgaacg ccacgttcct cggtatcacg cttcatctcc agcttatgct cgacgagaag 480

aagtacgaca ccagcgagtt ggatgagtcg gtcaacgagt tggagtttcc atgtttgact 540

cgtccttatc ctgttgaatg ccttccgtat cttttcattt ccaaggagtg gctacctttt 600

ttcatggacc aagcgagaag cttccgaaaa atgaagggta ttttggtcaa tacaattgct 660

gagctggagc ctcacgcttt ggaactcttc tccggtgacg gtgaccttcc tcgagcttat 720

ccggtgggac cggtgctgca tcttgaaagc ggatcggata attccaacga cgacgggaaa 780

caatcggaga ttctgcggtg gctagacgac caaccggcga agtccgtcgt gttcctctgt 840

tttgggagca tgggaggttt caacgaggag caaacaagag agatcgccgt cgcgttggat 900

cgaagcggtc accggtttct atggtctctc cgccgcgcat cgccggatat attaaagcag 960

ggacccggag attacacgga tgttgatgaa gttctcccgg agggattctt ggaccggaca 1020

ttggagagag ggaagataat cggatgggct ccgcaagtgg cggtgctagc gaagccggcc 1080

atcggcggct tcgtaactca ctgtggatgg aactcgatgc ttgagagctt gtggttcggc 1140

gttccgatgg tgacgtggcc gctatacgcc gagcagaagg taaatgcgtt cgagatggtg 1200

gaggagctgg gactggcggt ggagatacgg aggtctttga aaggagattt gatggccgga 1260

gggatggaaa cggttgcggc ggaggatatc gagagagcga taaggcgtgt gatggagcaa 1320

gatagtgacg tcaggaacag agtgaaggag atggctgaga agtgccacgt ggcgttaacg 1380

gacggcggat cttcgcaggt ggctttgcga aagtttatcc aagacgtgat cgagaatgtt 1440

gtggtctag 1449

<210> 2

<211> 482

<212>PRT

<213> Isatis tinctoria (Isatis indigotica fort.)

<400> 2

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

1 5 10 15

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

20 25 30

Leu Ser Ile Thr Val Ile Ile Ile Pro Arg Ser Ser Gly Gly Asp Ala

35 40 45

Thr Asp Ser Ala Gln Ile Ser Ser Leu Phe Ala Ala Ser Gln Val Arg

50 55 60

Leu Arg Tyr Glu Thr Ile Ser Val Ala Asp Glu Pro Thr Ala Asp Arg

65 70 75 80

Leu Pro Thr Gln Leu Tyr Ile Lys Asn Gln Lys Pro Gln Val Arg Asp

85 90 95

Ala Val Ala Lys Ile Leu Asp Pro Ala Arg Val Asp Ser Pro Pro Arg

100 105 110

Leu Ala Gly Phe Val Val Asp Met Phe Cys Thr Ser Met Ile Asp Leu

115 120 125

Ala Asp Glu Phe Gly Val Pro Thr Tyr Met Val Tyr Thr Ser Asn Ala

130 135 140

Thr Phe Leu Gly Ile Thr Leu His Leu Gln Leu Met Leu Asp Glu Lys

145 150 155 160

Lys Tyr Asp Thr Ser Glu Leu Asp Glu Ser Val Asn Glu Leu Glu Phe

165 170 175

Pro Cys Leu Thr Arg Pro Tyr Pro Val Glu Cys Leu Pro Tyr Leu Phe

180 185 190

Ile Ser Lys Glu Trp Leu Pro Phe Phe Met Asp Gln Ala Arg Ser Phe

195 200 205

Arg Lys Met Lys Gly Ile Leu Val Asn Thr Ile Ala Glu Leu Glu Pro

210 215 220

His Ala Leu Glu Leu Phe Ser Gly Asp Gly Asp Leu Pro Arg Ala Tyr

225 230 235 240

Pro Val Gly Pro Val Leu His Leu Glu Ser Gly Ser Asp Asn Ser Asn

245 250 255

Asp Asp Gly Lys Gln Ser Glu Ile Leu Arg Trp Leu Asp Asp Gln Pro

260 265 270

Ala Lys Ser Val Val Phe Leu Cys Phe Gly Ser Met Gly Gly Phe Asn

275 280 285

Glu Glu Gln Thr Arg Glu Ile Ala Val Ala Leu Asp Arg Ser Gly His

290 295 300

Arg Phe Leu Trp Ser Leu Arg Arg Ala Ser Pro Asp Ile Leu Lys Gln

305 310 315 320

Gly Pro Gly Asp Tyr Thr Asp Val Asp Glu Val Leu Pro Glu Gly Phe

325 330 335

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

340 345 350

Val Ala Val Leu Ala Lys Pro Ala Ile Gly Gly Phe Val Thr His Cys

355 360 365

Gly Trp Asn Ser Met Leu Glu Ser Leu Trp Phe Gly Val Pro Met Val

370 375 380

Thr Trp Pro Leu Tyr Ala Glu Gln Lys Val Asn Ala Phe Glu Met Val

385 390 395 400

Glu Glu Leu Gly Leu Ala Val Glu Ile Arg Arg Ser Leu Lys Gly Asp

405 410 415

Leu Met Ala Gly Gly Met Glu Thr Val Ala Ala Glu Asp Ile Glu Arg

420 425 430

Ala Ile Arg Arg Val Met Glu Gln Asp Ser Asp Val Arg Asn Arg Val

435 440 445

Lys Glu Met Ala Glu Lys Cys His Val Ala Leu Thr Asp Gly Gly Ser

450 455 460

Ser Gln Val Ala Leu Arg Lys Phe Ile Gln Asp Val Ile Glu Asn Val

465 470 475 480

Val Val

<210> 3

<211>6533

<212>DNA

<213>Artificial sequence

<400> 3

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540

tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat 600

tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660

actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc 720

gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780

aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc 840

agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900

cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960

aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat 1020

tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080

tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca 1140

taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac 1200

ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg 1260

tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca 1320

tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380

cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa 1440

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 1620

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 1740

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 1800

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 1860

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2040

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta 2340

caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400

ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 2460

gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520

gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580

gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 2640

aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700

ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760

acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 2820

ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880

tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 2940

tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000

cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060

gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120

ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180

catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 3240

ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300

gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 3360

gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420

ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480

atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540

cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600

tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 3660

ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720

aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780

atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840

cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900

gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 3960

tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020

agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080

gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140

ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200

catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 4260

tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320

tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380

gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440

ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500

tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 4560

catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 4620

cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680

tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 4740

ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800

ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 4860

cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920

gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980

aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 5040

ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac 5100

agcagcggcc tggtgccgcg cggcagccat atgaaaactg aagaaggtaa actggtaatc 5160

tggattaacg gcgataaagg ctataacggt ctcgctgaag tcggtaagaa attcgagaaa 5220

gataccggaa ttaaagtcac cgttgagcat ccggataaac tggaagagaa attcccacag 5280

gttgcggcaa ctggcgatgg ccctgacatt atcttctggg cacacgaccg ctttggtggc 5340

tacgctcaat ctggcctgtt ggctgaaatc accccggaca aagcgttcca ggacaagctg 5400

tatccgttta cctgggatgc cgtacgttac aacggcaagc tgattgctta cccgatcgct 5460

gttgaagcgt tatcgctgat ttataacaaa gatctgctgc cgaacccgcc aaaaacctgg 5520

gaagagatcc cggcgctgga taaagaactg aaagcgaaag gtaagagcgc gctgatgttc 5580

aacctgcaag aaccgtactt cacctggccg ctgattgctg ctgacggggg ttatgcgttc 5640

aagtatgaaa acggcaagta cgacattaaa gacgtgggcg tggataacgc tggcgcgaaa 5700

gcgggtctga ccttcctggt tgacctgatt aaaaacaaac acatgaatgc agacaccgat 5760

tactccatcg cagaagctgc ctttaataaa ggcgaaacag cgatgaccat caacggcccg 5820

tgggcatggt ccaacatcga caccagcaaa gtgaattatg gtgtaacggt actgccgacc 5880

ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga gcgcaggtat taacgccgcc 5940

agtccgaaca aagagctggc aaaagagttc ctcgaaaact atctgctgac tgatgaaggt 6000

ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag cgctgaagtc ttacgaggaa 6060

gagttggcga aagatccacg tattgccgcc actatggaaa acgcccagaa aggtgaaatc 6120

atgccgaaca tcccgcagat gtccgctttc tggtatgccg tgcgtactgc ggtgatcaac 6180

gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag acgcgcagac taattcgagc 6240

tcgaacaaca acaacaataa caataacaac aacctcgggg atgacgatga caaggtaccg 6300

ctggaagttc tgttccaggg gcccgaattc ggatccgaat tcgagctccg tcgacaagct 6360

tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg 6420

aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc 6480

ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gat 6533

Claims (9)

1. The application of the protein UGT236 is c 1) or c 2) as follows:

c1 Use as a glycosyltransferase;

c2 Use in the preparation of a product with glycosyltransferase function;

the glycosyltransferase has an activity of catalyzing phloretin to generate phlorizin and is not easy to generate byproducts;

the amino acid sequence of the protein UGT236 is shown in SEQ ID NO. 2.

2. Use of a nucleic acid molecule encoding the protein UGT236 according to claim 1, c 1) or c 2) as follows:

c1 Use as a glycosyltransferase;

c2 Use in the preparation of a product having a glycosyltransferase function;

the glycosyltransferase has an activity of catalyzing phloretin to generate phlorizin and is not easy to generate byproducts.

3. The use of claim 2, wherein: the nucleic acid molecule is a DNA molecule shown as SEQ ID NO. 1.

4. The use of the protein UGT236 according to claim 1, as c 3) or c 4) as follows:

c3 Use in the production of phlorizin;

c4 Use in the preparation of a product for the production of phlorizin;

the phlorizin production takes phloretin as a substrate;

the phlorizin is not easy to generate by-products during the production.

5. Use of a nucleic acid molecule encoding the protein UGT236 according to claim 1, c 3) or c 4) as follows:

c3 Use in the production of phlorizin);

c4 Use in the preparation of a product for the production of phlorizin;

the production of phlorizin takes phloretin as a substrate;

the phlorizin is not easy to generate by-products during the production.

6. The use of claim 5, wherein: the nucleic acid molecule is a DNA molecule shown in SEQ ID NO. 1.

7. The use of the protein UGT236 according to claim 1, being c 5) or c 6) as follows:

c5 Use in catalyzing phloretin;

c6 Use) for the preparation of a product for catalyzing phloretin.

8. Use of a nucleic acid molecule encoding the protein UGT236 according to claim 1, c 5) or c 6) as follows:

c5 Use in catalyzing phloretin;

c6 Application in the preparation of products for catalyzing phloretin.

9. The use of claim 8, wherein: the nucleic acid molecule is a DNA molecule shown in SEQ ID NO. 1.

Images