CN111073868B - Plant flavone methyltransferase protein and coding gene and application thereof - Google Patents

Abstract

The invention relates to a plant flavone methyltransferase protein and a coding gene and application thereof, and the invention shows that the enzyme activity experiment of IiOMT1 protein coded by IiOMT1 gene shows that the transgenic experiment of introducing isatis indigotica proves that the transgenic isatis indigotica expressing IiOMT1 gene can improve the content of 3' -O-methylflavone taking chrysoeriol and/or isogenistein as an example in plants compared with acceptor isatis indigotica; it is explained that IiOMT1 gene is a gene related to the content of 3 '-O-methylflavone such as chrysoeriol and/or isogenistein in plants, and IiOMT1 and its encoding gene can be used to increase the content of 3' -O-methylflavone such as chrysoeriol and/or isogenistein in plants.

Description

Plant flavone methyltransferase protein and coding gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a protein IiOMT1, and a coding gene and application thereof.

Background

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. The flavonoids are mainly present in the overground part of the leaves of isatis indigotica and are one of important substance bases for exerting pharmacological activity. The structural diversity of the flavonoid compound is enriched by the modification reactions such as oxidation degree, hydroxylation, methylation, glycosylation and the like of the carbon chain of the flavonoid compound. The hydroxyl oxygen methylation reaction improves the stability of the compound, enhances the solubility of the compound, promotes the absorption and improves the bioavailability, and is one of important post-modification reactions of the flavonoid compound. The oxygen methylation modification is catalyzed by methyl transferase, oxygen methyl transferase (O-methyl transferases) transfers methyl from an activated S-adenosyl-L-methionine (SAM) donor to a hydroxyl group to generate an oxygen methyl compound, which plays a key role in the biosynthesis and growth of plant flavonoids, but a coding gene of the methyl transferase catalyzing the key oxygen methylation modification in the isatis indigotica fort is still not identified. Therefore, the cloning of the coding gene of the isatis indigotica methyltransferase provides an important basis for clarifying the biosynthesis pathway and the regulation mechanism of flavonoid active ingredients, and improving the content of the flavonoid active ingredients in the isatis indigotica by utilizing the modes of gene engineering, metabolic engineering and the like.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: a protein having methyltransferase activity is provided.

The invention provides a protein, which is named IiOMT1, is derived from isatis tinctoria and is A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90% of identity with the protein shown in A1), and is related to the heat resistance and the disease resistance of plants;

A3) a fusion protein obtained by connecting protein tags at the N-terminal or/and the C-terminal of A1) or A2).

In the protein, the sequence 2 in the sequence table is composed of 257 amino acid residues.

The fusion protein in A3) is protein with an amino acid sequence of sequence 4 in the sequence table.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above proteins, the protein-tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Among the above proteins, IiOMT1 is derived from Isatis tinctoria.

Biological materials related to IiOMT1 are also within the scope of the invention.

The biological material related to the protein provided by the invention is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) said nucleic acid molecule, or a recombinant microorganism containing B2) said expression cassette, or a recombinant microorganism containing B3) said recombinant vector;

B5) a transgenic plant cell line comprising B1) the nucleic acid molecule or a transgenic plant cell line comprising B2) the expression cassette;

B6) transgenic plant tissue comprising the nucleic acid molecule of B1) or transgenic plant tissue comprising the expression cassette of B2);

B7) a transgenic plant organ containing the nucleic acid molecule of B1), or a transgenic plant organ containing the expression cassette of B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

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.

In the biological material, the nucleic acid molecule B1) is a DNA molecule shown in the following B1) or B2) or B3) or B4):

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule having 75% or more identity to the nucleotide sequence defined in b1) or b2), derived from Isatis tinctoria and encoding the protein IiOMT1 as defined in claim 1;

b4) a DNA molecule derived from Isatis tinctoria and encoding the protein IiOMT1 according to claim 1, which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID No. 1.

Wherein, the sequence 1 in the sequence table is composed of 774 nucleotides, the coding sequence is the sequence 1 in the sequence table, and the coding sequence is the protein shown in the sequence 2 in the sequence table.

In the above biological materials, the expression cassette containing a nucleic acid molecule encoding IiOMT1 (IiOMT1 gene expression cassette) described in B2) refers to DNA capable of expressing IiOMT1 in a host cell, which DNA may include not only a promoter which initiates transcription of the TaRHP1 gene, but also a terminator which terminates transcription of IiOMT 1. Further, the expression cassetteEnhancer sequences may also be included. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (U.S. patent 5,187,267); tetracycline inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I) ⁹⁸⁵ ) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the IiOMT1 gene expression cassette can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) and the like. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

The protein, or the biomaterial of claim 2 or 3, for any of the following applications c1-c8 also falls within the scope of the present invention:

c1) the use of said protein, or said biological material, in plant breeding;

c2) use of said protein, or said biological material, in the preparation of a product having methyltransferase function;

c3) the use of said protein, or said biomaterial, in the production of 3' -O-methylflavone;

c4) the use of said protein, or said biomaterial, in the preparation of a product for the production of 3' -O-methylflavone;

c5) the use of the protein, or the biomaterial, in the decomposition of ortho-dihydroxyflavone or ortho-methylhydroxyflavone;

c6) the use of the protein, or the biomaterial, in the preparation of a product for the breakdown of ortho-dihydroxyflavone or ortho-methylhydroxyflavone;

c7) use of said protein, or said biological material, for cultivating plants having an increased content of 3' -O-methylflavonoids.

Wherein, in the c3) or c4), the "production of 3' -O-methylflavone" uses ortho-dihydroxyflavone or ortho-methylhydroxyflavone and S-adenosyl-L-methionine as substrates.

The method for producing a transgenic plant according to the present invention comprises increasing the expression level of the protein of claim 1 or a gene encoding the protein and/or the activity of the protein in a target plant to obtain a transgenic plant; the transgenic plant has an increased 3' -O-methylflavone content compared to the plant of interest.

Wherein the "increasing the expression level of the protein of claim 1 or a gene encoding the protein" in a target plant is carried out by introducing a nucleic acid molecule encoding the protein of claim 1 into the target plant.

In the method, the coding gene of the protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

2) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) enhancer sequences such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

In the above methods, the transgenic plant is understood to include not only the first to second generation transgenic plants but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the use or the method, the plant or the target plant is a monocotyledon or a dicotyledon.

Further, the plant and the target plant are isatis tinctoria.

Further, the 3' -O-methylflavone may be chrysoeriol and/or isogenistein.

The invention has the beneficial effects that: the enzyme activity experiment of IiOMT1 protein coded by IiOMT1 gene shows that the transgenic experiment of Isatis Indigotica is introduced proves that the transgenic Isatis Indigotica expressing IiOMT1 gene can improve the content of 3' -O-methylflavone in plants, such as chrysoeriol and/or isogenistein, compared with the receptor Isatis indigotica; it is explained that IiOMT1 gene is a gene related to the content of 3 '-O-methylflavone such as chrysoeriol and/or isogenistein in plants, and IiOMT1 and its encoding gene can be used to increase the content of 3' -O-methylflavone such as chrysoeriol and/or isogenistein in plants.

Drawings

FIG. 1 is an OMT phylogenetic tree (adjacency) analysis.

FIG. 2 is a schematic diagram of the construction of recombinant plasmid pE-SUMO-IiOMT1 and SDS-PAGE of the expression product.

FIG. 3 shows the results of UPLC and Q-TOF-MS measurements in example 2.

FIG. 4 shows the results of UPLC and Q-TOF-MS measurements in example 2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The experimental procedures in the following examples are conventional 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.

Transfast Taq DNA polymerase, Escherichia coli Trans-T1(DE3) competent cells, pEASY-Uni Senamless Cloning and Assembly Kit, DNA gel recovery Kit were all products of Beijing Quanji Biotech Limited. PrimeScript 1st Strand cDNA Synthesis Kit is a product of Takara Shuzo (Dalian) engineering Co., Ltd. Restriction endonucleases, Prestained Protein Ladder, S-adenosyl-L-methionine are all products of the company NEB. Coli Rosetta (DE3) is a product well known to the century Biotechnology Ltd. IPTG and PMSF are both products from Sigma, USA.

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

The sequencing in the following examples was performed by Biotechnology Limited, Okagaku, Beijing.

pE-SUMO prokaryotic expression vectors are available from Lifesensors.

Example 1 preparation of recombinant methyltransferase

First, obtaining of Gene encoding Isatis tinctoria methyltransferase (IiOMT1 Gene)

The present inventors have found a gene encoding methyltransferase (IiOMT1 gene) in Isatis tinctoria through a large number of experiments. The nucleotide sequence of the IiOMT1 gene is shown as a sequence 1 in a sequence table. The IiOMT1 gene encodes protein IiOMT1, and the amino acid sequence of the protein IiOMT1 is shown as sequence 2 in the sequence table.

The amino acid sequence of protein IiOMT1 was subjected to a homology search in the Non-redundant GenBank CDS translation + PDB + Swissprot database using the BLAST program in the NCBI database.

The OMT phylogenetic tree (adjacency) is shown in FIG. 1. The results show that at the amino acid level protein IiOMT1 has a high homology with OMTs in other species, while having a typical active site domain.

Secondly, construction of recombinant plasmid pE-SUMO-IiOMT1

A schematic diagram of the construction of recombinant plasmid pE-SUMO-IiOMT1 is shown in FIG. 2A, and comprises the following steps:

1. total RNA of the Isatis tinctoria leaves was extracted, and then reverse transcription was performed using PrimeScript 1st Strand cDNA Synthesis Kit to obtain cDNA of Isatis tinctoria leaves.

2. Taking cDNA of isatis tinctoria leaves as a template, and adopting a primer F: 5' -CACCGCGAACAGATTGGAGGTATGGCGACGAC GACAACAG-3' and primer R: 5' -GCTCGAATTCGGATCCTCTAGTCAATTGATCCGACGGCAGATAnd carrying out PCR amplification on the primer pair consisting of the-3' to obtain a PCR amplification product. Wherein the reaction system is 50 μ L, 25 μ L

FastPfu PCR Supermix, 5. mu.L of primer F aqueous solution (concentration 10. mu. mol/L), 5. mu.L of primer R aqueous solution (concentration 10. mu. mol/L), 1. mu.L of Isatis tinctoria leaf cDNA aqueous solution (containing 20ng Isatis tinctoria leaf cDNA) and 14. mu.L of sterile double distilled water. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 34 cycles; extension at 72 ℃ for 7 min.

3. And (3) taking the PCR amplification product obtained in the step (1), carrying out agarose gel electrophoresis, and then recovering a DNA fragment of about 744bp by using a DNA gel recovery kit.

4. Taking pE-SUMO prokaryotic expression vector, using restriction enzyme BsaI to cut enzyme, recovering about 5.6kb vector skeleton.

5. The recovered gel product obtained in step 3 and the vector backbone obtained in step 4 were ligated using pEASY-Uni Seamless Cloning and Assembly kit to obtain recombinant plasmid pE-SUMO-IiOMT 1.

The recombinant plasmid pE-SUMO-IiOMT1 was sequenced.

The sequencing result shows that the recombinant plasmid pE-SUMO-IiOMT1 is a recombinant expression vector obtained by inserting a DNA molecule (protein of a coding sequence 2) with a nucleotide sequence shown as a sequence 1 in a sequence table into a restriction enzyme BsaI recognition sequence of a pE-SUMO prokaryotic expression vector and keeping other sequences of pE-SUMO unchanged. In the recombinant plasmid pE-SUMO-IiOMT1, a DNA molecule shown in a sequence 1 in a sequence table is fused with an SUMO label coding sequence on a carrier skeleton to form a fusion gene shown in a sequence 3 in the sequence table, and a fusion protein with an SUMO label shown in a sequence 4 in the sequence table is expressed.

Expression, separation and purification of recombinant methyltransferase

1. The recombinant plasmid pE-SUMO-IiOMT1 is transformed into competent cells of Escherichia coli Rosetta (DE3) to obtain recombinant bacterium A.

2. The recombinant formazan monoclonal was inoculated into 5mL of LB liquid medium (containing 50. mu.g/mL kanamycin), and cultured overnight at 37 ℃ and 250rpm with shaking to obtain a culture broth.

3. After completion of step 2, 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 concentration of IPTG was adjusted to 1mM in the system at a value of 0.4-0.6, followed by shaking culture at 250rpm at 16 ℃ for 16 hours and centrifugation at 5000g for 10min at 4 ℃ to collect the cells.

4. The cells obtained in step 3 were washed 2 times with precooled pure water, and then resuspended in 30mL of a 50mM Tris-Cl buffer solution (pH 7.4) containing 1mM EDTA, 1mM PMSF and 10% (v/v) glycerol to obtain a resuspension solution.

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 5min), 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 step 1 in the method, the recombinant plasmid pE-SUMO-IiOMT1 is replaced by pE-SUMO prokaryotic expression vector to obtain recombinant bacterium B (used as a reference bacterium), and then the step 2-5 is further carried out to obtain crude enzyme liquid of the recombinant bacterium B.

6. Taking 50% Ni-NTA-Agarose 10mL in a 10mL syringe, standing for 20min, washing with 50mL pure water, equilibrating the column with 50mL 20mM imidazole, 500mM NaCl, 0.2M phosphate buffer, and adjusting the flow rate to 50 mL/min ^-1 。

7. Taking 15mL of crude enzyme solution of the recombinant bacterium A obtained in the step 4, adding the crude enzyme solution into the syringe containing the activated balance Ni-NTA-Agarose obtained in the step 5, and adjusting the flow rate to be 50 mL/min ^-1 。

a) Eluting the hybrid protein by 50mL of buffer containing 20mM imidazole, 500mM NaCl and 0.2M phosphoric acid, and collecting the flow fraction;

b) the target protein was eluted with 25mL of a buffer containing 300mM imidazole, 500mM NaCl, 0.2M phosphate buffer (25 mL),

collecting the fraction;

c) eluting with 50mL of a buffer containing 500mM imidazole, 500mM NaCl, 0.2M phosphate, and finally eluting with 50mL of a buffer

Washing the column with mL ultrapure water;

8. transferring the fractions collected in the step b) and containing the target protein to an ultrafiltration tube for desalination and concentration, supplementing a desalination buffer solution during the desalination and concentration, continuing centrifugation, collecting a pure enzyme solution, adding 10% (v/v) of Glycerol, and preserving at-80 ℃ to obtain the pure enzyme solution of the recombinant bacteriacidum (induced by IPTG).

And performing SDS-PAGE on the crude enzyme solution (induced by IPTG) and the pure enzyme solution (induced by IPTG) of the recombinant bacterium A. The results are shown in B in FIG. 2, where M is Prestained Protein Ladder, 1 is crude enzyme solution of recombinant formazan, and 2 is pure enzyme solution of recombinant formazan (induced by IPTG). The result shows that the crude enzyme solution (induced by IPTG) of the recombinant bacterium A and the pure enzyme solution of the recombinant bacterium A both have an obvious specific protein expression band, the corresponding molecular weight is 41kD, and the size is consistent with the expected size.

Example 2 decomposition of luteolin by recombinant methyltransferase to Halloween

The crude enzyme solution of recombinant formazan prepared in example 1 (induced with IPTG) was used as a test solution.

1. And (4) preparing a reaction system. The reaction system consisted of 300. mu.L of the test solution, 1.5. mu.L of luteolin solution (solvent methanol, concentration 40mM) and 3. mu. L S-adenosyl-L-methionine (solvent methanol, concentration 32 mM).

2. Taking the reaction system prepared in the step 1, and carrying out water bath at 30 ℃ for 12 h.

3. And (3) taking the reaction 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. And (5) after the step 4 is completed, taking the supernatant, filtering the supernatant by using a filter membrane with the pore diameter of 0.22 mu M, and collecting filtrate to obtain filtrate A.

The filtrate a and eriodictyol standard were subjected to UPLC (ultra performance liquid chromatography) analysis.

The UPLC conditions are as follows: a chromatographic column: ACQUITY UPLC HSS T3(2.1 mm. times.50 mm, 1.7 μm); mobile phase: 0.1% formic acid-acetonitrile (A) and 0.1% formic acid-water (B); the elution gradient was: 10-21% A (0-3min), 35-45% A (5-7min), 55-65% A (9-12min), 95% A (12.5-14 min); flow rate: 0.4 mL/min; the detection wavelength is 350nm, the column temperature is 40 ℃, and the sample injection amount is 2 mu L. ()

The experimental results are shown in fig. 3, wherein IiOMT1+ luteolin is the filtrate a collected after the reaction of the crude enzyme solution of the recombinant bacterium A (induced by IPTG), and CK + luteolin is the filtrate collected after the reaction of the crude enzyme solution of the recombinant bacterium B). The result shows that the retention time of the chrysoeriol standard substance in the UPLC is 6.25 min; a characteristic peak exists in the filtrate collected after the crude enzyme solution (induced by IPTG) of the recombinant bacterium A reacts at the retention time of 6.25 min; and no corresponding characteristic peak is detected in the filtrate collected after the crude enzyme solution of the recombinant bacterium B reacts.

Qualitative analysis was performed on 1 characteristic peak and 1 non-characteristic peak of filtrate a, chrysoeriol standard and isogenistein standard using Q-TOF-MS (quadrupole-time of flight mass spectrometer). The mass spectrum conditions are as follows: waters Xevo G2-SQTOF-MS mass spectrometry with electrospray ionization source (ESI), 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 assay results for chrysoeriol standards correspond to Chryseoriol in fig. 3. Comparing the analysis result of filtrate a in fig. 3 with the analysis result of chrysoeriol standard, it was found that: the molecular ion peak of the product in the filtrate A is M/z 299.0531[ M-H [)] ^- With chrysoeriol ion peak M/z 299.0539[ M-H ]] ^- The molecular weights of the two are consistent, and the ion fragment splitting rules of the two are consistent, which indicates that a product in the filtrate A is obtained by adding a molecular methyl group on the basis of luteolin; and the peak time (6.33 min and 6.34min respectively) and the molecular weight (300.0531 and 300.0539 respectively) of the product in the filtrate A and the chrysoeriol standard are basically consistent, namelyThe product in filtrate a was chrysoeriol.

The above results indicate that the crude enzyme solution of recombinant bacteriabe (induced with IPTG) prepared in example 1 has methyltransferase activity and is capable of catalyzing luteolin to produce chrysoeriol.

Example 3 decomposition of isoorientin by recombinant methyltransferase to yield Genistein

Replacing 1.5. mu.L of luteolin solution (solvent methanol, concentration 40mM) from example 2 with 1.5. mu.L of isoerythroside solution (solvent methanol, concentration 40 mM); the other experimental procedures were the same as those in 1 to 5 of example 2 to obtain filtrate B.

And (4) carrying out UPLC (ultra performance liquid chromatography) analysis on the filtrate B and the isogenistein standard.

The UPLC conditions are as follows: a chromatographic column: ACQUITY UPLC HSS T3(2.1 mm. times.50 mm, 1.7 μm); mobile phase: 0.1% formic acid-acetonitrile (A) and 0.1% formic acid-water (B); the elution gradient was: 10-21% A (0-3min), 35-45% A (5-7min), 55-65% A (9-12min), 95% A (12.5-14 min); flow rate: 0.4 mL/min; the detection wavelength is 350nm, the column temperature is 40 ℃, and the sample injection amount is 2 mu L.

The experimental result is shown in FIG. 4, wherein IiOMT1+ Isoorintin is the filtrate B collected after the reaction of the crude enzyme solution of the recombinant bacterium A (induced by IPTG), and CK + Isoorintin is the filtrate collected after the reaction of the crude enzyme solution of the recombinant bacterium B. The result shows that the retention time of the isogenistein standard product in the UPLC is 3.77 min; a characteristic peak exists in the filtrate collected after the crude enzyme solution (induced by IPTG) of the recombinant bacterium A reacts at the retention time of 3.77 min; and no corresponding characteristic peak is detected in the filtrate collected after the crude enzyme solution of the recombinant bacterium B reacts.

And (3) carrying out qualitative analysis on 1 characteristic peak and 1 non-characteristic peak of the filtrate B and a laburnine standard substance by using a Q-TOF-MS (quadrupole-time of flight mass spectrometer). 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 results of the isogenistein standards analysis corresponded to isoascoparin in figure 4. Compare the analysis of filtrate B in figure 4 with that of the isogenistein standard: the molecular ion peak of the product in the filtrate B is M/z 461.1071[ M-H ] -, the molecular weight of the product is consistent with the molecular weight of the isogenistein ion peak M/z 461.1077[ M-H ] -, and the splitting rule of the ion fragments of the product and the isoorientin is consistent, which indicates that the product in the filtrate B is obtained by adding a molecular methyl on the basis of isoorientin; and the peak time (respectively 3.78min and 3.77min) and the molecular weight (respectively 462.1071 and 462.1077) of the product in the filtrate B are basically consistent with those of the isogenistein standard, which indicates that the product in the filtrate B is isogenistein.

The results show that the crude enzyme solution (induced by IPTG) of the recombinant bacterium A prepared in example 1 has the activity of methyltransferase and can catalyze isoorientin to generate isogenistein.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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

<120> plant flavone methyltransferase protein and coding gene and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 774

<212> DNA

<213> Isatis indigotica Fort.

<400> 1

atggcgacga cgacaacaga ggcaacgaag acatctacta atggagaaga taagcaatct 60

cagaatctcc gacaccaaga agtcggtcac aagagtctct tacagagcga cgatctctac 120

cagtatattc tggagacaag tgtgtatcca agagaaccag aatcaatgaa ggaactcagg 180

gaagtgacag caaaacaccc ttggaacata atgacaacat cagcagatga agggcagttt 240

ctgaacatgc tcatcaagct gattaacgcc aagaacacaa tggagatcgg cgtttacact 300

ggctactctc tcctcgccac cgctcttgct ctccccgaag acggcaaaat tctagccatg 360

gacgttaaca gagagaacta cgaattgggt ttgccgatca tcgagaaagc tggcgttgct 420

cacaagatcg atttcaggga aggccctgct cttcctgttc ttgatcaact cgttgctgac 480

gagaagaacc atggaacata tgacttcata ttcgttgatg ctgacaagga caactacatc 540

aactaccata aacgtttgat cgatcttgtc aaagttggag gtgtgatcgg ctacgacaac 600

actctgtgga acggttctgt cgtcgctgct cctgatgcac caatgaggaa gtacgttcgt 660

tactacagag actttgttct tgagctcaac aaggctctcg ctgctgaccc tcggatcgag 720

atatgcatgc tccctgtggg tgatggaatc actatctgcc gtcggatcaa ttga 774

<210> 2

<211> 257

<212> PRT

<213> Isatis indigotica Fort.

<400> 2

Met Ala Thr Thr Thr Thr Glu Ala Thr Lys Thr Ser Thr Asn Gly Glu

1 5 10 15

Asp Lys Gln Ser Gln Asn Leu Arg His Gln Glu Val Gly His Lys Ser

20 25 30

Leu Leu Gln Ser Asp Asp Leu Tyr Gln Tyr Ile Leu Glu Thr Ser Val

35 40 45

Tyr Pro Arg Glu Pro Glu Ser Met Lys Glu Leu Arg Glu Val Thr Ala

50 55 60

Lys His Pro Trp Asn Ile Met Thr Thr Ser Ala Asp Glu Gly Gln Phe

65 70 75 80

Leu Asn Met Leu Ile Lys Leu Ile Asn Ala Lys Asn Thr Met Glu Ile

85 90 95

Gly Val Tyr Thr Gly Tyr Ser Leu Leu Ala Thr Ala Leu Ala Leu Pro

100 105 110

Glu Asp Gly Lys Ile Leu Ala Met Asp Val Asn Arg Glu Asn Tyr Glu

115 120 125

Leu Gly Leu Pro Ile Ile Glu Lys Ala Gly Val Ala His Lys Ile Asp

130 135 140

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

145 150 155 160

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

165 170 175

Asp Asn Tyr Ile Asn Tyr His Lys Arg Leu Ile Asp Leu Val Lys Val

180 185 190

Gly Gly Val Ile Gly Tyr Asp Asn Thr Leu Trp Asn Gly Ser Val Val

195 200 205

Ala Ala Pro Asp Ala Pro Met Arg Lys Tyr Val Arg Tyr Tyr Arg Asp

210 215 220

Phe Val Leu Glu Leu Asn Lys Ala Leu Ala Ala Asp Pro Arg Ile Glu

225 230 235 240

Ile Cys Met Leu Pro Val Gly Asp Gly Ile Thr Ile Cys Arg Arg Ile

245 250 255

Asn

<210> 3

<211> 1098

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggtcatc accatcatca tcacgggtcc ctgcaggact cagaagtcaa tcaagaagct 60

aagccagagg tcaagccaga agtcaagcct gagactcaca tcaatttaaa ggtgtccgat 120

ggatcttcag agatcttctt caagatcaaa aagaccactc ctttaagaag gctgatggaa 180

gcgttcgcta aaagacaggg taaggaaatg gactccttaa gattcttgta cgacggtatt 240

agaattcaag ctgatcaggc ccctgaagat ttggacatgg aggataacga tattattgag 300

gctcaccgcg aacagattgg aggtatggcg acgacgacaa cagaggcaac gaagacatct 360

actaatggag aagataagca atctcagaat ctccgacacc aagaagtcgg tcacaagagt 420

ctcttacaga gcgacgatct ctaccagtat attctggaga caagtgtgta tccaagagaa 480

ccagaatcaa tgaaggaact cagggaagtg acagcaaaac acccttggaa cataatgaca 540

acatcagcag atgaagggca gtttctgaac atgctcatca agctgattaa cgccaagaac 600

acaatggaga tcggcgttta cactggctac tctctcctcg ccaccgctct tgctctcccc 660

gaagacggca aaattctagc catggacgtt aacagagaga actacgaatt gggtttgccg 720

atcatcgaga aagctggcgt tgctcacaag atcgatttca gggaaggccc tgctcttcct 780

gttcttgatc aactcgttgc tgacgagaag aaccatggaa catatgactt catattcgtt 840

gatgctgaca aggacaacta catcaactac cataaacgtt tgatcgatct tgtcaaagtt 900

ggaggtgtga tcggctacga caacactctg tggaacggtt ctgtcgtcgc tgctcctgat 960

gcaccaatga ggaagtacgt tcgttactac agagactttg ttcttgagct caacaaggct 1020

ctcgctgctg accctcggat cgagatatgc atgctccctg tgggtgatgg aatcactatc 1080

tgccgtcgga tcaattga 1098

<210> 4

<211> 365

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gly His His His His His His Gly Ser Leu Gln Asp Ser Glu Val

1 5 10 15

Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr

20 25 30

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

35 40 45

Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys

50 55 60

Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile

65 70 75 80

Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu Asp Met Glu Asp Asn

85 90 95

Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly Met Ala Thr Thr

100 105 110

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

115 120 125

Gln Asn Leu Arg His Gln Glu Val Gly His Lys Ser Leu Leu Gln Ser

130 135 140

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

145 150 155 160

Pro Glu Ser Met Lys Glu Leu Arg Glu Val Thr Ala Lys His Pro Trp

165 170 175

Asn Ile Met Thr Thr Ser Ala Asp Glu Gly Gln Phe Leu Asn Met Leu

180 185 190

Ile Lys Leu Ile Asn Ala Lys Asn Thr Met Glu Ile Gly Val Tyr Thr

195 200 205

Gly Tyr Ser Leu Leu Ala Thr Ala Leu Ala Leu Pro Glu Asp Gly Lys

210 215 220

Ile Leu Ala Met Asp Val Asn Arg Glu Asn Tyr Glu Leu Gly Leu Pro

225 230 235 240

Ile Ile Glu Lys Ala Gly Val Ala His Lys Ile Asp Phe Arg Glu Gly

245 250 255

Pro Ala Leu Pro Val Leu Asp Gln Leu Val Ala Asp Glu Lys Asn His

260 265 270

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

275 280 285

Asn Tyr His Lys Arg Leu Ile Asp Leu Val Lys Val Gly Gly Val Ile

290 295 300

Gly Tyr Asp Asn Thr Leu Trp Asn Gly Ser Val Val Ala Ala Pro Asp

305 310 315 320

Ala Pro Met Arg Lys Tyr Val Arg Tyr Tyr Arg Asp Phe Val Leu Glu

325 330 335

Leu Asn Lys Ala Leu Ala Ala Asp Pro Arg Ile Glu Ile Cys Met Leu

340 345 350

Pro Val Gly Asp Gly Ile Thr Ile Cys Arg Arg Ile Asn

355 360 365

Claims (5)

1. Use of a protein or protein-related biomaterial in any of the following c1) -c 3):

c1) use of a protein or biomaterial for the production of chrysoeriol and/or isogenistein;

c2) the use of a protein or biomaterial in the preparation of a chrysoeriol and/or isogenistein product;

c3) the application of protein or biological material in cultivating isatis tinctoria with increased content of chrysoeriol and/or isogenistein;

wherein the protein is the protein of A1) or A2) as follows:

A1) the amino acid sequence is protein of a sequence 2 in a sequence table;

A2) a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1);

the protein-related biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the protein of A1) or A2);

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

2. Use according to claim 1, characterized in that: B1) the nucleic acid molecule is a DNA molecule shown as b1) or b2) or b 3):

b1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3) a DNA molecule which hybridizes with the nucleotide sequence of SEQ ID No. 1 under stringent conditions and is derived from Isatis tinctoria and encodes the protein of claim 1.

3. The use as claimed in claim 1, wherein in c3), ortho-dihydroxyflavone or ortho-methylhydroxyflavone and S-adenosyl-L-methionine are used as substrates.

4. A method for producing a transgenic plant, comprising increasing the expression level of the protein of claim 1 or a gene encoding the protein and/or the activity of the protein in a plant of interest to obtain a transgenic plant; the transgenic plant has an increased content of chrysoeriol and/or genistein compared to the target plant, which is Isatis tinctoria.

5. The method according to claim 4, wherein the increase in the expression level of the protein of claim 1 or the gene encoding the protein is achieved by introducing a nucleic acid molecule encoding the protein of claim 1 into the plant.

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