A protein with O-isopentenyltransferase activity, related biomaterials and applications

Abstract

This invention discloses a protein with O-isopentenyltransferase activity, related biomaterials, and applications, belonging to the field of biotechnology. This invention screened and cloned an O-isopentenyltransferase gene (GlOPT), which can be used to prepare O-isopentenyltransferase. Experiments have confirmed that this O-isopentenyltransferase can catalyze the in vitro conversion of 8-hydroxybergamot lactone to arugulatin; catalyze the conversion of bergamotol to isoimperatorin; and catalyze the conversion of xanthotoxin to imperatorin. This provides technical support for the production of isopentenyl furanocoumarin-like active ingredients from arugula, lays the foundation for research on the biosynthetic pathways of coumarin compounds, and has significant theoretical and practical implications.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a protein having O-isopentenyltransferase activity, related biological materials and applications. Background Art

[0002] The traditional Chinese medicine Glehniae Radix is sweet, slightly bitter, slightly cold, and belongs to the lung and stomach meridians. Traditional Chinese medicine believes that Glehniae Radix has the effects of nourishing yin and moistening the lung, nourishing the stomach and promoting fluid production, relieving cough and resolving phlegm. Modern pharmacology shows that Glehniae Radix has activities such as regulating body immunity, anti-tumor, antibacterial, and anti-inflammatory. Glehnia littoralis Fr. Schmidt ex Miq. is the original plant of Glehniae Radix. Coumarin compounds are important active components of Glehniae Radix. Their biosynthesis originates from the phenylpropanoid metabolic pathway. Phenylalanine is enzymatically catalyzed to form the basic nucleus of coumarin, umbelliferone; then, under the action of C-prenyltransferase (CPT) and cyclase, linear or angular coumarin precursor substances are generated; among them, the linear coumarin psoralen is catalyzed by O-isopentenyltransferase (OPT), CYP450, and methyltransferase (MT) to form various isopentenyl coumarin components. Isopentenylation side-chain modification can significantly improve the pharmacological activity of coumarin, enhance its lipophilicity and bioavailability. However, the O-isopentenyltransferase gene and its function in Glehnia littoralis have not been cloned and identified yet. Summary of the Invention

[0003] The object of the present invention is to provide a protein having O-isopentenyltransferase activity, related biological materials and applications, so as to solve the problems existing in the above-mentioned prior art. The present invention screened and cloned an O-isopentenyltransferase gene (GlOPT), and an O-isopentenyltransferase can be prepared by using this gene. It has been experimentally confirmed that this O-isopentenyltransferase can catalyze the formation of phellopterin from 8-hydroxybergapten in vitro; catalyze the formation of isopimpinellin from bergaptol; catalyze the formation of imperatorin from xanthotoxol. It provides technical support for the production of Glehnia littoralis isopentenyl furanocoumarin active components, and lays a foundation for the study of the biosynthesis pathway of coumarin compounds, having important theoretical and practical significance.

[0004] To achieve the above object, the present invention provides the following scheme:

[0005] The present invention provides a protein having O-isopentenyltransferase activity, and the protein is any one of the following A1)-A4):

[0006] A1) A protein with the amino acid sequence shown in SEQ ID NO.4;

[0007] A2) A protein with the same function as A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence described in A1).

[0008] A3) Proteins that have 99%, 95%, 90%, 85%, or 80% sequence identity with the amino acid sequence described in A1) or A2) and have the same function;

[0009] A4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins described in A1)-A3).

[0010] The protein described in this invention is derived from *Glehnia littoralis* and possesses the function of O-isopentenyltransferase (GlOPT). This protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0011] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0012] The present invention also provides biomaterials related to the above-mentioned proteins, said biomaterials being any one of the following B1)-B7):

[0013] B1) The nucleic acid molecule that encodes the protein;

[0014] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0016] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0017] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0018] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2);

[0019] B7) Transgenic plant organs containing the nucleic acid molecules described in B1) or transgenic plant organs containing the expression cassette described in B2).

[0020] Those skilled in the art can mutate the nucleotide sequence of the nucleic acid molecule encoding the protein using directed evolution or point mutation. Nucleotides that are artificially modified and have 80% or more sequence identity with the nucleotide sequence of the protein isolated in this invention, as long as they encode and have the function of the protein, are all derived from and equivalent to the nucleotide sequence of this invention.

[0021] Furthermore, the nucleic acid molecule described in B1) is any one of the following b1)-b3):

[0022] b1) A cDNA molecule or DNA molecule whose coding sequence is SEQ ID NO.3;

[0023] b2) The nucleotide sequence of the coding strand is the cDNA molecule or DNA molecule of SEQ ID NO.3;

[0024] b3) A DNA molecule that has more than 80% sequence identity with the DNA molecule described in b1) or b2) and encodes the protein.

[0025] In this invention, the sequence consistency of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0026] This invention also provides the use of the above-described protein or biological material in any of the following P1)-P5):

[0027] The application of the protein or biomaterial described in P1) in the preparation of O-isopentenyltransferase;

[0028] P2) The application of the protein or the biomaterial described therein in the preparation of products with O-isopentenyltransferase activity;

[0029] The application of the protein or biomaterial described in P3) in the preparation of aralialin;

[0030] The application of the protein or biomaterial described in P4) in the preparation of isoimperatorin;

[0031] The application of the protein or biomaterial described in P5 in the preparation of imperatorin.

[0032] The present invention also provides a method for preparing O-isopentenyltransferase, comprising the step of expressing the encoding gene of the above-mentioned protein in a biological organism to obtain O-isopentenyltransferase.

[0033] Furthermore, the method for expressing the protein in a biological organism includes introducing the gene encoding the protein into a recipient microorganism to obtain a recombinant microorganism, which is then cultured and induced to express the protein to obtain an O-isopentenyltransferase.

[0034] Optionally, the recipient microorganism includes Saccharomyces cerevisiae.

[0035] The present invention also provides a product having O-isopentenyltransferase activity, wherein the product contains the above-mentioned protein.

[0036] The present invention also provides a method for preparing arugulatin, comprising the steps of using 8-hydroxybergamot lactone as a substrate and performing an enzymatic reaction with the above-mentioned protein to generate arugulatin.

[0037] Furthermore, the enzymatic reaction system contains dimethyl allyl pyrophosphate and Mg. 2+ .

[0038] The present invention also provides a method for preparing isoimperatorin, comprising the steps of using bergamot as a substrate and performing an enzymatic reaction with the above-mentioned protein to generate isoimperatorin.

[0039] Furthermore, the system of the enzymatic reaction contains dimethyl allyl pyrophosphate and Mg. 2+ .

[0040] The present invention also provides a method for preparing imperatorin, comprising the steps of using xanthotoxin as a substrate and performing an enzymatic reaction with the above-mentioned protein to generate imperatorin.

[0041] Furthermore, the system of the enzymatic reaction contains dimethyl allyl pyrophosphate and Mg. 2+ .

[0042] The present invention discloses the following technical effects:

[0043] This invention screened and cloned an O-isopentenyltransferase gene (GlOPT) from *Gnaphalium affine*, which can be used to prepare O-isopentenyltransferase. Experiments confirmed that this O-isopentenyltransferase possesses the characteristic structural domains of isopentenyltransferases and can catalyze the in vitro conversion of 8-hydroxybergamot lactone to isopentenyltransferase; the conversion of bergamotol to isopentenyltransferase; and the conversion of xanthotoxin to imperatorin. The research results of this invention provide significant technical support for the production of isopentenyl furanocoumarin-like active ingredients from *Gnaphalium affine*, lay the foundation for research on the biosynthetic pathways of coumarin compounds, and have important theoretical and practical significance. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 This is a schematic diagram of the biosynthetic pathway of coumarin compounds.

[0046] Figure 2 The image shows an agarose gel electrophoresis diagram of the O-isopentenyltransferase gene from the coral plant; where M is the DNA Marker (DL2000), and lanes 1 and 2 are the PCR amplification products of two replicates.

[0047] Figure 3 The results are PCR identification results of the recombinant plasmid pESC-His-GlOPT; where M is the DNA Marker (DL2000), and lanes 1-8 are the 8 transformants of pESC-His-GlOPT.

[0048] Figure 4 The results show the in vitro catalytic activity of GlOPT protein against 8-hydroxybergamot lactone; where A is the catalytic reaction process of GlOPT protein; B is the UPLC-UV detection result of the catalytic reaction, from top to bottom: arugulain standard, empty carrier protein, and GlOPT protein.

[0049] Figure 5 The results show the in vitro catalytic activity of GlOPT protein against bergamot; where A is the catalytic reaction process of GlOPT protein; B is the UPLC-UV detection result of the catalytic reaction, from top to bottom: isoimperatorin reference standard, empty carrier protein, and GlOPT protein.

[0050] Figure 6 The results show the in vitro catalytic activity of GlOPT protein against xanthotoxin; where A is the catalytic reaction process of GlOPT protein; B is the UPLC-UV detection result of the catalytic reaction, from top to bottom: imperatorin reference standard, empty carrier protein, and GlOPT protein. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0055] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0056] This invention screened and cloned an O-isopentenyltransferase gene (GlOPT) from *Glehnia littoralis* Fr. Schmidt ex Miq., the nucleotide sequence of which is shown in SEQ ID NO.3, encoding a protein with the sequence shown in SEQ ID NO.4. This invention prepared recombinant O-isopentenyltransferase protein (GlOPT) using a *Saccharomyces cerevisiae* expression system. Experimental verification showed that the GlOPT protein has the activity of catalyzing the in vitro synthesis of 8-hydroxybergamot lactone, bergamotol, and xanthotoxin into arugulatin, isoimperatorin, and imperatorin, respectively. The biosynthetic pathway of coumarin compounds in *Glehnia littoralis* is as follows: Figure 1 As shown, the GlOPT protein plays a crucial role in the biosynthetic pathway of coumarin compounds. The discovery of this gene and protein can provide important technical support for the in vitro synthesis and production of coumarin compounds.

[0057] The dimethyl allyl pyrophosphate (DMAPP) used in the following examples was synthesized by Tianjin WuXi AppTec New Drug Development Co., Ltd. 8-hydroxybergamot lactone was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 24043012; sennain was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 24112021; bergamotol was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 24031802; isoimperatorin was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 23010515; xanthotoxin was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 24051104; imperatorin was purchased from Beijing Bettercare Biopharmaceutical Technology Co., Ltd., catalog number: 23120403.

[0058] The *Saccharomyces cerevisiae* expression vector pESC-His was purchased from Beijing Huayueyang Biotechnology Co., Ltd., catalog number: VECT2220. The BY4742 yeast strain was also purchased from Beijing Huayueyang Biotechnology Co., Ltd., catalog number: huayueyang09372. - Blunt Zero vector was purchased from TransGold, catalog number: CB501-01. E. coli Trans1-T1 Phage Resistant Chemically Competent Cell was purchased from TransGold, catalog number: CD501-02. EZNA TM The Plant RNA kit was purchased from Omega, catalog number: R6827-01. The Gel Extraction Kit was purchased from Omega, item number: D2500-02. (PrimeScript) TM The lst Strand cDNASynthesis Kit was purchased from TaKaRa, catalog number: 6110A. Hot Start High-Fidelity DNA Polymerase was purchased from NEB, catalog number: #M0493L. In-Fusion HD Cloning Kit was purchased from TaKaRa, catalog number: 639649. Frozen-EZYeast Transformation II TM The reagent kit was purchased from Zymo Research, catalog number: T2001.

[0059] Example 1: Screening and Cloning of the O-Isopentenyltransferase Gene (GlOPT) from *Gynostemma pentaphyllum*

[0060] I. Screening of O-isopentenyltransferase genes in *Gnaphalium affine*

[0061] The screening of candidate genes was carried out according to the following procedure: (1) Blast search was performed on the CDS file of the *Solanum lyratum* transcriptome using the reported amino acid sequence of the isopentenyltransferase (AkPT1, LC557134), and candidate genes were initially selected in combination with the functional annotations of each Unigene in the transcriptome database; (2) Sequences with FPKM values ​​greater than 1 were selected; (3) Protein sequence alignment was performed between the candidate genes and the reference genes to find incomplete sequences with fewer amino acids. After manual modification, complete amino acid sequences were obtained, and online alignment was used to confirm that the candidate genes contained the conserved domain of isopentenyltransferase (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi); (4) Phylogenetic tree was constructed using the reported isopentenyltransferase protein sequence and the candidate gene protein sequence. Genes clustered in the same branch as the primary isopentenyltransferase were excluded by clustering information, and the coumarin GlOPT gene with possible function was identified by comprehensive judgment.

[0062] II. Cloning of the O-isopentenyltransferase gene from *Sargassum fusiforme*

[0063] 1. Obtaining cDNA from Coralwegium

[0064] Using EZNA TM Total RNA was extracted from the roots of *Gnaphalium affine* using the Plant RNAkit (Omega). Total RNA samples were subjected to agarose gel electrophoresis, and the difference in brightness between the 28S and 18S bands was compared. The quality of RNA extraction was determined by measuring the absorbance at 260, 280, and 230 nm using a micro-spectrophotometer. PrimeScript was used. TM cDNA from seaweed roots was obtained using the lst Strand cDNA Synthesis Kit (TaKaRa).

[0065] 2. Cloning and sequencing of full-length cDNA

[0066] Using first-strand cDNA as a template, The full-length protein-coding region (CDS) of the candidate gene for Hot Start High-Fidelity DNA Polymerase (NEB) cloning was amplified by PCR using the following primers:

[0067] GlOPT-F1: 5'-ATGATTCTGACTTCGTCCTT-3' (SEQ ID NO. 1);

[0068] GlOPT-R1: 5'-TCAACGTAGAAAATGAACAAGT-3' (SEQ ID NO. 2).

[0069] The amplification products were subjected to agarose gel electrophoresis, and the electrophoresis results showed that a specific fragment appeared at approximately 1100 bp. Figure 2 ),use The Gel Extraction Kit (Omega Bio-tek, Inc., Norcross, GA) was used to extract PCR products into gel-purified and ligated products. - The Blunt Zero vector (Full Gold) was transformed into Trans1-T1 Phage Resistant Chemically Competent Cell (Full Gold), and positive transformants were selected for sequencing (Shanghai Sangon Biotech).

[0070] Sequencing results showed that a DNA fragment with the nucleotide sequence shown in SEQ ID NO.3 was amplified using primer pair GlOPT-F1 / GlOPT-R1. The gene shown in SEQ ID NO.3 was named the GlOPT gene. SEQ ID NO.3 is a complete open reading frame encoding the protein shown in SEQ ID NO.4 (named GlOPT protein).

[0071] SEQ ID NO.3:

[0072]

[0073] SEQ ID NO.4:

[0074] MILTSFSLRSQHGFPTSLQSQRTQIHNSNKYKEYKSFMDFLPIASISKCSFGNSYRCKLVREQKVHSPTFCAQSTNEEFILQQKINHIITPQSNLWKNVDVFLRFCRLHSIIGGIVGLISISLLTVTSLGDLSPAFFTGLLKAIIPMALINTYTSCLNQVTDVEIDKVNKPHFVLASGEYTMEQGKAIGAALALTAF IMGILFNSPSLLLGIVAYFFVGTAYSVKLPLLRWKKNPFLAAFTIINILLIVPIVSFIHTQTYVLGRPLVFTKPFAFSLFGNTMFGIALALMKDIPDMEGDKAFGIQTFSLMHGKRKVFDICRSIMLTVYGSAMLIGALSSSPLNKLITVLGHGALGCILWTRSQSLNLDDVPAVESFYMFSWKLFYAEYLLVHFLR.

[0075] The recombinant vector containing the GlOPT gene was finally obtained through the above steps. -Blunt Zero-GlOPT.

[0076] Example 2: Expression and functional analysis of the O-isopentenyltransferase gene (GlOPT) in yeast from *Gynostemma pentaphyllum*

[0077] 1. Construction of yeast expression vector pESC-His-GlOPT

[0078] Based on the nucleotide sequence of the GlOPT gene (SEQ ID NO.3), primers GlOPT-F2 / GlOPT-R2 with EcoRI and NotI restriction sites were designed respectively.

[0079] GlOPT-F2: 5'-TTGAAAATTCGAATTCATGATTCTGACTTCGTCCTT-3' (SEQ ID NO.5);

[0080] GlOPT-R2: 5'-GATACTAGTGCGGCCGCTCAACGTAGAAAATGAACAAGT-3' (SEQ ID NO. 6).

[0081] The recombinant vector obtained in Example 1 Using Blunt Zero-GlOPT as a template and GlOPT-F2 / GlOPT-R2 as primers, PCR amplification was performed. After gel extraction, the fragments were ligated with the large backbone fragment of the yeast expression vector pESC-His, which had been digested with restriction endonucleases EcoR I and Not I, using the In-Fusion HD Cloning Kit (TaKaRa). The ligation reaction system was then transformed into Trans1-T1 Phage Resistant Chemically Competent Cell to obtain transformants.

[0082] The results of PCR detection of positive transformants are as follows Figure 3 As shown, the target band was present at approximately 1100 bp in all eight positive transformants. Sequencing confirmed that the recombinant expression vector pESC-His-GlOPT has the following structure: the DNA fragment shown in SEQ ID NO.3 was inserted between the EcoRI and NotI restriction sites of the pESC-His vector, resulting in a recombinant plasmid expressing the GlOPT protein. The recombinant expression vector pESC-His-GlOPT can express the protein with the amino acid sequence shown in SEQ ID NO.4.

[0083] 2. Induced expression of protein GlOPT

[0084] The expression vector pESC-His-GlOPT constructed in step 1 was used with Frozen-EZ YeastTransformation II. TM The kit was used to transform *Saccharomyces cerevisiae* strain BY4742, and transformants were then screened on SD-His (glucose) yeast-deficient medium. Simultaneously, the pESC-His empty vector was transformed into the expression host *Saccharomyces cerevisiae* strain BY4742 as a control strain.

[0085] The screened recombinant strain BY4742-pESC-His-GlOPT and the BY4742-pESC-His control strain were multiplied in SD-His (glucose) liquid medium at 30℃ and 200 rpm until OD200. 600 The value was 1.0, and then the cells were transferred to SD-His (galactose) induction medium and induced at 30℃ and 200rpm for about 18h. The cells were then collected by centrifugation at 10,000×g for 20min.

[0086] Add half a volume of disruption buffer (50 mM NaH2PO4-Na2HPO4, 1 mM EDTA, 5% glycerol, pH 7.4) and 10 mM DTT and 1 mM PMSF to the bacterial cells, resuspend the cells, and homogenize them continuously at approximately 900 bar for 4 min at 4°C using an autoclave. Then, centrifuge at 2,000 × g for 10 min at 4°C, transfer the supernatant to a 26.3 mL ultracentrifuge tube, centrifuge at 20,000 × g for 30 min, transfer the supernatant to a new 26.3 mL ultracentrifuge tube, centrifuge at 160,000 × g for 90 min, discard the supernatant, and a translucent yellow gelatinous microsomal protein precipitate will appear at the bottom of the tube. Dissolve the microsomal protein precipitate completely by repeatedly aspirating with 1 mL of reaction buffer (50 mM Tris-HCl, 10% glycerol, pH 7.5) using a 1 mL syringe to obtain GLOPT protein. Following the same procedure, empty vector protein was collected from the BY4742-pESC-His control strain. The protein concentration was adjusted to 10 mg / mL.

[0087] 3. Activity analysis of O-isopentenyltransferase in *Gnaphalium affine*

[0088] (1) Catalytic activity of 8-hydroxybergamot lactone

[0089] Using 8-hydroxybergamot lactone buffer as a substrate, the total buffer system was 100 μL, comprising 50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, 400 μM DMAPP, and 200 μM 8-hydroxybergamot lactone. The GLOPT protein obtained in step 2 and the empty vector protein were added separately to bring the final concentration of GLOPT protein in the buffer to 2 mg / mL. The catalytic reaction was carried out at 30 °C for 60 min. The reaction was terminated by adding 200 μL of chromatographic acetonitrile, vortexed for 20 s, centrifuged at 16,000 × g for 15 min, and the supernatant was collected and filtered through a syringe filter (equipped with a 0.2 μm PTFE membrane) (Jinteng) for subsequent UPLC-UV detection.

[0090] The UPLC-UV instrument was a Waters Acquity UPLC-PDA ultra-high performance liquid chromatography system. The chromatographic column was ACQUITYUPLC HSS T3 (2.1×100mm, 1.8μm, Waters); mobile phase: 0.1% formic acid water (A)-acetonitrile (B); elution gradient: 0-5.5min, 10%-100%B; 5.5-7.5min, 100%B; 7.5-8.0min, 100%-10%B; 8.0-10.0min, 10% (% represents volume percentage); flow rate: 0.5mL / min; column temperature: 37℃; injection volume: 2μL; detection wavelength: 254nm.

[0091] When 8-hydroxybergamot lactone is used as a substrate, GlOPT protein can react with dimethylallyl pyrophosphate (the donor of isopentenyl) and Mg... 2+ In its presence, it is converted into aurantin ( Figure 4 A). UPLC-UV detection results are as follows Figure 4 As shown in B, the product was determined to be aurantin by comparison with the standard, and no aurantin was generated in the empty carrier protein reaction.

[0092] (2) Catalytic activity of bergamot

[0093] Using bergamot hydrate buffer as a substrate, the total buffer system was 100 μL, comprising 50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, 400 μM DMAPP, and 200 μM bergamot hydrate. The GluOPT protein obtained in step 2 and the empty vector protein were added separately to bring the final concentration of GluOPT protein in the buffer to 2 mg / mL. The catalytic reaction was carried out at 30 °C for 60 min. The reaction was terminated by adding 200 μL of chromatographic acetonitrile, vortexed for 20 s, centrifuged at 16,000 × g for 15 min, and the supernatant was collected and filtered through a syringe filter (equipped with a 0.2 μm PTFE membrane) (Jinteng) for subsequent UPLC-UV detection.

[0094] When bergamot is used as a substrate, GLOPT protein can react with dimethylallyl pyrophosphate (the donor of isopentenyl) and Mg... 2+ In its presence, it is converted into isoimperatorin ( Figure 5 A). UPLC-UV detection results are as follows Figure 5 As shown in B, the product was determined to be isoimperatorin by comparison with the standard, and no isoimperatorin was generated in the empty carrier protein reaction.

[0095] (3) Catalytic activity of zanthoxylum phenol

[0096] Using a 100 μL buffer solution containing xanthocyanin as a substrate, the buffer system comprised 50 mM Tris-HCl (pH 7.5), 10% glycerol, 5 mM MgCl2, 400 μM DMAPP, and 200 μM xanthocyanin. The GLOPT protein obtained in step 2 and the empty vector protein were added separately to bring the final concentration of GLOPT protein in the buffer solution to 2 mg / mL. The catalytic reaction was carried out at 30 °C for 60 min. The reaction was terminated by adding 200 μL of chromatographic acetonitrile, vortexed for 20 s, centrifuged at 16,000 × g for 15 min, and the supernatant was collected. The supernatant was filtered through a syringe filter (equipped with a 0.2 μm PTFE membrane) (Jinteng) for subsequent UPLC-UV detection.

[0097] When xanthotoxin is used as a substrate, the GlOPT protein can react with dimethylallyl pyrophosphate (the donor of isopentenyl) and Mg. 2+ In its presence, it is converted into imperatorin ( Figure 6 A). UPLC-UV detection results are as follows Figure 6 As shown in B, the product was determined to be imperatorin by comparison with the standard, and no imperatorin was generated in the empty carrier protein reaction.

[0098] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A protein possessing O-isopentenyltransferase activity, characterized in that, The protein is either A1 or A2 as follows: A1) A protein with the amino acid sequence shown in SEQ ID NO.4; A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in A1).

2. A biomaterial relating to the protein of claim 1, characterized in that, The biomaterial is any one of the following B1)-B7): B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

3. The biomaterial according to claim 2, characterized in that, B1) The nucleic acid molecule described is as follows (b1) or (b2): b1) A cDNA molecule or DNA molecule whose coding sequence is SEQ ID NO.3; b2) A DNA molecule that has more than 80% sequence identity with the DNA molecule described in b1) and encodes the protein described therein.

4. The use of the protein of claim 1 or the biomaterial of claim 2 or 3 in any of the following P1)-P4): P1) The application of the protein or the biomaterial described therein in the preparation of O-isopentenyltransferase; P2) Application of the protein or biomaterial described therein in the preparation of aralialin; The application of the protein or biomaterial described in P3 in the preparation of isoimperatorin; The application of the protein or biomaterial described in P4 in the preparation of imperatorin.

5. A method for preparing O-isopentenyltransferase, characterized in that, The method includes the step of expressing the gene encoding the protein of claim 1 in a biological organism to obtain O-isopentenyltransferase.

6. The method according to claim 5, characterized in that, The method for expressing the protein in a biological organism includes introducing the gene encoding the protein into a recipient microorganism to obtain a recombinant microorganism, which is then cultured and induced to express the protein to obtain an O-isopentenyltransferase.

7. A method for preparing coralberry extract, characterized in that, The method includes the step of using 8-hydroxybergamot lactone as a substrate and performing an enzymatic reaction with the protein described in claim 1 to generate arugulatin.

8. A method for preparing isoimperatorin, characterized in that, The method includes the step of using bergamot as a substrate and performing an enzymatic reaction with the protein described in claim 1 to generate isoimperatorin.

9. A method for preparing imperatorin, characterized in that, The method includes the step of using xanthotoxin as a substrate and conducting an enzymatic reaction with the protein described in claim 1 to generate imperatorin.

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