Prunella vulgaris glycosyltransferase and application thereof in biocatalytic synthesis of isorosmarinic acid glycoside

Abstract

The invention relates to the technical field of genetic engineering, and particularly discloses a novel glycosyl transferase PvUGT3910 derived from Prunella vulgaris L. and an application of the novel glycosyl transferase PvUGT3910 in biocatalytic synthesis of isorosmarinic acid glycoside. The amino acid sequence of the enzyme is shown as SEQ ID NO: 1, the enzyme has specific glucosylation catalytic activity on hydroxyl at C3 site of rosmarinic acid, and a high-activity product isorosmarinic acid glycoside is generated. According to the invention, the UGT enzyme capable of specifically catalyzing the C3-site glycosylation of rosmarinic acid is cloned from selfheal for the first time, the enzyme activity is verified through a prokaryotic and eukaryotic dual system, and the industrial pain point problem that isorosmarinic acid glycoside is difficult to extract is solved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and enzyme catalysis technology, and specifically relates to a method for preparing a novel enzyme derived from Prunella vulgaris ( Prunella vulgaris The present invention relates to a novel glycosyltransferase PvUGT3910 isolated from Psoralea corylifolia L., a gene encoding the same and applications thereof, and in particular to the use of the enzyme in the biocatalytic synthesis of isorosmarinic acid glycosides (rosmarinic acid-3-O-glucoside). Background Art

[0002] Prunella vulgaris ( PrunellavμLgaris Prunella vulgaris L. is a perennial herbaceous plant of the genus Prunellae in the Lamiaceae family. Its mature fruit clusters are used as medicine, boasting benefits such as clearing heat and detoxifying, eliminating carbuncles and draining pus, and promoting diuresis and relieving stranguria. Prunella vulgaris is rich in flavonoids, phenolic acids, and triterpenes. Rosmarinic acid is a key phenolic acid compound and a marker compound used in Chinese Pharmacopoeia to assess Prunella vulgaris quality. Isorosmarinic acid glycoside (rosmarinic acid-3-O-glucoside) is a downstream metabolite of rosmarinic acid glycosylation. Glycosylation increases the stability and bioavailability of rosmarinic acid. It is primarily found in the seeds of Prunella vulgaris fruit clusters. Current research suggests its potential for treating breast cancer and insomnia. Currently, isorosmarinic acid glycosides are primarily extracted from Prunella vulgaris seeds, a difficult, time-consuming, and inefficient method. This severely limits Prunella vulgaris germplasm resources and the development of new drugs. Currently, there are no research reports on the biosynthesis of isorosmarinic acid glycosides in Prunella vulgaris. Therefore, the discovery and application of key genes for the biosynthesis of isorosmarinic acid glycosides in Prunella vulgaris is of great significance. Summary of the Invention

[0003] The first object of the present invention is to provide an isolated Prunella vulgaris glycosyltransferase for catalyzing the glucosylation of rosmarinic acid at the C3 position to produce isorosmarinic acid. The Prunella vulgaris glycosyltransferase has an amino acid sequence as shown in SEQ ID NO: 1, or a variant of the amino acid sequence as shown in SEQ ID NO: 1 that retains catalytic activity for the glucosylation of rosmarinic acid at the C3 position, obtained by substituting and / or deleting and / or adding one or more amino acid residues.

[0004] The second object of the present invention is to provide a gene encoding the glycosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0005] The third object of the present invention is to provide a specific primer pair for amplifying the Prunella vulgaris glycosyltransferase gene, which primer pair consists of an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

[0006] The fourth object of the present invention is to provide a recombinant expression vector comprising the Prunella vulgaris glycosyltransferase gene, wherein the vector is a prokaryotic expression vector or a plant expression vector.

[0007] The fifth object of the present invention is to provide a host cell containing a recombinant expression vector of the Prunella vulgaris glycosyltransferase gene, wherein the host cell is Escherichia coli or Agrobacterium.

[0008] The sixth object of the present invention is to provide a use of the glycosyltransferase or its encoding gene, or a recombinant expression vector or host cell containing the encoding gene in catalyzing the glucosylation of rosmarinic acid at the C3 position to biosynthesize isosmarinic acid glycosides.

[0009] The seventh object of the present invention is to provide a method for catalytically synthesizing isosmarinic acid glycosides, comprising: using rosmarinic acid and UDP-glucose as reaction starting materials, and utilizing the glycosyltransferase to catalyze the glucosylation of rosmarinic acid at the C3 position.

[0010] Furthermore, the glycosyltransferase is produced through an Agrobacterium-mediated tobacco transient expression system, and rosmarinic acid and UDP-glucose are injected into tobacco leaves for in vivo catalysis.

[0011] The eighth object of the present invention is to provide a biocatalyst comprising the glycosyltransferase for catalyzing the synthesis of isorosmarinic acid glycosides.

[0012] The ninth object of the present invention is to provide a transgenic tobacco plant, the genome of which is integrated with the Prunella vulgaris glycosyltransferase gene, which is expressed in leaf tissue and can catalyze rosmarinic acid to produce isorosmarinic acid glycosides.

[0013] The beneficial effects of the present invention are: The present invention screened the glycosyltransferase obtained for the first time from the transcriptome data of leaves, flowers, stems, seeds and ears of Prunella vulgaris, successfully expressed it in both a prokaryotic expression system and a tobacco transient expression system, and was able to catalyze rosmarinic acid to produce the corresponding isosmarinic acid glycoside.

[0014] Compared with the existing chemical synthesis of isorosmarinic acid glycosides, the present invention utilizes glycosyltransferase and can efficiently catalyze the generation of corresponding glycosylation products by adding substrates. The entire reaction process has the advantages of simple operation, high safety factor, short production cycle, high efficiency, and environmental friendliness.

[0015] The invention fills the blank in the synthesis pathway of phenolic glycosides in Prunella vulgaris and provides an optional enzymatic element for the glycosylation modification of similar compounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : The structural formula and conversion diagram of rosmarinic acid and isosmarinic acid glycosides.

[0017] Figure 2 : Schematic diagram of the tertiary structure of PvUGT3910 protein.

[0018] Figure 3 : Agarose gel electrophoresis detection of Prunella vulgaris total RNA.

[0019] Figure 4 : Agarose gel electrophoresis of PvUGT3910 gene amplification.

[0020] Figure 5 : Plasmid map of the prokaryotic expression vector pet28a-PvUGT3910.

[0021] Figure 6 : SDS-PAGE electrophoresis of pet28a-PvUGT3910 protein expression.

[0022] Figure 7 : LC-MS / MS detection results of the product of rosmarinic acid catalyzed by pet28a-PvUGT3910 protein.

[0023] Figure 8 : Plasmid map of the recombinant transient expression vector PCAMBIA1300-35S-GFP-PvUGT3910.

[0024] Figure 9 : LC-MS / MS detection results of the product of rosmarinic acid catalyzed by transient expression of the recombinant transient expression vector PCAMBIA1300-35S-GFP-PvUGT3910 in tobacco. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art on the basis of the following examples should also be deemed to fall within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the following examples are generally implemented according to conventional conditions or reference books such as "Molecular Cloning Laboratory Manual" (New York: Cold Spring Harbor Laboratory, 2017), or implemented according to the methods recommended in the operating manual provided by the manufacturer. The materials whose sources are not specified in the examples are all commonly used materials well known in the art, which can be constructed by themselves according to literature reports or obtained through commercial channels.

[0026] Applicant used HMMMER to perform pfam annotation on the transcriptome data of Prunella vulgaris, identified all glycosyltransferases, screened out sequences with high expression level, and obtained a gene PvUGT3910 possibly involved in biosynthesis of isorosmarin by constructing phylogenetic tree and BLAST comparison. This work was completed by using TBtools.

[0027] Amino acid sequence of glycosyltransferase PvUGT3910 (SEQ ID NO. 1): MAHGHMIPAIDMAKLFTSRGLKATIVSTPSFSDPVKKAQDCGFQIDLIILDFPHRESGLPDHIVSLDQVTSEQQLLPKFLKALSLLQEPVEELIRRHNPDCLVSDMFLPWTADSAAASGIPRLVFHGTSYFSLCVSEEMRRHKPFNNVVSDSEPFLIPNLPHQVRLVKTQVSDYNLHENKDDFTRWIQRMKEADARSYGVVVNSFYELEPSYADHYRRVLGRKAWSLGPFSIQGQNKQQVAIKDGHYCLEWLDSKKANSVVFVCFGSMAKFTPPQLREIAGGLENSGQDFIWIVREAGDKEEKWLPEKYEERVKGRGLIVRGWAPQVAILEHEAVGAFVTHCGWNSTLEGVCAGVPMITWPLFAEQFFNEKLLTEVLRIGVSVGNKKWQRGANEGVGGEALAKAVQVVMVGETAAEMRKRAAECKEMGREAVQQGGSSYDDLTALMEEIATYSPPSPTKHHDHAHLLI Nucleotide sequence of glycosyltransferase PvUGT3910 (SEQ ID NO. 2): The tertiary structure of P. vulgaris PvUGT3910 protein is as follows Figure 2 Analysis of the protein structure, combined with its sequence, revealed that PvUGT3910 achieves efficient and specific glycosylation of rosmarinic acid at the C3 position through a GT-B folding framework, a PSPG motif anchoring the sugar donor, a hydrophobic / polar complementary pocket recognizing rosmarinic acid, and a catalytic triad-mediated nucleophilic attack.

[0028] The PvUGT3910 protein, a member of the glycosyltransferase family, has a sequence length of 334 amino acids, a theoretical molecular weight of approximately 37.8 kDa (based on the ExPASy ProtParam tool), and an isoelectric point (pI) of 5.89, indicating a significant net negative surface charge at physiological pH, suggesting that it may preferentially bind cationic substrates through electrostatic interactions. Hydrophobicity analysis (GRAVY index: -0.32) further confirmed that it is a typical hydrophilic protein, suggesting that it is primarily localized in the cytoplasm or functions through peripheral membrane association. Sequence composition analysis showed that high-frequency residues include leucine (L, 9.0%), alanine (A, 7.8%) and glycine (G, 7.5%). The enrichment of such small molecules / flexible amino acids may confer conformational adjustability to the catalytic core, thereby adapting to substrate diversity and dynamic catalytic requirements; in addition, local aggregation of proline (P, 5.7%) and serine (S, 5.4%) may participate in mediating specific hydrogen bond networks in the substrate recognition process and conformational rearrangements in glycosyl transfer reactions. Secondary structure prediction (JPred4) indicates that the protein consists of 34% α-helices (densely distributed at positions 1-50 of the N-terminus and positions 150-200 of the central region, likely maintaining catalytic domain rigidity through a helical bundle), 21% β-sheets (dispersed throughout the domain, forming the β-barrel backbone of the substrate-binding pocket), and 45% random coils (particularly enriched near the C-terminal PSPG motif). This suggests that the C-terminus may regulate the binding and release of the UDP-sugar donor through a highly dynamic random coil conformation, thereby coordinating with the N-terminal domain to complete the glycosylation cycle. These physicochemical and structural features collectively support the catalytic mechanism of PvUGT3910 as a typical GT-B fold glycosyltransferase, namely, a two-domain structure (Rossmann fold) forming a substrate channel and relying on the PSPG motif to dynamically coordinate the specific binding of the donor and acceptor.

[0029] Example 1: Gene cloning and expression vector construction After RNA was extracted from Prunella vulgaris, reverse transcription was performed on cDNA, and then the full-length target fragment was obtained by PCR, which was constructed into the pet28a expression vector by seamless cloning. The specific steps are as follows.

[0030] 1. Gene cloning and cDNA synthesis Total RNA was extracted using the Biotec Polysaccharide and Polyphenol Plant Total RNA Rapid Extraction Kit. Fresh Prunella vulgaris seed tissue was ground into powder under liquid nitrogen for extraction. Total RNA content and quality were determined using an ultramicro spectrophotometer and 1% agarose gel electrophoresis, respectively. Figure 3 After passing the test, the samples were stored at -80°C for future use. Total RNA was used as a template and reverse transcribed into cDNA using the PrimeScript™ Ⅱ 1st Strand cDNA Synthesis Kit (TAKARA, 6210A) and stored at -20°C.

[0031] Based on the CDS sequence of the gene, forward and reverse primers containing the complete open reading frame were designed using Snapgene software. PCR amplification was performed using the cDNA as a template. The primer sequences and reaction conditions are as follows.

[0032] Primer sequences: F:5'-atgggtcgcggatccgaattcATGGCCCACGGCCACATG-3' R:5'-ctcgagtgcggccgcaagcttTTAAATTAGTAGATGAGCGTGGTCATG-3'.

[0033] PCR reaction system (50 µL): cDNA 2 µL, upstream primer: 2 µL, downstream primer: 2 µL, 2 × Phanta Flash Master Mix 25 µL, ddH2O 19 µL.

[0034] PCR reaction conditions: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, 60℃ annealing for 5s, 72℃ extension for 30s, 35 cycles; 72℃ 1min to terminate the reaction, store at 4℃, expand the system for gel cutting and recovery to obtain the target fragment ( Figure 4 ).

[0035] 2. Linearized Vector Preparation Double enzyme digestion was used to completely linearize the vector and reduce the transformation background (false positive clones). The enzyme digestion sites were selected on the vector (EcoRI / HindIII). The Thermo Fisher fast endonuclease Fermentas FastDigest EcoRI (FD0274) and Fermentas FastDigest HindIII (FD0504) were used in the following reaction system:

[0036] The enzyme was digested at 37°C for 30 minutes and inactivated at 80°C for 15 minutes. The digestion effect was detected by 1% agarose gel electrophoresis. The system was expanded for gel excision and recovery to obtain the linearized vector.

[0037] 3. Ligation of the vector by homologous recombination The amount of DNA required for the recombination reaction was calculated according to the formula using the Invitrogen® ClonExpress® II One Step Cloning Kit (C112). To ensure accuracy of pipetting, the linearized vector and insert were diluted appropriately before the recombination reaction was prepared. The amount of each component added was not less than 1 μl.

[0038] Optimal amount of vector used = [0.02 x number of base pairs of the vector] ng (0.03 pmol) Optimal amount of insert used = [0.04 x number of base pairs of the insert] ng (0.06 pmol) The following reaction was prepared on ice:

[0039] The reaction was mixed gently by pipetting up and down (do not vortex) and then briefly centrifuged to collect the reaction at the bottom of the tube. The reaction was incubated at 37°C for 30 min; then it was cooled at 4°C or immediately on ice.

[0040] 4. Transformation of the recombination product The DH5a competent cells were thawed on ice, 10 μl of the recombination product was added to 100 μl of the competent cells, the tube was mixed by flicking (do not vortex), and the mixture was incubated on ice for 30 min. The mixture was then heated at 42°C for 45 sec, immediately cooled on ice for 2-3 min, and 900 μl of LB medium (without antibiotics) was added. The mixture was incubated at 37°C for 1 h with shaking at 200-250 rpm. The appropriate LB solid medium containing the correct antibiotic was preheated in a 37°C incubator, the mixture was centrifuged at 5,000 rpm (2,400 x g) for 5 min, the supernatant was discarded, and the bacterial cells were resuspended with the remaining medium. The cells were spread gently on the plate containing the correct antibiotic using a sterile spreader, and the plate was incubated in a 37°C incubator for 12-16 h until colonies appeared.

[0041] 5. Identification of the recombination product Several colonies from the transformation plate were picked for colony PCR identification using at least one of the universal sequencing primers on the vector. If the cloning was correct, a band slightly larger than the size of the insert should appear. The colonies that were positive in the colony PCR were inoculated into liquid LB medium containing the appropriate antibiotic and incubated overnight. The plasmid was extracted and sequenced. 500 μl of the correct sequencing colonies were inoculated into 30 ml of LB liquid medium containing Kanamycin and incubated at 37°C for 10 h.

[0042] The recombinant plasmid was extracted according to the TIAN prep Mini Plasmid Kit (DP103) and stored at -20°C. Figure 5 shown.

[0043] Example 2: Expression and purification of recombinant protein The recombinant plasmid pet28a-PvUGT3910 was transformed into prokaryotic Escherichia coli BL21 (DE3) competent cells for induction expression. The specific method is as follows.

[0044] BL21 (DE3) competent cells were removed from -80°C and thawed on ice. 25 µL aliquots were dispensed into ice-cold 1.5 mL microcentrifuge tubes. 50 ng of vector was added and gently pipetted several times. The tubes were incubated on ice for 5 minutes, snap-frozen in liquid nitrogen for 5 minutes, heat-shocked at 37°C for 5 minutes, and then incubated on ice for 5 minutes. In a laminar flow hood, 700 µL of LB medium was added and the cells were recovered at 28°C at 200 rpm for 1 hour. Centrifuged at 12,000 rpm for 1 minute, 700 µL of the supernatant was aspirated, and the remaining liquid and precipitate were mixed with a pipette. The cells were evenly plated onto LB medium supplemented with 50 mg / L Kan and incubated in an inverted tube at 37°C. Single colonies were identified and PCR products were analyzed by gel electrophoresis. Positive clones were cultured in 20 mL of LB medium supplemented with Kan at 37°C at 200 rpm to an OD value of 0.5-0.6. 1 mL of bacterial culture was centrifuged at 8000 rpm for 5 minutes to collect the cells. The supernatant was discarded, and 250 μL of Tris-HCl lysis buffer was added to the pellet and stored at 4°C for subsequent gel running. The remaining bacterial culture was treated with IPTG to a final concentration of 0.1 mM and cultured at 28°C for 20 hours. A pet28a empty vector was treated under the same conditions as a blank control. 16 mL of bacterial culture was centrifuged at 8000 rpm for 5 minutes to collect the cells. The supernatant was discarded, and 4 mL of Tris-HCl lysis buffer was added to the pellet. The cells were disrupted using an ultrasonic cell disruptor (Ningbo Xinzhi, JY96-IIN) for 10 minutes. 500 μL of the disrupted cells was stored at 4°C for subsequent gel running. The remaining 3500 μL of disrupted cells was centrifuged at 8000 rpm for 5 minutes at 4°C. The supernatant was aspirated, and the pellet was resuspended in 3500 μL of Tris-HCl lysis buffer.

[0045] Take 40 μL of induced empty load, uninduced, induced crude enzyme, supernatant and precipitate, add 10 μL of loading buffer, mix well, boil for 10 minutes, load and perform 10% SDS-PAGE electrophoresis analysis, stain with Coomassie Brilliant Blue solution, decolorize with pure water for observation and analysis. The results showed that the protein was successfully expressed in E. coli ( Figure 6 ). Example 3: In vitro enzyme activity and functional identification of recombinant protein 1. In vitro enzymatic assay of recombinant proteins Rosmarinic acid was used as a substrate for an in vitro enzymatic reaction. The system was as follows:

[0046] Add substrates rosmarinic acid, UDP-glucose, and crude enzyme solution; react at 37°C for 2 hours, add 100 μl of methanol to terminate the reaction, centrifuge at 12000 ppm for 15 minutes at 4°C, collect the supernatant, filter through a 0.22 μm microporous membrane, and perform LC-MS analysis.

[0047] 2. Ultra-High Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS / MS) Analysis of Enzymatic Reactions (1) Standard preparation: accurately weigh 0.524 mg of isorosmarinic acid glycoside standard and dissolve it in 10 ml of ultrapure water to a final concentration of 0.0524 mg / mL; accurately weigh 0.360 mg of rosmarinic acid standard and dissolve it in 10 ml of ultrapure water to a final concentration of 0.036 mg / mL.

[0048] (2) Preparation of mixed standard intermediate solution (10 μg / mL): Take 0.191 mL of isorosmarinic acid glycoside standard stock solution and 0.278 mL of rosmarinic acid standard stock solution in a 1 mL volumetric flask and dilute to the mark with methanol. (3) Preparation of mixed standard working solution: Take 0.02 mL, 0.05 mL, 0.1 mL, 0.2 mL and 0.5 mL of the standard intermediate solution into five 1 mL volumetric flasks, and dilute to the mark with methanol. The mixed standard solution with the contents of isorusmarinic acid glycosides and rosmarinic acid is 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL and 5.0 μg / mL, respectively.

[0049] (4) Liquid chromatography conditions A Waters ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used with a mobile phase of 0.1% formic acid (A)-acetonitrile (B) at a flow rate of 0.4 mL / min. Gradient elution was performed as follows: 95% to 70% A (0–15 min); 70% to 50% A (15–19.5 min); 50% to 5% A (19.5–20.5 min); 5% A (20.5–21.5 min); 5% to 95% A (21.5–22 min); and 95% A (22–25 min). The column temperature was 45°C, the injector temperature was 12°C, and the injection volume was 1 μL.

[0050] (5) Mass spectrometry conditions Compounds were detected using an electrospray ionization source in negative ionization and multiple reaction monitoring (MRM) mode. Two sets of ion pairs were used for quantitative and qualitative analysis, respectively, and quantification was performed using an external standard method. The capillary voltage was 3 kV, the evaporation temperature was 500°C, and the gas flow rate was 750 L / Hr.

[0051] Ion source: electrospray ion source (ESI source); Monitoring mode: negative ion monitoring mode. The monitoring ion pairs and related parameter settings are shown in the table below:

[0052] 3. Results Analysis Using rosmarinic acid as a substrate and UDP-glucose as a glycosyl donor, the catalytic results were detected by an in vitro enzyme expression system combined with liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The results showed that within the specified reaction time, compared with the control group, PvUGT3910 could catalyze the O-glucosylation of rosmarinic acid to produce isosmarinic acid glycosides ( Figure 7 ), but its catalytic activity was weak, which may be related to some inherent defects of prokaryotic expression, such as the lack of post-translational modification, protein folding, and protein solubility in the prokaryotic system. Therefore, we used tobacco transient expression to further verify PvUGT3910.

[0053] Example 4: Tobacco transient expression function verification This part of the vector construction was carried out by Wuhan Jinkairui Biological Co., Ltd. After the target gene and vector were fully synthesized, the stop codon was removed and the construct was constructed into the PCAMBIA1300-35S-GFP vector for fusion expression. The specific steps are as follows: 1. Gene Amplification PCR amplification reaction system:

[0054] PCR amplification reaction conditions:

[0055] For electrophoresis detection and recovery of amplified fragments, refer to the kit instructions.

[0056] 2. Enzyme digestion of vector and target gene

[0057] 3. Ligation of the vector and target gene

[0058] 16°C for 0.5-1 h, -20°C storage.

[0059] 4. Transformation Add the DNA fragment to be transformed to a tube containing TOP10 competent cells (25 ng of DNA is required for 50 μl of competent cells), the volume should not exceed 5% of the competent cells, mix the contents gently by rotating several times, place the centrifuge tube mixture in a circulating water bath heated to 42°C, heat shock for 90 s, do not shake the tube. Quickly transfer the tube to an ice bath to cool the cells for 1-2 min. Add 200 μl of SOC liquid medium to each tube, warm the medium to 37°C in a water bath, then transfer the tube to a shaker set to 37°C, 220 rpm, incubate for 45 min to allow the cells to recover and express the plasmid-encoded resistance marker gene. Transfer the appropriate volume of transformed competent cells to LB medium containing the corresponding antibiotic, invert the plate, and incubate at 37°C, 12-16 hours later, bacterial colonies will appear.

[0060] 5. Colony PCR verification and sequencing After the colonies grow on the plate, randomly pick several for colony PCR verification to detect the transformants. Positive clones are sent to the company's sequencing platform for sequencing verification, and finally obtain a plant expression vector containing a gene expression cassette. The plasmid map is shown in Figure 6 .

[0061] 6. Agrobacterium transformation and infection of tobacco and in vivo enzymatic Take 1 μL of recombinant PCAMBIA1300-35S-GFP-PvUGT3910 plasmid, heat shock into Agrobacterium GV3101 (pSoup) competent cells for transformation, pick single colonies for PCR identification, take positive colonies to expand culture to OD600 of about 0.6-0.8, centrifugal collection of bacterial cells, resuspended with equal volume of infection liquid (10 mmol·L -1 MES, 10 mmol·L -1 MgCl2, 200 mmol·L -1 ACS), stand for 2 h, then inject tobacco, then cultivate in the dark for 48 h, then inject 1 mL of equal volume of 1 mM rosemary acid and 1.5 mM UDP-glucose mixture, continue to cultivate in the dark for 4 days, weigh 1.0 g of fresh sample, cut and place in a 50 mL EP tube, add 20 mL of 70% methanol, 400 W ultrasonic extraction for 1 h, pass the extract through a 0.22 um microporous filter to obtain the sample solution to be tested, and the detection method is the same as in Example 3.

[0062] 7. Tobacco in vivo enzymatic and LC-MS / MS detection Through tobacco in vivo enzyme activity expression system combined with liquid chromatography-tandem mass spectrometry detection, whether the protein encoded by PvUGT3910 gene has the function of catalyzing rosmarinic acid to generate isorosmarinoside was analyzed. By comparison with the standard product, it was confirmed that the protein encoded by PvUGT3910 gene had the ability to catalyze rosmarinic acid to generate isorosmarinoside, and the catalytic efficiency was improved from 1.59% of the prokaryotic system to 42.57%, which was significantly higher than that of the prokaryotic expression system. The above tests proved that the protein encoded by PvUGT3910 gene could catalyze the hydroxyl group at C(3) of rosmarinic acid to be modified by glucosylation, and finally generate isorosmarinoside.

Claims

1. An isolated glycosyltransferase having an amino acid sequence as shown in SEQ ID NO: 1, or a variant of the amino acid sequence as shown in SEQ ID NO: 1 obtained by substitution and / or deletion and / or addition of one or more amino acid residues, which retains the catalytic activity of the glucosyl group at the C3 position of rosmarinic acid.

2. A gene encoding the glycosyltransferase according to claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID NO:

2.

3. A specific primer pair for amplifying the gene according to claim 2, consisting of an upstream primer as shown in SEQ ID NO. 3 and a downstream primer as shown in SEQ ID NO.

4.

4. A recombinant expression vector comprising the gene according to claim 2, wherein the vector is a prokaryotic expression vector or a plant expression vector.

5. A host cell comprising the recombinant expression vector according to claim 4, wherein the host cell is Escherichia coli or Agrobacterium.

6. Use of the glycosyltransferase according to claim 1 or its encoding gene, or a recombinant expression vector or host cell containing the encoding gene, in catalyzing the glucosylation of rosmarinic acid at the C3 position to biosynthesize isosmarinic acid.

7. A method for catalytically synthesizing isorosmarinic acid glycosides, comprising: Rosmarinic acid and UDP-glucose are used as reaction starting materials, and the glycosyltransferase according to claim 1 is used to catalyze the glucosylation of rosmarinic acid at the C3 position.

8. The method according to claim 7, wherein: The glycosyltransferase is produced by an Agrobacterium-mediated tobacco transient expression system, and rosmarinic acid and UDP-glucose are injected into tobacco leaves for in vivo catalysis.

9. A biocatalyst comprising the glycosyltransferase according to claim 1, for catalyzing the synthesis of isorosmarinic acid glycosides.

10. A transgenic tobacco plant having the gene according to claim 2 integrated into its genome, wherein the gene is expressed in leaf tissue and can catalyze rosmarinic acid to produce isosmarinic acid glycosides.

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