A glycosyltransferase gene for barley flavonoids and its applications

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a glycosyltransferase gene for flavonoid compounds in highland barley and its applications. This invention discovers a novel glycosyltransferase gene, HOVUSG6091200, in highland barley. The protein expressed by this gene can efficiently catalyze the glycosylation modification of various flavonoid compounds, thereby preparing flavonoid glucosides and enhancing the health benefits of highland barley. This invention also transfers this gene into tobacco, enabling tobacco plants to express the glycosyltransferase gene for flavonoid compounds, further producing flavonoid glucosides and increasing the value of tobacco plants. The novel gene provided by this invention, along with its recombinant vector, recombinant bacteria, and transgenic plants, all have promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a glycosyltransferase gene of barley flavonoids and its uses. Background Technology

[0002] Barley, a cold-resistant crop unique to the Qinghai-Tibet Plateau region, has long been adapted to the extreme environment of high altitude, low oxygen, and strong ultraviolet radiation. Its seeds and stems are rich in flavonoids, secondary metabolites that play a crucial role in plant stress resistance, antioxidant activity, and interactions with soil microorganisms. However, the biological activity and stability of natural flavonoids are easily affected by environmental factors, and their function within the plant depends on the regulation of glycosylation modifications.

[0003] Flavonoid glycosylation modification significantly enhances the water solubility, chemical stability, and transmembrane transport capacity of flavonoids by adding glycosyl groups (such as glucose and rhamnose) to the flavonoid backbone. Furthermore, glycosylation modification can modulate the bioactivity of flavonoids, affecting their functional specificity in plant disease resistance, ultraviolet absorption, and signal transduction. Although the role of glycosyltransferases (GTs) in plant secondary metabolism has been extensively studied, the enzymatic mechanisms underlying flavonoid glycosylation modification in barley remain to be explored.

[0004] In recent years, transcriptomic and metabolomics analyses have identified several glycosyltransferase genes (such as HvGT1-4) involved in flavonoid glycosylation modification in highland barley. However, current research has largely focused on single-substrate-specific glycosyltransferases, and multifunctional glycosyltransferases capable of achieving efficient glycosylation modification on multiple flavonoid substrates have not yet been discovered. This technological gap limits the biosynthetic efficiency of highland barley flavonoids and their application potential in functional foods, pharmaceutical intermediates, and other fields. Summary of the Invention

[0005] To address the problems of existing technologies, this invention provides a glycosyltransferase gene for barley flavonoids and its uses.

[0006] The use of the gene with the nucleotide sequence shown in SEQ ID NO.3 in the preparation of flavonoid glucoside; wherein the flavonoid glucoside is selected from at least one of senna-7-O-glucoside, luteolin-7-O-glucoside, sennaol-7-O-glucoside, apigenin-7-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, and anthocyanin-3-O-glucoside.

[0007] The present invention also provides the use of a recombinant vector containing a gene with a nucleotide sequence as shown in SEQ ID NO.3 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of senna-7-O-glucoside, luteolin-7-O-glucoside, senna-7-O-glucoside, apigenin-7-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, and anthocyanin-3-O-glucoside.

[0008] Preferably, the recombinant vector is recombinant pGEX-6P-1 or recombinant pEAQ.

[0009] The present invention also provides the use of recombinant bacteria containing a gene with a nucleotide sequence as shown in SEQ ID NO.3 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of senna-7-O-glucoside, luteolin-7-O-glucoside, senna-7-O-glucoside, apigenin-7-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, and anthocyanin-3-O-glucoside.

[0010] Preferably, the recombinant bacteria are recombinant Escherichia coli or recombinant Agrobacterium;

[0011] Preferably, the recombinant bacteria is recombinant Transetta or recombinant Agrobacterium EHA105.

[0012] The present invention also provides the use of a protein with an amino acid sequence as shown in SEQ ID NO.4 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of senna-7-O-glucoside, luteolin-7-O-glucoside, senna-7-O-glucoside, apigenin-7-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, and anthocyanin-3-O-glucoside.

[0013] The present invention also provides a method for preparing flavonoid glucoside, which uses a protein with an amino acid sequence as shown in SEQ ID NO.4, with glucose as a glycosyl donor and flavonoids as glycosyl acceptors to prepare flavonoid glucoside; wherein the flavonoids are selected from at least one of succinate, luteolin, succinate, quercetin, apigenin, kaempferol, and anthocyanins.

[0014] This invention also provides a method for constructing a transgenic plant that produces flavonoid glucosides, comprising the following steps:

[0015] The gene with the nucleotide sequence shown in SEQ ID NO.3 was transferred into a plant to obtain a plant expressing the protein with the amino acid sequence shown in SEQ ID NO.4; the plant was barley or tobacco.

[0016] Preferably, the method for transferring the substance into the plant is one of Agrobacterium-mediated transformation, gene gun method, electroporation, PEG-mediated transformation, liposome method, and calcium phosphate-DNA coprecipitation method.

[0017] The present invention also provides the use of a gene with a nucleotide sequence as shown in SEQ ID NO.3, a recombinant vector containing the gene with a nucleotide sequence as shown in SEQ ID NO.3, and a recombinant bacterium containing the gene with a nucleotide sequence as shown in SEQ ID NO.3 in the preparation of transgenic plants for producing flavonoid glucosides; wherein the plant is barley or tobacco; and the flavonoid glucosides are selected from at least one of senna-7-O-glucoside, luteolin-7-O-glucoside, sennaol-7-O-glucoside, apigenin-7-O-glucoside, quercetin-7-O-glucoside, kaempferol-3-O-glucoside, and anthocyanin-3-O-glucoside.

[0018] This invention provides a broad-spectrum substrate-adaptive glycosyltransferase gene, HOVUSG6091200, derived from highland barley. The protein expressed by this gene—a highland barley flavonoid glycosyltransferase—can convert various flavonoids into flavonoid glucosides, enhancing the health benefits of highland barley and promoting its targeted improvement. This invention utilizes this gene fragment for in vitro expression to obtain the highland barley flavonoid glycosyltransferase. In the in vitro reaction, glucose was used as the glycosyl donor and flavonoids as the glycosyl acceptor, successfully preparing flavonoid glucosides. This invention also transfers this gene into tobacco, enabling tobacco plants to express the highland barley flavonoid glycosyltransferase gene, further producing flavonoid glucosides and increasing the value of tobacco plants. The novel gene provided by this invention, along with its recombinant vector, recombinant bacteria, and transgenic plants, all have promising application prospects.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 For the purification analysis of HvUGT71A2 protein, HvUGT71A2 protein was purified using an E. coli expression system. SP (supernatant) is the total soluble protein after cell lysis and centrifugation; FT (flowthrough) is the protein not bound to the resin; W2, W3, and W4 are the collection solutions of the second, third, and fourth elution fractions, respectively; E1 and E2 are the first and second elution fractions, respectively; M is the protein molecular weight marker (protein ladder), Marker: 100, 70, 55, 40, 35, 25 kDa.

[0022] Figure 2 A. HvUGT71A2 catalyzes anthocyanin glycosylation. Enzymatic reaction results show that HvUGT71A2 can catalyze the O-glucosylation of Cyanidin at the 3-hydroxyl group. B. Schematic diagram of the one-step reaction mechanism of HvUGT71A2 catalyzing anthocyanin modification.

[0023] Figure 3 To investigate the glycosylation activity of HvUGT71A2 on various flavonoids, enzymatic reaction results showed that HvUGT71A2 can catalyze the O-glucosylation of Chrysoeriol (Chr), Luteolin (Lut), Eriodictyol (Eri), Quercetin (Que), and Apigenin (Api) at the 7-hydroxyl position, as well as the O-glucosylation of Kaempferol (Kae) at the 3-hydroxyl position.

[0024] Figure 4 The reaction procedure for verifying the in vitro enzyme activity of HvUGT71A2 was hypothesized. All experiments were based on three independent replicates (n=3), and data are expressed as mean ± standard deviation.

[0025] Figure 5 Mass spectra of Chrysoeriol 3-O-glucoside, Chrysoeriol 7-O-glucoside, Luteolin 7-O-glucoside, Eriodictyol 7-O-glucoside, Kaempferol 3-O-glucoside, Quercet in 7-O-glucoside, and Apigenin 7-O-glucoside. Detailed Implementation

[0026] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0027] Example 1: Construction of the HOVUSG6091200 gene and vector, and its prokaryotic expression.

[0028] This embodiment mainly describes the method for obtaining the HvUGT71A2 (HOVUSG6091200) gene, constructing the vector, and expressing it in prokaryotes.

[0029] 1. Obtaining the target gene HOVUSG6091200

[0030] Weigh 2 grams of fresh barley leaves, extract barley RNA, synthesize cDNA using Thermo Fisher's M-MLV Reverse Transcriptase, and design primers as follows:

[0031] F:ATGGCGCCCCCGC(SEQ ID NO.1)

[0032] R:TCACACCCGACAAACTATCTCGACA(SEQ ID NO.2)

[0033] Perform PCR amplification to obtain a fragment of the target band size (results as shown in the image). Figure 1 (As shown). The PCR products were purified using a gel extraction kit (Gel Extraction Kit D2500-02, OMEGA).

[0034] The nucleotide sequence (SEQ ID NO.3) of the amplified target fragment HOVUSG6091200 gene is as follows:

[0035]

[0036] The HOVUSG6091200 gene described in the aforementioned nucleotide sequence can be obtained using the above method or synthesized directly.

[0037] 2. Carrier Construction

[0038] The gene HOVUSG6091200 was transferred into the vector pGEX-6P-1 to obtain the recombinant vector.

[0039] 3. Construction of recombinant strains

[0040] The recombinant vector was transformed into the Transetta(DE3) strain to obtain a recombinant strain containing the target fragment.

[0041] 4. Expression of the target gene

[0042] (1) PCR detection of positive clones, plasmid extraction and sequencing.

[0043] (2) Transform Escherichia coli transeta(DE3) with the correctly sequenced plasmid vector by heat shock and resistant CN.

[0044] (3) Randomly select two normal-sized clones and incubate them in 5 mL of LB medium containing ampicillin (Amp) at 37°C for 7 hours. Select one of them and add 4 mL of activated bacterial solution (concentration of 1×10⁻⁶). 6 ~10 7 (CFU / ml) was transferred at a ratio of 1:50 to 200 mL of LB medium and incubated on a large shaker at 37°C and 200 rpm. After 3–4 hours, 2 μL of 1 M IPTG inducer was added to the 200 mL medium, and incubated overnight at 20°C and 160 rpm. The remaining 1 mL of bacterial culture was used for preservation.

[0045] (4) Collect the bacterial cells the next morning, put them into a 500mL centrifuge bottle, and centrifuge at 4000rpm for 10min.

[0046] (5) Resuspend the bacterial cells in 50 mL Lysis buffer, vortex to mix, transfer to 50 mL centrifuge tubes, add 50 μL L PMSF and 10 μL β-mercaptoethanol respectively, mix well and place on ice.

[0047] (6) The Escherichia coli cell disruption experiment was conducted using a high-pressure disruptor.

[0048] (7) After the sample is disrupted, take 20 μL as the total protein sample. Then take 1 mL of the disrupted sample and centrifuge at 13,000 rpm for 10 min at 4°C. Take 20 μL of the supernatant as the supernatant sample. Add an equal volume of 2*Loading buffer to the supernatant sample, boil for 5 min, and detect protein expression by SDS-PAGE electrophoresis. The remaining supernatant can be temporarily frozen at -20°C. The remaining uncentrifuged sample can be frozen at -80°C.

[0049] (8) After SDS-PAGE electrophoresis, add Coomassie Brilliant Blue staining solution, microwave for 1 minute, then stain for half an hour, and add destaining solution to destain. Change the destaining solution every 1 hour until the protein bands are clear, then transfer to distilled water.

[0050] (9) Purification of GST-tagged fusion protein. Centrifuge all lysed but not centrifuged samples. Mix the supernatant with 1 mL of resin at 4°C for 3 h. After mixing, pass the mixture through the chromatography column twice. First, rinse the resin (Glutathione Sepharose™ 4B, GE) with pre-cooled Lysis buffer, and simultaneously check the eluent with Bradford Assay until the blue color disappears, indicating that the contaminating protein has been washed away. Then, elute the target protein with 15 mmol / L reduced glutathione solution (0.09 g dissolved in 20 mL lysis buffer), adding 1 mL each time. Collect approximately 1 mL from the bottom of the chromatography column using 1.5 mL centrifuge tubes, labeling each tube as E1, E2, E3, E4, E5, and E6, until Bradford Assay indicates that the eluent contains no protein. Continue eluting the resin with any remaining reduced glutathione solution, then rinse with Lysis buffer, ddH2O, and 20% ethanol, and store in 20% ethanol.

[0051] (10) The collected protein was detected by SDS-PAGE, yielding a 76 kDa band. Figure 1 The molecular weight of the GST tag is 26 kDa, and the molecular weight of the remaining target protein is approximately 50 kDa, which is the same as the molecular weight calculated from the amino acids. This indicates that the present invention has prepared a target protein with a GST tag.

[0052] The amino acid sequence of the target protein (SEQ ID NO.4) is as follows:

[0053] MAPPPPHIAVVAFPFSSHAAVLFSFARALAAAAPAGTSLSFLTTADNAAQLRKAGALPGNLRFVEVPDGVPPGETSWLSPPRRMELFMAAAEAGGVRAGLEAACASAGGARVSC VVGDAFVWMAADAASAAGAPWVAVWTAASCALLAHLRTDALRRDVRDQAASRADELLTAHAGLGGYRVRDLPDGVVSGDFNYVISLLVHRQAQRLPKAATAVALNTFPGLDPPD LTAALAAELPNCQPLGPYHLLPGAEPTADTNEAPADPHGCLAWLDRRPARSVAYVSFGTNATARPDELQELAAGLEASGAPFLWSLREESWPLLPPGFLERAPGLVVPWAPQVG VLRHAAVGAFVTHAGWASVMEGVSSGVPMACRPFFGDQTMNARSVASVWGFGTAFDGPMTRGAVANAVATLLRGEDGERMRAKAQELQAMVGKAFEPDGGCRKNFDEFVEIVCRV

[0054] Example 2: Construction of transgenic tobacco

[0055] ① The transient expression vector containing the target gene HOVUSG6091200 (transient expression vector pEAQ, from John Innes Centre) was transformed into Agrobacterium (EHA105);

[0056] ② Pick positive Agrobacterium clones and place them in 500 μL of LB containing the corresponding antibiotic (kn) and incubate for 20-24 hours;

[0057] ③ Transfer 200ul to 5ml of LB containing the corresponding antibiotic (kn), and shake at 220rpm on a 28°C shaker until OD = 2.0.

[0058] ④ Collect the bacterial cells by centrifugation at 10000 rpm at room temperature for 2 min, resuspend the bacterial cells in pre-prepared transformation buffer, and shake on a shaker for 3 h; the working solution composition and concentration of the buffer are as follows: 10 mM MES (pH 5.7), 10 mM MgCl2, 100 μUDP-glucose.

[0059] ⑤ Take a prepared 1ml syringe, remove the needle, select a syringe with a smooth nozzle, draw in the bacterial solution, take a 1-month-old Nicotiana benthamiana, hold the leaf with your hand, and inject from the underside of the leaf to allow Agrobacterium to penetrate. Mark each injected tobacco plant by circling the area where Agrobacterium penetrates on the leaf; use an equal volume of transformation buffer to inject tobacco as a control.

[0060] ⑥ Tobacco injected with Agrobacterium was cultured in the dark for 24 hours, and then transferred to a tobacco incubator for culture under light for 24-48 hours to obtain transgenic tobacco.

[0061] The following experimental examples illustrate the beneficial effects of the present invention:

[0062] Experiment 1: Enzyme activity detection of HOWUS G6091200 protein

[0063] 1. Experimental Methods

[0064] 1.1 Obtaining HOVUSG6091200 protein

[0065] The target protein with a molecular weight of 76 kDa and a GST tag was prepared according to the method in Example 1.

[0066] 1.2 Enzyme activity detection

[0067] In vitro glycosyltransferase assays were performed in 100 μL of Tris-HCl buffer (100 mM, pH 7.4) containing 200 μM anthocyanins (Cyanidin, Cya), chrysoeriol (Chr), luteolin (Lut), eriodictyol (Eri), quercetin (Que), apigenin (Api), and kaempferol (Kae) as glycosyl acceptors, 100 μM UDP-glucose as glycosyl donors, and 500 ng of purified protein. After incubation for 10 min, 300 μL of ice-cold methanol was added to stop the reaction. The reaction mixture was then filtered through a 0.2 μm filter (micropores) and used for LC-MS analysis.

[0068] 2. Experimental Results

[0069] Figure 2 To investigate the glycosylation of anthocyanins (Cyanidin) catalyzed by HvUGT71A2, the enzymatic reaction results showed that HvUGT71A2 can catalyze the O-glucosylation of Cyanidin at the 3-hydroxyl position. Figure 3 , 4To investigate the glycosylation activity of HvUGT71A2 on various flavonoids, enzymatic reaction results showed that HvUGT71A2 can catalyze the O-glucosylation of Chrysoeriol (Chr), Luteolin (Lut), Eriodictyol (Eri), Quercetin (Que), and Apigenin (Api) at the 7-hydroxyl position, as well as the O-glucosylation of Kaempferol (Kae) at the 3-hydroxyl position. Figure 5 Mass spectrometry analysis of the in vitro enzyme activity products demonstrates that the HOVUSG6091200 protein of this invention has the ability to catalyze the glycosylation of Chrysoeriol (Chr), Luteolin (Lut), Eriodictyol (Eri), Quercetin (Que), Apigenin (Api), and Kaempferol (Kae) into Chrysoeriol 7-O-glucoside, Luteolin 7-O-glucoside, Eriodictyol 7-O-glucoside, Quercetin 7-O-glucoside, Apigenin 7-O-glucoside, and Kaempferol 3-O-glucoside, indicating a promising market application prospect.

[0070] In summary, this invention provides a broad-spectrum substrate-adaptive glycosyltransferase gene HOVUSG6091200 derived from highland barley. The protein expressed by this gene—a highland barley flavonoid glycosyltransferase—can convert various flavonoids into flavonoid glucosides, enhancing the health benefits of highland barley and promoting its targeted improvement. This invention uses this gene fragment for in vitro expression to obtain the highland barley flavonoid glycosyltransferase. In the in vitro reaction, glucose is used as the glycosyl donor and flavonoids as the glycosyl acceptor, successfully preparing flavonoid glucosides. This invention also transfers this gene into tobacco, enabling tobacco plants to express the highland barley flavonoid glycosyltransferase gene, further producing flavonoid glucosides and increasing the value of tobacco plants. The novel gene provided by this invention, along with its recombinant vector, recombinant bacteria, and transgenic plants, all have promising application prospects.

Claims

1. The use of a gene with the nucleotide sequence shown in SEQ ID NO. 3 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of ginsenoside-7-O-glucoside and anthocyanin-3-O-glucoside.

2. The use of a recombinant vector containing a gene with a nucleotide sequence as shown in SEQ ID NO. 3 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of ginsenoside-7-O-glucoside and anthocyanin-3-O-glucoside.

3. The use according to claim 2, characterized in that: The recombinant vector is either recombinant pGEX-6P-1 or recombinant pEAQ.

4. The use of recombinant bacteria containing a gene with a nucleotide sequence as shown in SEQ ID NO. 3 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of ginsenoside-7-O-glucoside and anthocyanin-3-O-glucoside.

5. The use according to claim 4, characterized in that: The recombinant bacteria are recombinant Escherichia coli or recombinant Agrobacterium.

6. The use according to claim 5, characterized in that: The recombinant bacteria are recombinant Transetta or recombinant Agrobacterium EHA105.

7. Use of a protein with the amino acid sequence shown in SEQ ID NO. 4 in the preparation of flavonoid glucosides; wherein the flavonoid glucosides are selected from at least one of ginsenoside-7-O-glucoside and anthocyanin-3-O-glucoside.

8. A method for preparing flavonoid glucosides, characterized in that: It uses a protein with an amino acid sequence as shown in SEQ ID NO. 4, glucose as a glycosyl donor, and flavonoids as glycosyl acceptors to prepare flavonoid glucosides; the flavonoids are selected from at least one of ginsenosides and anthocyanins.

9. The use of a gene with the nucleotide sequence shown in SEQ ID NO. 3, a recombinant vector containing the gene with the nucleotide sequence shown in SEQ ID NO. 3, and a recombinant bacterium containing the gene with the nucleotide sequence shown in SEQ ID NO. 3 in the preparation of transgenic plants for the production of flavonoid glucosides; wherein the plant is barley or tobacco; and the flavonoid glucosides are selected from at least one of ginsenoside-7-O-glucoside and anthocyanin-3-O-glucoside.

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