Flavonoid glycosyltransferase and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to flavonoid glycosyl transferase and application thereof. The invention relates to a flavonoid glycosyl transferase gene, wherein the nucleotide sequence of the flavonoid glycosyl transferase gene is shown as SEQ ID NO. 1. By focusing the rice drought resistance related gene and predicting and coding the flavonoid glycosyl transferase, through corresponding experiments, key biochemical evidences are provided for clarification of a molecular mechanism of the rice drought resistance related gene in drought response, a foundation is laid for analysis of a mechanism of glycosylation modification regulation and control of rice drought resistance, and the application of the flavonoid glycosyl transferase to rice drought resistance regulation and control is developed. The method has important theoretical value and application potential for crop stress resistance genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a flavonoid glycosyltransferase and its applications. Background Technology

[0002] Rice (Oryza sativa L.) is the staple food for more than half of the world's population, especially in Asia, providing approximately 80% of daily calorie intake. Its safe production is crucial for ensuring food security. However, global warming has led to frequent droughts, severely restricting rice yields and causing huge economic losses. In recent years, global rice production has been significantly reduced due to drought, and the accumulation of reactive oxygen species induced by drought stress leads to oxidative damage in plants. Flavonoid metabolites, such as flavonols, can effectively scavenge reactive oxygen species.

[0003] Flavonoid metabolites, also known as flavonoid compounds, refer to a class of plant secondary metabolites with a 2-phenylchromone (2-phenylchromone) core structure. They are widely present in the roots, stems, leaves, flowers, and fruits of higher plants in either bound (e.g., flavonoid glycosides) or free (e.g., flavonoid aglycones). As products of plant secondary metabolism, flavonoids are polyphenolic compounds biosynthesized via the phenylalanine metabolic pathway. They participate in various physiological processes during plant growth and development, such as resisting environmental stresses like pathogens, herbivores, and ultraviolet radiation. Chemically, flavonoids typically consist of two benzene rings linked by a central three-carbon chain. Their basic skeleton can derive various subclasses, such as flavonols, anthocyanins, and flavans. These compounds not only impart color characteristics to plants but also possess various biological activities, including antioxidant, anti-inflammatory, and antitumor effects.

[0004] Glycosyltransferases enhance the stability, bioactivity, and water solubility of metabolic substrates such as flavonols through glycosylation. They can also catalyze the formation of flavonol glycosides and other secondary metabolites with unknown functions, thus affecting the drought resistance of rice. Therefore, further research on glycosyltransferases has significant theoretical value and application potential for the genetic improvement of crop stress resistance. Summary of the Invention

[0005] This invention aims to provide a flavonoid glycosyltransferase and its application. By focusing on the rice drought-related gene RESPONSE TO DROUGHT 1 (RTD1) and predicting that it encodes a flavonoid glycosyltransferase, this invention, through corresponding experiments, not only provides key biochemical evidence for elucidating the molecular mechanism of RTD1 in drought response, but also lays the foundation for understanding the mechanism by which glycosylation modification regulates rice drought resistance. This has significant theoretical value and application potential for the genetic improvement of crop stress resistance.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a flavonoid glycosyltransferase gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0007] The present invention also claims protection for a flavonoid glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0008] The present invention also claims protection for the use of a flavonoid glycosyltransferase gene in the synthesis of flavonoid glycosides, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0009] The present invention also claims protection for the use of a flavonoid glycosyltransferase in the synthesis of flavonoid glycosides, the amino acid sequence of which is shown in SEQ ID NO. 3.

[0010] In one embodiment of the present invention, the flavonoid glycosides include at least one of rutin, quercetin, luteolin, and genistein.

[0011] This invention also claims protection for a method for preparing flavonoid glycosides, comprising the following steps: S1. Obtain a flavonoid glycosyltransferase containing the amino acid sequence shown in SEQ ID NO. 3; S2. Using the flavonoid glycosyltransferase described in step S1 to catalyze the substrate, synthesize flavonoid glycoside compounds.

[0012] In one embodiment of the present invention, the substrate includes a flavonoid receptor and UDP-glucose; the flavonoid glycoside compound includes at least one of rutin, quercetin, luteolin, and genistein.

[0013] In one embodiment of the present invention, the flavonoid receptors include quercetin, luteolin, and genistein.

[0014] This invention also claims a method for simultaneously detecting seven flavonoid components in the reaction products of the said flavonoid glycosyltransferase, comprising the following steps: S1. Preparation of standard solution: Weigh rutin, quercetin, quercetin glycoside, luteolin, luteolin glycoside, genistein, and genistein, dissolve them in methanol and dimethyl sulfoxide and make up to volume to prepare a stock solution with a concentration of 10 mg / mL, and store it at -20℃ in the dark. S2. Preparation of test solution: Add 0.1% formic acid alcohol solution to the sample to be tested, filter, and take the filtrate as the test solution; S3. High-performance liquid chromatography (HPLC) was used for detection. The detection conditions were as follows: column: C18; mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile; gradient elution program: 0-25 min 5%-30% B; 25-30 min 30% B; 30-32 min 30%-80% B; 32-38 min 80%-5% B; 38-58 min 5% B; flow rate: 0.4 mL / min. -1 Column temperature: 35℃; injection volume: 20mL; detection wavelength: 254 nm.

[0015] In one embodiment of the present invention, the concentration of the methanol solution in step S1 is 80%.

[0016] In one embodiment of the present invention, the volume ratio of methanol solution and dimethyl sulfoxide in step S1 is 1:1.

[0017] In one embodiment of the present invention, the filtration in step S2 is performed using a 0.22 mm filter membrane.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention focuses on the rice drought-resistance-related gene RESPONSE TO DROUGHT 1 (RTD1), which is predicted to encode a flavonoid glycosyltransferase that plays a negative regulatory role in rice drought resistance. To determine whether it is a glycosyltransferase and its specific reaction substrate, the RTD1 protein was transferred into a PGEX-MBP vector and cultured in *E. coli* BL21. After IPTG induction, recombinant RTD1 protein was obtained using MBP tag affinity chromatography. SDS-PAGE and Western blotting confirmed the acquisition of a high-purity, soluble in vitro recombinant protein with a molecular weight of approximately 93 kDa. Based on this, a sensitive analytical method based on high-performance liquid chromatography (HPLC) was established to verify that RTD1 is a glycosyltransferase that specifically catalyzes quercetin, luteolin, and genistein by detecting substrate consumption and product formation in the in vitro enzymatic reaction. This invention not only provides key biochemical evidence for elucidating the molecular mechanism of RTD1 in drought response, but also lays the foundation for understanding the mechanism by which glycosylation modification regulates drought resistance in rice. It has important theoretical value and application potential for crop stress resistance genetic improvement and for providing molecular targets for rice stress resistance breeding, and has potential application prospects in sustainable agricultural development. Attached Figure Description

[0019] Figure 1The figures show the results of RTD1 recombinant protein testing in this embodiment of the invention. (a), (b), and (c) show the recombinant protein size detected using Coomassie Brilliant Blue staining, and (d) shows the MBP-RTD1 recombinant protein tested using a Western blot assay with MBP antibody. kDa represents protein size, and GroEL represents a bacterial chaperone protein.

[0020] Figure 2 The following are liquid chromatograms of seven flavonoid standards in this embodiment of the invention. (a) Rutin; (b) Quercetin; (c) Quercetin; (d) Luteolin; (e) Gentiana; (f) Luteolin; (g) Gentiana.

[0021] Figure 3 This is a standard curve diagram of the seven types of flavonoid mixed standards in the embodiments of the present invention.

[0022] Figure 4 These are liquid chromatograms of the in vitro enzyme activity reaction in this embodiment of the invention. (a) is the chromatogram of the control group (containing quercetin, quercetin, and rutin) of mixture group 1; (b) is the chromatogram of mixture group 1 after RTD1 catalysis; (c) is the chromatogram of the control group (containing luteolin, luteolinoside, genistein, and genistein) of mixture group 2; and (d) is the chromatogram of mixture group 2 after RTD1 catalysis. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0025] The experimental materials used in the embodiments of this invention are as follows: Experimental reagents: Standards include rutin, quercetin, isoquercitrin, luteolin, luteoloside, genistein, and genistin. Other ingredients include MBPbeads, maltose, Tris-HCl (pH 7.5), Amp, IPTG, protein marker (RM19001), DTT, glycerol, MgCl2, formic acid, methanol, acetonitrile, and DMSO.

[0026] Experimental instruments and equipment: Ultra-low temperature refrigerators, pH meters, electrophoresis apparatus, pipettes, vortex mixers, benchtop low-temperature high-speed centrifuges, water baths, shakers, autoclaves, high-performance liquid chromatographs, cell ultrasonic disruptors, etc.

[0027] Example 1: Obtaining the flavonoid glycosyltransferase RTD1 gene 1. Candidate RTD1 Analysis First, plant-derived UDP-glycosyltransferase (UGT) family protein sequences were obtained from the UniProt database, and an initial candidate sequence set of 985 sequences was established. Then, MAFFT was used to perform multiple sequence alignment on these sequences, and the region containing the ATG8 binding motif (AIM) was extracted from the alignment results. A sequence conservation map of this region was constructed using WebLogo to determine the conserved amino acid characteristics of the AIM region.

[0028] After preliminary sequence analysis, a phylogenetic tree was constructed based on the full-length sequence multiple sequence alignment results to determine the phylogenetic position of candidate sequences. The analysis showed that the RTD1 sequence clusters in the typical UGT73 lineage, which is known to be associated with flavonoid metabolism. Furthermore, RTD1 shows high phylogenetic similarity with several annotated UGT73 members, indicating good conservation across species. Multiple sequence alignment analysis of the domain composition of RTD1 and related homologous proteins revealed that RTD1 contains a complete PSPG box, consistent with the structural characteristics of typical plant UGTs. Additionally, the AIM motif is clearly identifiable in the RTD1 sequence, and its position and amino acid composition are consistent across different species, suggesting that this sequence possesses potential ATG8 binding capacity.

[0029] Based on the above analysis, members without detected AIM motifs were removed from the initial sequence set, retaining candidate sequences containing AIM, resulting in 233 sequences for subsequent analysis. KEGG annotation was performed on this candidate set, and members related to abiotic stress responses or flavonol metabolism were screened, yielding 11 further candidate sequences. Based on this, combined with glycosylation modification site predictions and publicly available literature data, RTD1 was ultimately identified as the target gene.

[0030] 2. Cloning of candidate RTD1 genes 2.1 Extraction of total RNA from rice (1) Cut an appropriate amount of fresh rice leaves (Zhonghua 11, a rice variety bred by the Institute of Crop Science, Chinese Academy of Agricultural Sciences) and place them in a pre-cooled mortar (the mortar has been sterilized in advance). Add liquid nitrogen and grind them quickly and thoroughly in the liquid nitrogen. Weigh 100 mg of the ground powder into a 1.5 mL centrifuge tube without RNase, add 1 mL of Trizol, and vortex to mix. Alternatively, the sample can be cut into pieces and ground directly in Trizol. Let the lysed sample or homogenate stand at room temperature for 5-10 min to completely separate the nucleoprotein from the nucleic acid.

[0031] (2) Add 0.2 mL of chloroform, vortex for 30 s, and let stand at room temperature for 5 min. Then centrifuge at 12000 r / min and 4 ℃ for 11 min.

[0032] (3) Transfer the top aqueous phase to a new 1.5 mL RNase-free centrifuge tube, add an equal volume of isopropanol solution, shake to mix, and let stand at room temperature for 15 min.

[0033] (4) Centrifuge at 12000 r / min and 4 ℃ for 11 min, and discard the supernatant.

[0034] (5) Add 700 μL of 75% ethanol to wash the precipitate. Centrifuge at 12000 r / min and 4 ℃ for 3 min, and discard the supernatant. Dry at room temperature for 5-10 min (do not heat, and do not dry it too much, as RNA is difficult to dissolve after it is completely dried).

[0035] (6) Add 30-50 μL of RNAase-free ddH2O to fully dissolve the RNA. Store the resulting RNA solution at -80℃ or use it for subsequent experiments.

[0036] 2.2 Synthesis of the first strand of cDNA This experiment used the Evo M-MLV Reverse Transcription Kit II (Accurate Biotechnology) to reverse transcribe the previously extracted RNA to obtain the desired cDNA. The specific procedures are as follows: (1) Take the extracted RNA out of the -80 ℃ freezer, place it on ice to thaw, and then measure the concentration of RNA.

[0037] (2) The amount of RNA used in the system, X, is calculated as 1 μg RNA / concentration × volume. GDNA Clean Reagent is used to remove any residual DNA from the RNA. The reaction system is shown in Table 1: Table 1. First-step reverse transcription reaction system The reaction mixture was thoroughly mixed using a pipette, then briefly centrifuged in a centrifuge before being transferred to a PCR instrument. The reaction was performed at 42 °C for 2 min. The mixture was then immediately placed on ice for the next experimental step.

[0038] (3) The above reaction solution was further reverse transcribed into cDNA. The mixed reaction system was pipetted and centrifuged briefly before being placed in a PCR instrument. PCR reaction conditions: 37 ℃, 15 min; 85 ℃, 5 s. After the reaction, the system was stored at -20 ℃ for later use. The reaction system is shown in Table 2. Table 2. Second step reverse transcription reaction system 2.3 Target gene amplification Using cDNA extracted from Zhonghua 11 as the amplification template, specific cloning primers were designed based on the target gene sequence. The specific primer information is as follows: CdsF: 5'ATGGCAGCTGAGTCCAC 3'; CdsR: 5'CTATTCCACTCCAGTTTGCG 3'. The target gene fragment was obtained by PCR amplification. The amplification reaction system is shown in Table 3, and the amplification program is shown in Table 4. After amplification, the reaction product was added to an agarose gel containing 1% nucleic acid dye and electrophoresed for 10 min.

[0039] Table 3 Target gene amplification system Table 4. Target gene amplification procedure The nucleotide sequence of RTD1 is shown in SEQ ID NO. 1. The CDS sequence is shown in SEQ ID NO. 2.

[0040] SEQ ID NO. 1 SEQ ID NO. 2 is as follows: CDS sequence GCGTGGCTCGACAAGCAGGCCACCTGCTCCGTCGTCTACGTCGGCTTCGGCAGCGTCCTGCGAAAGCTTCCGAAGCACCTGTCCGAGGTCGGCCATGGCCTCGAGGACTCCGGCAAGCCGTTCCTCTGGGTGGTGAAGGAGTCGGAAGCTTCGTCCAGGCCGGAGGTGCAGGAATGGCTGGACGAGTTCATGGCGCGAACCGCGACGCGCGGCCTCGTGGTGCGCGGGTGGGCGCCGCAGGTGACCATCCTGTCGCACCACGCCGTCGGTGGCTTCCTCACGCACTGCGGGTGGAACTCGCTGCTGGAGGCCATCGCCCGTGGCGTGCCCGTGGCGACGTGGCCACACTTCGCCGACCAGTTCCTGAACGAGCGGCTCGCCGTGGACGTGCTCGGCGTCGGCGTGCCGATCGGCGTGACGGCGCCGGTGAGCATGTTGAACGAGGAGTACTTGACAGTTGATCGGGGTGACGTCGCGCGGGTGGTGTCGGTGCTGATGGACGGCGGCGGCGAGGAGGCCGAGGAGAGGAGGAGGAAGGCCAAGGAGTACGGTGAGCAAGCTCGAAGGGCCATGGCGAAAGGAGGCTCCTCGTATGAGAAC SEQ ID NO. 3 is as follows: Amino acid sequence Protein >LOC_Os02g11110.2 MAAESTAQAPAQPHFVLAPLAAHGHLIPMVDLAGLLAAHGARASLVTTPLNATWLRGVAGKAAREKLPLEIVELPFSPAVAGLPPDYQSADKLSENEQFTPFVKAMRGLDAPFEAYVRALERR PSCIISDWCNTWAAGVARSLGIPRLFFHGPSCFYSLCDLNAVVHGLHEQIAAAADADDEQETYVVPGMPVRVTVTKGTVPGFYNAPGCEALRDEAIEAMLAADGVVVNTFLDLEAQFVACYEA ALGKPVWTLGPLCLHNRDDEAMASTDQRAITAWLDKQATCSVVYVGFGSVLRKLPKHLSEVGHGLEDSGKPFLWVVKESEASSRPEVQEWLDEFMARTATRGLVVRGWAPQVTILSHHAVGGF LTHCGWNSLLEAIARGVPVATWPHFADQFLNERLAVDVLGVGVPIGVTAPVSMLNEEYLTVDRGDVARVVSVLMDGGGEEAEERRRKAKEYGEQARRAMAKGGSSYENVMRLIARFTQTGVE* Example 2: Induction and purification of recombinant RDT1 protein Induced expression of RDT1 recombinant protein (1) Carrier preparation The required vector bacterial strains were picked from the clean bench and added to culture medium containing the corresponding antibiotics. The bacterial solution was then placed in a constant temperature shaker at 180 r / min and 37 ℃ for 15 h overnight. Subsequently, plasmids were extracted using a small plasmid extraction kit, and the purity and concentration of the extracted plasmids were determined. 3 μg of the extracted plasmids, such as pRHVcGFP, pRTVcRFP, pMetYCgate, pCAMBIA1300-nLUC, and pCAMBIA1300-cLUC, were placed in 1.5 mL centrifuge tubes and double-digested with restriction endonucleases to prepare linearized vectors. After the enzyme digestion reaction system was prepared, it was digested for 15 min in a constant temperature water bath at 37 ℃. The enzyme digestion reaction system is shown in Table 5. Table 5 Enzyme digestion reaction system After the enzyme digestion reaction was completed, the digestion products were purified and recovered using a gel recovery kit, and the concentration and purity of the linear vector were determined. High-quality linear vectors were selected for subsequent experiments to improve the success rate of the experiments.

[0041] (2) Carrier recombination A one-step cloning method was used to ligate the enzyme-digested linear vector with the target gene fragment obtained by PCR amplification. The target fragment and linear vector were added to a 1.5 mL centrifuge tube at a ratio of 2:1, followed by the addition of a one-step cloning enzyme. The reaction system is detailed in Table 6. After thorough mixing, the mixture was briefly centrifuged and then incubated in a 50 °C metal bath for 20 min to complete the ligation of the target fragment and linear vector. After the reaction, the mixture was immediately cooled on ice. The reaction product was then stored at -20 °C for later use or directly used for transformation.

[0042] Table 6 Linear Carrier Recombination Reaction System (3) Transformation of the linker products Remove competent DH5α cells from the -80 ℃ freezer and thaw on ice. Then, add 50 μL of completely thawed DH5α competent cells to each tube of the ligation product and mix gently. Incubate the mixture on ice for 5 min, then heat shock it in a 42 ℃ water bath for 45-60 s, and then quickly return it to ice for 2 min. In a clean bench, add 700 μL of antibiotic-free liquid LB medium to each tube of reaction solution, and then incubate on a shaker at 180 r / min and 37 ℃ for 20 min. After resuscitation, the bacterial mixture was centrifuged at 6000 r / min for 30 s, most of the supernatant was discarded, and only 50-100 μL of culture medium was retained for resuspending the bacterial cells. In a clean bench, the resuspended bacterial solution was evenly spread on the surface of LB solid medium containing the corresponding antibiotic using a sterile spreader. The culture dish was then sealed with sealing film and inverted in a 37 ℃ constant temperature incubator for overnight incubation for 15 h.

[0043] (4) Screening of positive clones Observe the growth status of colonies in the transformed and cultured LB solid medium. Label larger monoclonal colonies. In a clean bench, use an autoclaved toothpick to pick up the labeled monoclonal colonies and add them to the PCR reaction system. To confirm whether the selected colonies are positive monoclonal colonies, specific primers (JDF: 5'ACAACGCTCCGGGTTGTGA 3'; JDR: 5'CTGCCGAAGCCGACGTAGA 3') need to be designed in advance for detection. The specific PCR reaction system is detailed in Table 7. The PCR amplification procedure for positive identification is shown in Table 8. Table 7 Colony PCR Reaction System Table 8 Identification PCR Amplification Procedure After the PCR reaction, the reaction products were added to a 1% agarose gel for electrophoresis analysis, and the target band was observed using a gel electrophoresis apparatus. If the expected band appeared, it indicated that the single colony was a positive clone. Subsequently, the positive colony was inoculated into liquid culture medium containing the corresponding antibiotic and cultured overnight. The bacterial culture was then sent to Guangzhou Youkang Technology Co., Ltd. or Guangzhou Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results need to be further sequence alignment analysis to determine their accuracy.

[0044] (5) Induced expression of recombinant RDT1 protein Take 20-100 mL of the above bacterial culture and inoculate it into 10 mL of liquid LB medium containing ampicillin (Amp). Incubate overnight under suitable conditions with shaking. Transfer the entire overnight culture to 400 mL of liquid LB medium containing ampicillin (Amp) and incubate at 37°C with shaking until OD reaches the target value. 600 When the concentration is 0.4-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to the bacterial culture until the final concentration is 0.1-1 mM (the normal working concentration is 0.2 mM), and continue to induce culture in a constant temperature shaker at 37℃ for 3.5 h.

[0045] 2. MBP affinity chromatography purification Transfer the bacterial culture obtained from the above steps to 50 mL centrifuge tubes and cool on ice. Centrifuge at 8000 rpm for 3 min at 4 °C, discard the supernatant, and collect the bacterial pellet. Add 30 mL of MBP equilibration buffer to each bacterial pellet and vortex thoroughly to resuspend. Under ice bath conditions, sonicate (parameters: power 10%-20%, sonication 10 s, interval 15 s, total duration 30 min) until the bacterial culture becomes clear. Centrifuge at 12000 rpm for 10 min at 4 °C, collect the supernatant, and transfer it to a new tube for later use. Take 500 mL of linear starch resin (MBP beads), add 500 mL of equilibration buffer, and gently resuspend. Centrifuge at 5000 rpm for 30 s, discard the supernatant, and repeat the washing twice. Add the pretreated MBP beads to 1 mL of equilibration buffer and mix well. Add the mixture to the lysis supernatant and vortex at 4 °C for 1 h. Rinse the filter column 2-3 times with 10 mL of equilibration buffer. Transfer the mixture to the filter column and wash with equilibration buffer to remove unbound proteins. Elute the protein with elution buffer (containing 10 mM maltose) at 200, 400, and 200 mL intervals, respectively. Label the collection tubes (strain name, date). Rinse the resin with equilibration buffer and finally soak in 20% ethanol. Store at 4°C to obtain the soluble in vitro recombinant protein RTD1.

[0046] Example 3: Identification of RDT1 recombinant protein 1. SDS-PAGE electrophoresis Prepare a 10% separating gel according to the specified ratio. After pouring the gel, cover it with anhydrous methanol and press the interface flat. Discard the gel after it solidifies. Prepare a stacking gel, insert a comb to avoid air bubbles, and remove the comb after it solidifies. Moisten the gel with electrophoresis buffer before removing the comb. Mix the protein sample with loading buffer 1:1 and incubate at 95°C for 8 min. Load 20 mL of protein into each well and perform electrophoresis at a constant voltage of 110 V for 1 h 40 min. Perform electrophoresis of the purified protein on SDS-PAGE and then stain with Coomassie Brilliant Blue to confirm the protein size.

[0047] 2. Western Blot The PVDF membrane was activated in methanol for 2 min, then placed in transfer buffer for later use. The transfer clamp was assembled in the following order: negative electrode → sponge → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge → positive electrode. Air bubbles were removed from each layer using a glass rod, ensuring tight adhesion between the gel and membrane. The clamp was placed in the transfer tank, filled with pre-cooled transfer buffer at 4°C, and covered with an ice pack. Transfer was performed at a constant current of 260 mA for 2 h. After transfer, the membrane was immersed in 5% skim milk / TBST and blocked on a shaker at room temperature for at least 1.5 h. The primary antibody was diluted 1:1000 with blocking buffer. After the membrane was blotted dry, it was immersed in the primary antibody solution and incubated overnight at 4°C (or 1.5 h at room temperature), sealed to protect from light. After completion, the membrane was washed 3 times with TBST, 5 min each time. The secondary antibody was diluted 1:5000-1:10000 with blocking buffer and incubated on a shaker at room temperature for 1 h. The membrane was washed using the same method as above. Prepare the developer solution by mixing solution A (luminol) and solution B (peroxide) in a 1:1 ratio. After the membrane is dried, place it in the imager, add the developer solution evenly, and expose to capture the signal.

[0048] Based on Coomassie Brilliant Blue staining, the experiment used a series of gradient volumes of equilibration buffer to wash the protein. The results showed that when 70 mL of equilibration buffer and 10 mM elution buffer were added, there were fewer contaminating proteins, and the recombinant protein had high purity. The recombinant protein size is estimated to be approximately 100 kDa. The lower band represents the MBP empty vector, approximately 40 kDa (Figure 1abc).

[0049] The successful induction of soluble recombinant protein RTD1 in vitro was confirmed using a Western blot assay with MBP antibody. The band of the MBP-RTD1 recombinant protein was found at 93.08 kDa (Figure 1d), indicating that the target protein RTD1 is approximately 53 kDa. Below this band, a faint band, known as the bacterial chaperone (GroEL), is present, which is typically observed during the induction of recombinant glycosyltransferase genes.

[0050] Example 4: In vitro enzyme activity detection 1. In vitro enzyme activity reaction system First, prepare the prediluted substrate working solution. Take 10 mL of 10 mg / mL flavonoid stock solution, add 90 mL of DMSO solution, vortex for 1 minute, and store on ice until use. Prepare 5 mL of 5X reaction buffer according to Table 9-10, and use immediately. Set up an experimental group (containing all reaction system components) and an enzyme-free control (using an equal volume of ultrapure water instead of RTD1 enzyme solution), with 3 replicates for each group.

[0051] In a sterile 1.5 mL EP tube, add ultrapure water, 5X reaction buffer, and UDP-glucose stock solution sequentially, followed by the appropriate volume of prediluted substrate working solution. Gently vortex to mix. The experimental preparation should be performed on ice. Add RTD1 enzyme solution and immediately mix with a pipette. Then, quickly transfer the reaction tube to a 37°C water bath and time the reaction precisely for 1 h. After the reaction is complete, immediately add an equal volume of 0.1% formic acid alcohol solution to terminate the reaction, vortex to mix, filter through a 0.22 mm filter membrane, and transfer 100 mL to the inner tube of the sample vial for HPLC analysis.

[0052] Table 9 Reaction system formulation (final volume 100 mL) Table 10. Volume of each substrate added and corresponding ultrapure water volume 2. Chromatographic conditions Chromatographic column: C18; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile; Gradient elution program: 0-25 min 5%-30% B; 25-30 min 30% B; 30-32 min 30%-80% B; 32-38 min 80%-5% B; 38-58 min 5% B; Flow rate: 0.4 mL / min -1 Column temperature: 35℃; injection volume: 20 mL; detection wavelength: 254 nm.

[0053] 3. Preparation of flavonoid standard solution Accurately weigh rutin, quercetin, quercetin glycoside, luteolin, luteolin glycoside, genistein, and genistein glycoside, dissolve and dilute to volume with 80% methanol and dimethyl sulfoxide (v / v=1:1) to prepare a stock solution with a concentration of 10 mg / mL, and store at -20℃ protected from light.

[0054] 4. Plotting the standard curve Take an appropriate amount of the stock solution and prepare working solutions of each single standard at a concentration of 100 ppm. Given that the chromatographic retention times of quercetin and luteolin, and quercetin and luteolinoside are relatively close, to avoid co-elution interference and ensure accurate quantification, the standards are divided into two groups and mixed standard solutions are prepared independently: the first group contains rutin, quercetin, and quercetin; the second group contains luteolin, luteolinoside, genistein, and genistein. These are serially diluted with 0.1% formic acid alcohol solution to prepare mixed standard solutions of 50, 100, 150, 200, and 250 μM. Under the established chromatographic conditions, inject 20 μL of each concentration standard solution for analysis.

[0055] By analyzing the single-standard working solution, the retention times of each component under the given chromatographic conditions were experimentally determined. The experimental results are as follows: Figure 2 As shown. After obtaining the analytical results of two sets of series concentration mixed standard working solutions, a linear regression was performed on the peak area (Y) of the target compound against the mass concentration (X, mM), resulting in a good standard curve. The regression equations and correlation coefficients (R²) for each compound are shown below. 2 See Table 11 for details; the standard curve is shown below. Figure 3 As shown.

[0056] Table 11 Regression equations and correlation coefficients of the flavonoid standard curve 5. Test Results HPLC analysis results ( Figure 4 This clearly demonstrates that the RTD1 protein possesses catalytic activity towards a variety of flavonoid glycoside substrates. Compared to the control group ( Figure 4 Compared to group a, 4c), the enzyme reaction group ( Figure 4 The chromatograms of b and 4d showed significant changes: the peak areas of the three substrate peaks (quercetin, luteolin, and genistein) decreased significantly, while the peak areas of their corresponding products (quercetin, luteolin, and genistein) increased significantly in sync, and rutin showed no significant change.

[0057] Furthermore, the relative magnitudes of the peak area changes varied significantly, as shown in Table 12. Under the same initial substrate concentration, the increase in product peak area was: quercetin > luteolin > genistein. Correspondingly, the decrease in substrate peak area was: quercetin > luteolin > genistein, with corresponding product concentration changes of 45.0483 mM, 37.2731 mM, and 14.9668 mM, respectively.

[0058] Table 12 Changes in peak area and concentration of in vitro enzyme activity reaction In vitro enzyme activity experiments confirmed that RTD1 protein can catalyze the formation of three flavonoid aglycones—quercetin, luteolin, and genistein—into their corresponding glycoside products. Figure 4 This indicates that RTD1 possesses glycosyltransferase function and exhibits catalytic activity against a variety of structurally similar substrates. Analysis of peak area changes in the chromatogram further reveals a significant catalytic preference of RTD1 for different substrates. The consistent relative magnitudes of product production and substrate consumption indicate that the enzyme has the highest catalytic efficiency for quercetin, followed by luteolin, and the relatively weakest catalytic efficiency for genistein. Figure 4 b, d).

[0059] The experimental results above demonstrate that this invention qualitatively identified the glycosyltransferase activity of RTD1 and preliminarily clarified its substrate preference. These results provide crucial evidence for elucidating the biological function of RTD1 in plant secondary metabolism.

[0060] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A flavonoid glycosyltransferase gene, characterized in that, The nucleotide sequence of the flavonoid glycosyltransferase gene is shown in SEQ ID NO.

1.

2. A flavonoid glycosyltransferase, characterized in that, The amino acid sequence of the flavonoid glycosyltransferase is shown in SEQ ID NO.

3.

3. The application of a flavonoid glycosyltransferase gene in the synthesis of flavonoid glycosides, characterized in that, The nucleotide sequence of the flavonoid glycosyltransferase gene is shown in SEQ ID NO.

1.

4. The application of a flavonoid glycosyltransferase in the synthesis of flavonoid glycosides, characterized in that, The amino acid sequence of the flavonoid glycosyltransferase is shown in SEQ ID NO.

3.

5. The application as described in claim 3 or 4, characterized in that, The flavonoid glycosides include at least one of rutin, quercetin, luteolin, and genistein.

6. A method for preparing flavonoid glycosides, characterized in that, Includes the following steps: S1. Obtain a flavonoid glycosyltransferase containing the amino acid sequence shown in SEQ ID NO. 3; S2. Using the flavonoid glycosyltransferase described in step S1 to catalyze the substrate, synthesize flavonoid glycoside compounds.

7. The method for preparing flavonoid glycosides as described in claim 6, characterized in that, The substrate includes flavonoid receptors and UDP-glucose; the flavonoid glycosides include at least one of rutin, quercetin, luteolin, and genistein.

8. The method for preparing flavonoid glycosides as described in claim 7, characterized in that, The flavonoid receptors include quercetin, luteolin, and genistein.

9. A method for simultaneously detecting seven flavonoid components in the reaction product of the flavonoid glycosyltransferase as described in claim 2, characterized in that, Includes the following steps: S1. Preparation of standard solution: Weigh rutin, quercetin, quercetin glycoside, luteolin, luteolin glycoside, genistein, and genistein, dissolve them in methanol and dimethyl sulfoxide and make up to volume to prepare a stock solution with a concentration of 10 mg / mL, and store it at -20℃ in the dark. S2. Preparation of test solution: Add 0.1% formic acid alcohol solution to the sample to be tested, filter, and take the filtrate as the test solution; S3. High performance liquid chromatography was used for detection, and the detection conditions were as follows: column: C18; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile; Gradient elution program: 0-25 min 5%-30% B; 25-30 min 30% B; 30-32 min 30%-80% B; 32-38 min 80%-5% B; 38-58 min 5% B; Flow rate: 0.4 mL / min -1 Column temperature: 35℃; injection volume: 20 mL; detection wavelength: 254 nm.

10. The method as described in claim 9, characterized in that, The concentration of the methanol solution in step S1 is 80%; The volume ratio of the methanol solution and dimethyl sulfoxide in step S1 is 1:1; The filtration described in step S2 is performed using a 0.22 mm filter membrane.

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