Use of a poigt79b1 protein and its coding gene in regulating flavonoid and / or fatty acid content in seeds

Abstract

This invention relates to the field of biotechnology, and in particular to the application of a PoUGT79B1 protein and its encoding gene in regulating the content of flavonoids and / or fatty acids in seeds. This invention provides a PoUGT79B1 protein whose encoding gene contains a conserved PSPG motif with glutamine at the end, suggesting its specificity for the UDP-Glc donor. The PoUGT79B1 gene exhibits optimal affinity for Qu and Lu; PoUGT79B1 can catalyze three aglycones; and PoUGT79B1 is located in the nucleus and cytoplasm. Results from specific embodiments of this invention show that overexpression of the PoUGT79B1 gene promotes the accumulation of flavonoids and fatty acids in seeds, verifying its functional conservation in the regulation of flavonoid and lipid metabolism. This invention provides a theoretical basis and gene resources for the high-value utilization of peony germplasm resources and also offers new ideas for improving the quality of woody oil crops.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a PoUGT79B1 protein and an application of a coding gene thereof in regulating flavonoids and / or fatty acid content in seeds. BACKGROUND

[0003] Recent studies have shown that the accumulation of fatty acids in peony seeds has obvious spatiotemporal specificity. In previous studies, the seed development process of Paeonia suffruticosa was divided into four key periods: S1 (20 days after pollination, seed formation and rapid volume increase), S2 (95 days after pollination, seed volume reaches maximum), S3 (110 days after pollination, seeds begin to turn brown), and S4 (125 days after pollination, seed coat turns black). The oil accumulation pattern shows that the period from S1 to S2 is a rapid synthesis period of unsaturated fatty acids, and ALA reaches a peak value (107.96 mg / g FW) in S2. Wang et al. revealed through transcriptomic research that 53-88 DAP (days after pollination, DAP) is a rapid biosynthesis period of fatty acids, and key genes such as PDAT, FAD2, and FAD3 are highly expressed during this period. However, current research on the regulation of flavonoids and fatty acid synthesis in peony seeds is still relatively scarce. Therefore, it is necessary to explore and utilize key regulatory genes. SUMMARY

[0004] The present application aims to provide a PoUGT79B1 protein and an application of a coding gene thereof in regulating flavonoids and / or fatty acid content in seeds, in order to solve the problems existing in the prior art.

[0005] To achieve the above-mentioned purpose, the present application provides the following solutions. The present application provides a PoUGT79B1 protein, and the amino acid sequence of the PoUGT79B1 protein is shown in SEQ ID NO. 2.

[0006] The present application provides a coding gene of the above-mentioned PoUGT79B1 protein, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

[0007] The present application provides a recombinant vector containing the above-mentioned coding gene.

[0008] The present application provides a recombinant bacterium containing the above-mentioned recombinant vector.

[0009] The present application provides an application of the above-mentioned PoUGT79B1 protein, the above-mentioned coding gene, the above-mentioned recombinant vector, or the above-mentioned recombinant bacterium in any one of the following, (1) regulating flavonoid content in plant seeds; (2) preparing a preparation for regulating flavonoid content in seeds; (3) regulating fatty acid content in plant seeds; (4) preparing a preparation for regulating fatty acid content in plant seeds.

[0010] Optionally, the flavonoid content and / or fatty acid content in plant seeds is increased by increasing the expression level of the PoUGT79B1 protein or the expression amount of the encoding gene in the plant.

[0011] Optionally, the flavonoids include quercetin-3-O-glucose-7-rhamnoside, quercetin-3-O-rhamnose-7-rhamnoside and quercetin-3-O-rhamnoside; and the fatty acids include palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, eicosadienoic acid and erucic acid.

[0012] Optionally, the plants include peony and Arabidopsis.

[0013] The present application provides a method for regulating flavonoid content in plant seeds, which increases the flavonoid content in plant seeds by increasing the expression level of the PoUGT79B1 protein or the expression amount of the encoding gene in the plant. The amino acid sequence of the PoUGT79B1 protein is shown as SEQ ID NO. 2.

[0014] Optionally, the plants include peony and Arabidopsis.

[0015] The present application provides a method for regulating fatty acid content in plant seeds, which increases the fatty acid content in plant seeds by increasing the expression level of the PoUGT79B1 protein or the expression amount of the encoding gene in the plant. The amino acid sequence of the PoUGT79B1 protein is shown as SEQ ID NO. 2.

[0016] Optionally, the plants include peony and Arabidopsis.

[0017] The present application discloses the following technical effects: This invention provides a PoUGT79B1 protein. The gene encoding this protein contains a conserved PSPG motif with glutamine (Q) at the end, suggesting its specificity for UDP-Glc donors. The PoUGT79B1 gene exhibits the best affinity for Qu and Lu. PoUGT79B1 has the broadest substrate scope (catalyzing three aglycones). PoUGT79B1 is located in both the nucleus and cytoplasm. Specific embodiments of this invention demonstrate that overexpression of the PoUGT79B1 gene promotes the accumulation of flavonoids and fatty acids in seeds, verifying its functional conservation in the regulation of flavonoid and lipid metabolism. This invention provides a theoretical basis and gene resources for the high-value utilization of peony germplasm resources and also offers new ideas for improving the quality of woody oil crops. Attached Figure Description

[0018] 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.

[0019] Figure 1 SDS-PAGE analysis of PoUGT79B1 protein expression under different induction conditions; Figure 2 Western blot analysis of purified PoUGT79B1 protein; Figure 3 For in vitro enzyme activity analysis of PoUGT79B1 protein; Figure 4 Subcellular localization of the PoUGT79B1 protein; the experiment was independently repeated three times, and representative results are shown in the figure; scale bar = 5 μm; Figure 5 RT-qPCR validation of the expression level of the PoUGT79B1 gene in Arabidopsis thaliana overexpression lines; Figure 6 To determine the flavonoid content in the seeds of the PoUGT79B1-overexpressing Arabidopsis thaliana line; among which, QG-3-R-7: quercetin-3-O-glucose-7-rhamnoside; QR-3-R-7: quercetin-3-O-rhamnoside; QR-3: quercetin-3-O-rhamnoside; Figure 7The fatty acid content in the seeds of the PoUGT79B1 overexpressing Arabidopsis thaliana line was determined; the fatty acid content was as follows: palmitic acid: C16:0; stearic acid: C18:0; oleic acid: C18:1; linoleic acid: C18:2; linolenic acid: C18:3; eicosenoic acid: C20:1; eicosadienoic acid: C20:2; erucic acid: C22:1. Detailed Implementation

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] 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.

[0025] In the early stages of this invention, metabolomics analysis was performed on seeds at different developmental stages. Based on transcriptomics and broad-target metabolomics, key genes in the flavonoid and succinate biosynthetic pathways were identified. Subsequently, structural modeling and docking analysis were performed on the screened genes. The PoUGT79B1 gene showed high similarity to the template UGT structure (PDB code: 9ufi), with a sequence similarity of 48.88% and a GMQE value of 0.83, exhibiting the same α-helix and β-chain regions. Docking results showed that five amino acids in PoUGT79B1 (VAL144, ARG222, TYR142, ILE145, and PHE124) may interact with UDP-Glc. PHE124, TYR142, VAL144, and ILE145 were predicted to interact with uridine groups, while ILE145 was thought to interact with diphosphate groups. ARG222 interacted with glucose. The docking score between PoUGT79B1 and UDP-Glc was -5.602. kCal / mol; Substrate prediction analysis showed that six amino acids of PoUGT79B1 (SER281, GLN342, PHE360, SER362, ASP381, and GLU365) interact with He; six amino acids of PoUGT79B1 (TRP21, TYR200, ASP381, ASP381, THR192, and HIS26) interact with Km; seven amino acids of PoUGT79B1 (SER281, G...) interact with Km. The amino acids LN342, PHE360, SER362, GLU365, ASP381, and HIS357 of PoUGT79B1 interact with Lu; six amino acids of PoUGT79B1 (SER281, GLN342, PHE360, SER362, ASP381, and HIS357) interact with Qu; and five amino acids of PoUGT79B1 (ARG222, GLN342, THR343, GLU365, and HIS32) interact with Sy. The docking scores of PoUGT79B1+UDP-Glc with He, Km, Lu, Qu, and Sy were -7.755, -7.25, -8.063, -8.075, and -7.339 kCal / mol, respectively, indicating that the protein has a better affinity for Qu, followed by Lu. In summary, this invention ultimately screened a UFGT (UDP-flavonoid glycosyltransferase) – 276225_c0_g1 – and named it the PoUGT79B1 gene. Based on this, specific embodiments of this invention further validate its function.

[0026] The CDS sequence of the PoUGT79B1 gene is shown in SEQ ID NO.1, specifically as follows:

[0027] The amino acid sequence of the PoUGT79B1 protein is shown in SEQ ID NO.2, specifically: MARAESNQLHFTMFPWFAFGHMNPYLHLANKLAERGHIVSFLLPRGAQPRIQQLSLHPALISFYPLDVPHVDGLPPGAETASDVPFPDHTHLCTAFDQTQNQVEPILTKLKPD FILFDFGFWVPALVGHLGTKSVYYAVVSAAAYAWAVPALCVKDDSTLAETMHPPPGYPSSVIKIRPHENKQFLGVFKGMGSGLTLHERLTSGMKNCDAIAMRTCQEIEGRFCD YIAQQYSKSVLLTGPVLSPVASLDEQWANWLSTFKPGSVVFCALGSQIVLQKEDFQELILGFELAQLPFLVALTPPQGCVTIKEVLPQGFEERVRGRGLVHGGWVPQPEMLKH PSIGCFVNHCGYGSMWESLISDCQIVLLPFLADQCINTKLMVEELKVAVEIPREGSGQFTKENLSQAIVSVTDDGEVATLIRANHRKWKDIVLSRDKQGVYIDSFIQNMKKMQE .

[0028] The full sequence of the PoUGT79B1 gene is shown in SEQ ID NO.3, specifically:

[0029] Experimental materials: Seeds of *P. ostii* were collected in 2021 from the Peony Resource Nursery of the Institute of Botany, Chinese Academy of Sciences, and seeds of *Paeonia suffruticosa* were collected in Lhasa, Tibet in 2025. Seeds from two developmental stages, S2 (95 days after pollination) and S4 (125 days after pollination), were selected, and the kernels and seed coats were separated. Three biological replicates were set up for each sample group, and a total of 24 samples were analyzed for metabolomics. Wild-type Arabidopsis thaliana (Columbia); Nicotiana benthamiana; The above materials were provided by the Institute of Botany, Chinese Academy of Sciences, and the applicant promises to distribute them to the public for 20 years from the date of application.

[0030] Experimental reagents and standards: The following reagents were purchased: RNAprep Pure Plant Kit (universal total RNA extraction kit), All-in-one RT Master (reverse transcription kit), 1.1×S4 Fidelity PCR Mix, 2×GS Taq pcr mix, Uniclone One Step Seamless Cloning Kit (Beijing Jinsha Biotechnology Co., Ltd.); Gel Extraction Kit and MolPure® Plasmid Mini Kit (DNA gel extraction kit), Plasmid Maxprep Kit (plasmid extraction and purification kit), Plasmid Maxprep Kit (plasmid extraction and purification kit), Vigras Biotechnology (Beijing) Co., Ltd.; Taq Pro Universal SYBR qPCR Master Mix (fluorescence quantitative reagent kit), Novizan Biotechnology Co., Ltd.; TaKaRa restriction endonuclease, TaKaRa, Beijing Liuhetong Economic and Trade Co., Ltd.; DNA Marker (100-5000). BP), DH5α competent cells, GV3101 competent cells, and Rosetta (DE3) competent cells were purchased from Beijing Bomeide Gene Technology Co., Ltd.; ampicillin solution (100 mg / mL), kanamycin stock solution (100 mg / mL), and rifampicin stock solution (50 mg / mL) were purchased from Beijing Coollab Technology Co., Ltd.; Gst-Bind packing material (GLUTATHIONE SEPHAROSE 4B), affinity chromatography empty column (Solarbio), Amicon® Ultra filter (Merck Millipore), color pre-stained standard molecular weight protein markers (10-180 kD), precast gel (SDS-PAGE Precast Protein Gel 12% 15 Wells), SDS-PAGE sample buffer, protein gel rapid staining solution, TBST, anti-GST-Tag mouse monoclonal antibody, and goat anti-mouse secondary antibody (Goat Anti-Mouse IgG (H&L)-HRP) Conjugated protein, Western blotting buffer, Western blotting-specific skim milk powder, and ultrasensitive ECL luminescent solution were purchased from Beijing Bio-Tech Co., Ltd.; the protein concentration assay kit (Bradford) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0031] Luteolin (Lu), quercetin (Qu), hesperitin (He), kaempferol (Km), syringetin (Sy), quercetin-3-O-glucoside, and uridine diphosphate glucose (UDP-Glc) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] The primers used in the experiment are shown in Table 1.

[0033] Table 1. Specific information about the primers Example 1 1. RNA extraction RNA was extracted using a universal plant total RNA extraction kit (RNAprep Pure Plant Kit) under RNase-free conditions, following the kit instructions. After extraction, the concentration and purity were determined using an Implen ultra-micro spectrophotometer. The RNA was diluted to 250 ng / μL and stored at -80°C for later use.

[0034] 2. RNA reverse transcription Reverse transcription was performed using an All-in-one RT Master kit, and the reaction system is shown in Table 2.

[0035] Table 2 RNA reverse transcription system The above reaction system was reacted in a PCR instrument at 50℃ for 5 min and 80℃ for 5 sec. The cDNA obtained after the reaction was stored in a -20℃ freezer for later use.

[0036] 3. Cloning of the target gene Glycosyltransferases were screened from differentially expressed genes in the *Pteris vittata* seed transcriptome, and the candidate gene PoUGT79B1 was identified using quantitative real-time PCR results. The candidate gene was cloned using a high-fidelity PCR kit (1.1×S4 Fidelity PCR Mix) with *Pteris vittata* seed cDNA as a template. Primers used for gene cloning are shown in Table 1. The 50 μL PCR amplification reaction system is shown in Table 3.

[0037] Table 3 PCR amplification reaction system The PCR amplification program was as follows: pre-denaturation: 98℃, 2 min; amplification: 98℃, 10 s; 57℃, 10 s; 72℃, 45 s; for a total of 35 cycles; final extension: 72℃, 5 min.

[0038] The products were detected by 1% agarose gel electrophoresis, and bands of the correct length were extracted and recovered using a DNA gel extraction kit.

[0039] The following steps were performed: 4 μL of the recovered product and 1 μL of pEASY-Blunt Simple Cloning Vector were added to a 0.2 mL tube, and the PCR was run at 37°C for 15 min. Add 5 μL of the product to 30-50 μL of T1 competent cells, gently tumble to mix, incubate on ice for 20-30 min, incubate at 42℃ for 30 s, immediately place on ice for 2 min, add 500 μL of LB medium to the tube, incubate at 200 rpm and 37℃ for 1 h, centrifuge at 1500 g for 1 min, discard part of the supernatant, mix the bacterial culture, spread evenly on solid medium containing antibiotics, and seal after the bacterial culture dries. Incubate overnight at 37℃ in an inverted petri dish. Add 500 μL of LB medium containing Kans to a 1.5 mL centrifuge tube, pick white single colonies and add them to the centrifuge tube, shake at 200 rpm and 37℃ for at least 5 h until the bacteria are turbid. Perform bacterial PCR using Taq enzyme according to the following reaction system: 2×DNA Taq Mix 5 μL, M13F (10 μM) 0.5 μL, M13R (10 μM) 0.5 μL. μL of bacterial culture, 1.0 μL of ddH2O, and 3 μL of ddH2O were used. The PCR amplification program was as follows: pre-denaturation: 94℃, 3 min. Amplification: 94℃, 30 s; 55℃, 30 s; 72℃, 1 min; for a total of 30 cycles. Final extension: 72℃, 10 min.

[0040] PCR products were detected using 1% agarose gel electrophoresis, and bacterial cultures with correct bands were sent to Biomed Sequencing for sequencing.

[0041] 4. Carrier Construction Construction of overexpression vector: The pSUPER1300 vector with a GFP tag was used as the overexpression vector (this vector is disclosed in the literature "ABSCISIC ACID-INSENSITIVE 5-ω3 FATTY ACID DESATURASE3 moduleregulates unsaturated fatty acids biosynthesis in Paeonia stii" (Li Y, Wang X, Zhang X, et al. ABSCISIC ACID-INSENSITIVE 5-ω3 FATTY ACID DESATURASE3 moduleregulates unsaturated fatty acids biosynthesis in Paeonia stii[J]. PlantScience, 2022, 317:111189.), in which the vector name is pSuper1300). The overexpression vector was double-digested with restriction endonucleases XbaⅠ and SacⅠ. The double digestion reaction system is shown in Table 4.

[0042] Table 4. Double enzyme digestion reaction system The reaction was carried out at 37℃ for 15 min, and the double enzyme digestion products were identified by gel electrophoresis and then recovered by gel extraction.

[0043] Using the complete CDS sequence (SEQ ID NO.1) successfully identified in step "1. Cloning of the target gene" as a template, the homologous arm sequence was cloned using a high-fidelity enzyme. The target gene with homologous arms was ligated to the vector using the Uniclone One StepSeamless Cloning Kit. The primer sequences (S1300-PoUGT79B1-F and S1300-PoUGT79B1-R) for constructing homologous arms are shown in Table 1.

[0044] Thaw DH5α competent cells on ice. Add 50 μL of DH5α competent cells and 5 μL of ligation product to a centrifuge tube, gently swirl to mix, and immediately place on ice for 30 min. Heat shock the centrifuge tube in a 42°C water bath for 90 s, then quickly transfer it to an ice bath to cool the cells for 2 min, without shaking the centrifuge tube. In a clean bench, add 500 μL of LB liquid medium (antibiotic-free) to the centrifuge tube, mix well, and incubate at 37°C with shaking at 200 rpm for 1 h to revive the cells. In a clean bench, evenly spread the bacterial suspension onto LB solid medium containing Kan (100 μg / mL) until the suspension is completely absorbed. Inverted plates were incubated overnight at 37°C for 12-16 h. Single colonies were picked and transferred to centrifuge tubes containing 600 μL LB liquid medium (Kan, 100 μg / mL). After shaking and incubating for 5 h at 37°C and 200 rpm, bacterial PCR was performed. Successfully identified bacterial cultures were sent to Biomed Sequencing for sequencing.

[0045] Construction of prokaryotic expression vector: pGEX-4T-2 vector was used for prokaryotic expression. The overexpression vector was double-digested with restriction endonucleases BamHI and SacHI. The double digestion system is shown in Table 4. The method of ligation with the CDS sequence of the target gene and transformation of DH5α competent cells is the same as above. The primers for cloning the homologous arm of the target gene are shown in Table 1.

[0046] 5. Protein expression (1) Transform the plasmid containing the target gene pGEX vector (i.e. the prokaryotic expression vector obtained in step “2, vector construction”) into competent cells of protein-induced strain Rosetta (DE3), identify positive strains by PCR, and use the same PCR amplification program and system as in step “1, cloning of the target gene”. (2) Take 2 mL of overnight culture and add it to a 500 mL Erlenmeyer flask containing 150 mL of LB culture medium (containing 50 ug / mL Amp). Stir at 37℃ and 200 rpm for about 2 h until OD = 0.5-0.8. (3) To explore the optimal induction conditions, two final IPTG concentrations were set: 0.2 mM and 0.4 mM; the induction temperatures were set at 16℃ and 28℃, the rotation speed was 120 rpm, and the induction time was 24 h. (4) Centrifuge at 4℃, 8000 rpm for 5 min to collect bacteria; (5) Add 15 mL PBS (containing 1 mM PMSF benzyl sulfonyl fluoride protease inhibitor, 100 μL 100 mM stock solution added to 10 mL PBS; 1 mM DTT, 10 μL 1 M DTT stock solution added to 10 mL PBS), and gently aspirate until the cells are evenly suspended. (6) On ice, use ultrasonic breaking for 15 min, power 10-20%, ultrasonic for 3 s, stop for 5 s, add 1 mM PMSF and DTT; (7) Centrifuge at 4℃ and 8000 rpm for 20 min; (8) Transfer the supernatant to a new centrifuge tube and add 1 mM PMSF and DTT.

[0047] 6. Protein purification Protein purification using column chromatography: Prepare the buffer solutions: Equilibration / wash buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4; Elution buffer: Prepare 10 mM reduced glutathione using the equilibration buffer (prepare fresh). 10 mM DTT can be added to both the equilibration and elution buffers. Filter the buffer solution through a 0.22 μm or 0.45 μm filter before use.

[0048] Equilibration of glutathione-agarose medium: Gently invert the container containing GST-Bind packing material (GLUTATHIONESEPHAROSE 4B) to homogenize the resin. Prepare 1 mL of resin for every 100 mL of cell lysate (5, obtained in protein expression (6)), pack it into the chromatography column, and open the cap at the bottom of the purification column to allow the liquid in the chromatography column to flow out naturally; Resuspend the equilibration medium in 5 times the volume of equilibration buffer, and repeat the equilibration process three times. Add the supernatant (5, obtained from protein expression (7)), resuspend it evenly with the resin, and gently shake it on a side-shaking shaker at 4℃ for 30 minutes to allow it to fully combine; (1) Open the cap at the bottom of the purification column to allow the liquid in the chromatography column to flow out naturally. Collect and save the eluent for SDS-PAGE electrophoresis. (2) Add 10 times the volume of resin washing buffer, shake gently at 4°C for 15 min to wash away the impurities that are not bound to the resin. Repeat the washing process 4-5 times, collect and store the washing buffer for SDS-PAGE electrophoresis. (3) Add 0.5 mL of elution buffer to each milliliter of resin, incubate at room temperature for 15 min, collect and store the elution buffer, and repeat the elution 3-5 times. This elution buffer is the purified GST-labeled protein sample. (4) Concentrate the protein sample using an Amicon® Ultra filter (Merck millipore); (5) Determine the protein content and perform SDS-polyacrylamide electrophoresis to detect the protein.

[0049] 7. In vitro enzyme activity test The enzyme reaction system (50 μL) contained 3 μg of purified protein, 2 mM UDP-Glc, 100 mM Tris-HCl and 0.1 mM of each receptor substrate (Km, Qu, Lu, He and Sy).

[0050] Determination of optimal pH for the reaction: Tris-HCl enzyme activity buffer solutions with pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 were prepared. Using quercetin as the substrate, the reaction solutions were prepared according to the above reaction system, and the reactions were carried out at 37°C for 30 min. Each reaction was repeated 3-5 times.

[0051] Determination of the optimal reaction temperature: Using quercetin as the substrate, the reaction solution was prepared according to the above reaction system using Tris-HCl enzyme activity buffer at pH 7.0. Reactions were carried out at 25℃, 30℃, 37℃, 42℃, 50℃, 60℃, and 70℃ for 60 min each. Each reaction was repeated 3-5 times. After the above reactions were completed, 50 μL of methanol was added to terminate the reaction, and the results were analyzed using UPLC-DAD. The UPLC-DAD method was based on Dr. Yin Qinggang's dissertation (2016).

[0052] Enzyme kinetic parameters were determined under the optimal enzyme-catalyzed reaction conditions defined above. 3 μg of purified and concentrated recombinant protein was added to each sample at concentrations of 25, 50, 75, 100, 125, 150, 200, and 400 μM, with each sample reacting for 60 min. Each protein was reacted in 3-5 replicates at each concentration. After the reaction was complete, 50 μL of methanol was added to terminate the reaction, and the results were analyzed using UPLC-DAD.

[0053] Enzyme kinetic parameters were calculated using Hyper 32 software. The double reciprocal curve method (Lineweaver-Burkplots) was selected to calculate the Km and Vmax values. The kcat value was calculated according to the formula kcat=Vmax / amount of protein, and then the kcat / Km value was calculated.

[0054] 8. Transformation of Arabidopsis thaliana Arabidopsis thaliana Columbia ecotype (Col-0) was used as the wild-type material and grown under the following conditions: 23℃, 16 h light / 8 h dark photoperiod, and 60% relative humidity. The overexpression vector prepared in step "4. Vector Construction" was transformed into Agrobacterium tumefaciens strain GV3101 to obtain Agrobacterium tumefaciens strain GV3101 carrying the overexpression vector. Col-0 inflorescences were transformed with Agrobacterium tumefaciens strain GV3101 carrying the overexpression vector, and stable T3 generation transgenic lines were obtained through screening for subsequent analysis. The expression level of the target gene was detected by RT-qPCR, and the flavonol content was determined by HPLC. Fatty acid content was determined according to the method described in the literature "Study on the PoKO gene for dwarfing oilseed lines synthesized from 'Fengdan' gibberellin". The primer sequences used for RT-qPCR are shown in Table 1.

[0055] 9. Subcellular localization of tobacco Tobacco Bunsenii plants were grown in pots under conditions of 23°C, 16 h light / 8 h dark light cycle, and approximately 60% relative humidity. The constructed overexpression vectors were combined with either the nuclear marker Super1300::NF-YA4-mCherry or the endoplasmic reticulum marker pCAMBIA1300::HDEL-mCherry (purchased from Zhuangmeng Biotechnology Co., Ltd.). The Agrobacterium tumefaciens strain GV3101 was used to co-infect tobacco leaves. The specific method was described in the literature "PhUGT78A22, a novel glycosyltransferase in paeonia “he xie”, can catalyze the transfer of glucose to glucosylated anthocyanins during petal blotch formation" (Li Y, Kong F, Liu Z, et al. PhUGT78A22, a novel glycosyltransferase in paeonia “he xie”, can catalyze the transfer of glucose to glucosylated anthocyanins during petal blotch formation[J]. Bmc Plant Biology, 2022a, 22(1):405.). Three days after infection, GFP and mCherry fluorescence signals were detected using a confocal microscope (Olympus FV1000MPE, Japan).

[0056] 10. Results and Analysis 10.1 Cloning of the target gene The agarose gel electrophoresis results showed that the band size was basically consistent with the theoretical length, indicating that the target fragment had been effectively amplified.

[0057] 10.2 PoUGT79B1 protein induction and purification In this embodiment, the PoUGT79B1 gene was successfully cloned into the pGEX-4T-2 vector. After transformation into Rosetta (DE3) competent cells, the induction conditions were optimized, with two IPTG concentration gradients of 0.2 mM and 0.4 mM, induction temperatures of 16℃ and 28℃, and a constant rotation speed of 120 rpm. SDS-PAGE analysis results showed ( Figure 1 The optimal induction conditions for the PoUGT79B1 gene were 0.2 mM IPTG at 28°C. Western blot results confirmed that all candidate UFGTs were successfully induced to express. Figure 2 The recombinant proteins were further purified by Ni-NTA affinity chromatography. SDS-PAGE analysis showed that the target protein band size was consistent with the expectation and the purity was high, meeting the requirements for subsequent enzyme activity studies (Table 4').

[0058] Table 4' PoUGT79B1 protein purification concentration 10.3. In vitro enzyme activity analysis of PoUGT79B1 protein Using UDP-Glc as the sugar donor and Km, Qu, Lu, He, and Sy as acceptor substrates, an in vitro enzymatic reaction was carried out at 37℃ and pH=7. The formation of glycosylated products was confirmed by HPLC analysis and comparison with standards. The results showed that PoUGT79B1 protein can catalyze the glycosylation of four flavonoids, Qu, Lu, and He. Figure 3 ).

[0059] To further investigate the catalytic efficiency for different substrates, enzyme kinetics were analyzed under reaction conditions of 37℃ and pH=7. Three μg of purified and concentrated recombinant protein was added to the aforementioned active substrate at 10 concentration gradients: 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μM. Three replicates were performed for each protein at each concentration. The affinity, catalytic activity, and overall catalytic efficiency of the PoUGT79B1 protein for specific substrates were evaluated by measuring the Michaelis constant (Km, reflecting enzyme-substrate affinity; lower values ​​indicate stronger affinity), maximum reaction rate (Vmax, reflecting enzyme catalytic ability), catalytic constant (Kcat, the conversion constant, representing the number of substrate molecules converted per unit time at each enzyme active site), and catalytic efficiency (Kcat / Km, a key indicator for comprehensively evaluating enzyme catalytic efficiency and specificity) (Table 5). The results showed that different proteins exhibited significant differences in kinetic properties for the same substrate and the same protein for different substrates, revealing the diversity of their substrate selectivity and catalytic mechanisms.

[0060] Table 5 Enzymatic kinetic parameters of PoUGT79B1 protein PoUGT79B1 has the strongest affinity for protein Lu, and its Km The lowest value (26.28 μM) indicates the tightest binding, although its catalytic rate ( Kcat =10.44 s -1 The overall catalytic efficiency (397.3 ml) is relatively low, but the overall catalytic efficiency is low. -1 s -1 It still has the highest affinity for the three, making it the most suitable substrate for proteins. Its affinity for quercetin is moderate. Km =38.34 μM, but with the strongest catalytic activity ( Kcat =14.49 s -1 It showed high conversion potential, but due to its slightly weaker binding, the catalytic efficiency (377.91 ml) was low. -1 s -1 Slightly lower than Lu. The weakest affinity for He ( ). Km =39.88 μM), while its catalytic rate ( Kcat =14.21 s -1 Its catalytic efficiency is similar to that of Qu, but due to insufficient binding capacity, its catalytic efficiency (356.39 ml) is lower. -1 s -1The concentration of Lu was the lowest among the three substrates. Overall, the catalytic advantage of the PoUGT79B1 protein for Lu mainly stems from its extremely high binding affinity. Although it is not the most efficient in terms of conversion efficiency at a single active site, it can achieve an overall improvement in catalytic efficiency through more efficient substrate binding.

[0061] 10.4. In vivo functional validation of PoUGT79B1 protein 10.4.1 Subcellular localization of the PoUGT79B1 gene To clarify the subcellular localization of the PoUGT79B1 gene, the overexpression vector was co-expressed with the nuclear marker gene (Super 1300::NF-YA4-mCherry) and the endoplasmic reticulum marker gene (pCAMBIA1300::HDEL-mCherry) in the epidermal cells of Nicotiana benthamiana leaves. Laser confocal microscopy revealed that the GFP signal of the PoUGT79B1 gene was mainly localized in the nucleus and cytoplasm. Figure 4 ).

[0062] 10.4.2 Validation function of heterologous overexpression of PoUGT79B1 protein To further investigate the biological function of the candidate PoUGT79B1 gene in peony plants, this study constructed PoUGT79B1 overexpression vectors and transformed them into Arabidopsis thaliana (A. thaliana) Col-0 ecotype, obtaining multiple stable transgenic overexpression lines (OE). The expression level of the target gene in each line was verified by qRT-PCR, and the types and contents of flavonoids in mature transgenic Arabidopsis seeds, as well as the composition and accumulation levels of fatty acids in the seeds, were systematically analyzed to reveal the potential regulatory functions of these genes in flavonoid and lipid metabolism.

[0063] (1) Detection of PoUGT79B1 gene expression level in overexpression lines The relative expression levels of the corresponding genes in each overexpression line were detected by qRT-PCR. The results showed that ( Figure 5 Compared to wild-type (WT), the expression levels of PoUGT79B1 in overexpression lines (OE 2-8, OE 5-7, and OE 6-7) were 142.2-288.0 times higher than those in WT (OE 6-7 was particularly high). This demonstrates that the PoUGT79B1 overexpression lines were successfully constructed, and independent lines with high expression levels were obtained, providing reliable genetic material for subsequent metabolic phenotypic analysis.

[0064] (2) Effects of overexpression of PoUGT79B1 on flavonoid metabolism in Arabidopsis thaliana In seeds, overexpression of PoUGT79B1 generally promoted the accumulation of quercetin derivatives. Figure 6All OE lines significantly promoted the accumulation of quercetin derivatives. QG-3-R-7 (1.11~1.23 times) and QR-3-R-7 (1.22~1.30 times) were significantly higher than WT in all three lines. QR-3 was also significantly increased in OE 2-8 (1.15 times). Although there was no significant difference from other lines, the values ​​were still higher than WT.

[0065] (3) Effects of overexpression of PoUGT79B1 on fatty acid metabolism in Arabidopsis thaliana Overexpression of PoUGT79B1 significantly promoted the accumulation of fatty acids in seeds, but differences existed among lines. OE 2-8 showed the strongest promoting effect, with the highest levels of C16:0 (26.49 mg / g), C18:0 (4.83 mg / g), C18:1 (45.54 mg / g), C18:2 (90.36 mg / g), C20:1 (52.10 mg / g), and C22:1 (5.55 mg / g). OE 5-7 and 6-7 showed weaker promoting effects, with fatty acid contents significantly higher than WT but slightly lower than OE 2-8. Figure 7 ).

[0066] Based on the above analysis of the metabolic phenotypes of Arabidopsis thaliana overexpression, the results showed that overexpression of the PoUGT79B1 gene generally promoted the accumulation of fatty acids in seeds.

[0067] 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 PoUGT79B1 protein, characterized in that, The amino acid sequence of the PoUGT79B1 protein is shown in SEQ ID NO.

2.

2. The gene encoding the PoUGT79B1 protein according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

3. A recombinant vector containing the encoding gene of claim 2.

4. A recombinant bacterium containing the recombinant vector of claim 3.

5. The use of the PoUGT79B1 protein of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, or the recombinant bacteria of claim 4 in any of the following: (1) Regulate the flavonoid content in plant seeds; (2) Preparation of formulations to regulate the flavonoid content in seeds; (3) Regulate the fatty acid content in plant seeds; (4) Prepare a formulation to regulate the fatty acid content in plant seeds.

6. The application according to claim 5, characterized in that, By increasing the expression level of the PoUGT79B1 protein or the expression level of the encoding gene in plants, the content of flavonoids and / or fatty acids in plant seeds can be increased.

7. The application according to claim 5, characterized in that, The flavonoids include quercetin-3-O-glucose-7-rhamnoside, quercetin-3-O-rhamnoside-7-rhamnoside, and quercetin-3-O-rhamnoside; the fatty acids include palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, eicosapentaenoic acid, eicosadienoic acid, and erucic acid.

8. The application according to claim 5, characterized in that, The plants mentioned include peony and Arabidopsis thaliana.

9. A method for regulating the flavonoid content in plant seeds, characterized in that, By increasing the expression level of the PoUGT79B1 protein or the expression level of the encoding gene in plants, the flavonoid content in plant seeds can be increased. The amino acid sequence of the PoUGT79B1 protein is shown in SEQ ID NO.

2.

10. A method for regulating the fatty acid content in plant seeds, characterized in that, By increasing the expression level of the PoUGT79B1 protein or the expression level of the encoding gene in plants, the fatty acid content of plant seeds can be increased. The amino acid sequence of the PoUGT79B1 protein is shown in SEQ ID NO.2.

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