Cloning and functional identification of liverwort flavone synthetase gene

Abstract

The invention discloses cloning and functional identification of a liverwort flavone synthetase gene, and belongs to the technical field of gene engineering. According to the invention, one flavone synthase I (flavones synthase I, FNS I) is obtained through screening, and is named as MpFNS I; a prokaryotic expression system is utilized, an MpFNS I recombinant protein is obtained through purification, and the result of in-vitro enzyme activity identification shows that an MpFNS I enzyme substrate is relatively wide in selectivity, has a flavone synthetase function and also has a part of flavonol synthetase function. The liverwort flavonoid has a significant difference on affinity and catalytic efficiency of different substrates, the in-vivo function of the liverwort flavonoid is verified by utilizing heterologous expression of arabidopsis thaliana, experimental evidence is provided for comprehensively analyzing a biosynthetic pathway of the liverwort flavonoid, and meanwhile, a research target is provided for deeply researching a mechanism for synthesizing and responding to UV-B radiation of the liverwort flavonoid.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the cloning and functional identification of a Marchantia polyphylla flavonoid synthase gene. Background Art

[0002] Bryophytes are the earliest extant terrestrial higher plants and are divided into three branches: mosses, liverworts, and hornworts. Bryophytes produce structurally diverse secondary metabolites, mainly terpenes, aromatics, alkaloids, etc. Among them, the aromatic metabolites in liverworts are mainly bibenzyl and bisbibenzyl compounds, as well as phenylpropanoids and flavonoids. The aromatic compounds of mosses are mainly flavonoids. As the first model liverwort to complete whole genome sequencing, Marchantia polymorpha has the characteristics of a small genome (about 220Mb), fast growth, and strong reproductive capacity. In recent years, transformation technology and targeted genome modification have been successfully applied to liverwort, making it a multi-purpose model plant with a special evolutionary status, making it suitable for solving evolutionary, molecular, cellular and developmental problems.

[0003] The flavonoid biosynthesis pathway is one of the most important secondary metabolic pathways in plants. Modern plant chemistry and molecular biology studies have found that flavonoids are ubiquitous in terrestrial higher plants, including mosses (hornworts have not yet been found). Flavonoids play an important role in the adaptation of mosses to terrestrial environmental stresses such as water shortage, high temperature, high-intensity light and UV-B radiation. However, the expression of the FNS gene responsible for flavonoid biosynthesis in the liverwort plant Marchantia polymorpha has not been reported. Identifying and analyzing the key enzyme FNS in the liverwort flavonoid biosynthesis pathway, studying its in vitro and in vivo functions, and clarifying the liverwort flavonoid biosynthesis pathway have important biological significance for a deeper understanding of the evolutionary process of the flavonoid metabolic pathway in terrestrial higher plants. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide a cloning and functional identification of a Marchantia polyphylla flavonoid synthase gene, which lays a foundation for further in-depth research on the synthesis and regulatory mechanism of Marchantia polyphylla flavonoids.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a Marchantia polymorpha flavonoids synthase MpFNS I. The amino acid sequence of the Marchantia polymorpha flavonoids synthase MpFNS I is shown in SEQ ID NO. 1, and is specifically as follows:

[0007] SEQ ID NO.1:

[0008] MAPPAAESVGIASEKKNVVPYSVMKLSDSQMDVPEKFVKAMGERPTVAYNDYCKEIPVISLKNITSSDADRARIVAEIGLACAEWGIFQVVDHGVPEELMTKMAANSMEFFKLPLEEKLKYATKPGGFPVGYASGSHRSDDDILDWRELMVHRTLPSSIREQDINIWPESPDTYRQVLADYS DNVDTLVTSLLGLISELGLPTDYIKNAVGGEKTEQKILANYYPQCPQPDLTLGLRSHTDYGTITILQQEVGGLQAYKEDRDLWVTVEPIRGALVVNLGDQIQILSNAKYCSVEHQAVVNSNETRLTLVVFANPNSLSQMGPAPELLSEENPAKYRSYTFKDYLPICYAKQTKKHYDALAI.

[0009] In a second aspect of the present invention, a gene encoding the Marchantia polymorpha flavonoid synthase MpFNS I is provided. The CDS nucleotide sequence of the gene encoding the Marchantia polymorpha flavonoid synthase MpFNS I is shown in SEQ ID NO. 2, and is specifically as follows:

[0010] SEQ ID NO.2:

[0011]

[0012] The third aspect of the present invention provides the use of the Marchantia polyphylla flavonoid synthase MpFNS I in the following (1) or (2):

[0013] (1) Catalytic substrate;

[0014] (2) Regulate the expression of flavonoid synthesis genes.

[0015] The optimal pH value of the substrate catalyzed by the Marchantia polyphylla flavonoid synthase MpFNS I is distributed in the range of 7.0-7.5.

[0016] The affinity and catalytic efficiency of Marchantia polyphylla flavonoid synthase MpFNS I to different substrates are significantly different. m The values ranged from 4.28–29.66 μM.

[0017] In a fourth aspect, the present invention provides a recombinant expression vector or genetically engineered bacteria containing the gene encoding the Marchantia polyphylla flavonoid synthase MpFNS I.

[0018] In a fifth aspect, the present invention provides a method for catalyzing flavanones in a substrate to produce flavonoids, wherein the method comprises adding Marchantia polyphylla flavonoid synthase MpFNS I to the substrate to catalyze the production of flavonoids from flavanones.

[0019] The flavanone in the substrate is naringenin, hesperetin, liquiritigenin, chondrostenone or eriodictyol, and the generated flavonoids are apigenin, diosgenin 7,4'-dihydroxyflavone, chrysin or luteolin.

[0020] In a sixth aspect, the present invention provides a method for catalyzing dihydroflavonol to produce flavonols, wherein the method comprises adding Marchantia polyphylla flavonoid synthase MpFNS I to a substrate to catalyze dihydroflavonol to produce flavonols.

[0021] The Marchantia polyphylla flavonoid synthase MpFNS I catalyzes substrates and exhibits a wide range of substrate selectivity. It is a bifunctional enzyme of flavonoid synthase and flavonol synthase. It can use naringenin, hesperetin, liquiritigenin, chondrostenol, and eriodictyol as substrates to catalyze the production of apigenin, diosgenin, 7,4′-dihydroxyflavone, chrysin, and luteolin, respectively. It can also catalyze dihydroflavonols to produce flavonols.

[0022] Beneficial effects of the present invention:

[0023] 1. The present invention screened and isolated a Marchantine polymorpha flavones synthase I (FNS I), named MpFNS I. Amino acid sequence alignment results showed that MpFNS I shared approximately 27–67% amino acid sequence similarity with FNS Is from other plants. The recombinant MpFNS I protein was successfully expressed in Escherichia coli using a prokaryotic expression system. The recombinant protein was purified by nickel affinity chromatography and assayed for in vitro enzymatic activity, demonstrating that MpFNS I exhibited broad substrate selectivity, possessing both flavonoid synthase and partial flavonol synthase functions.

[0024] 2. The catalytic properties of MpFNS I towards different substrates were analyzed, and the optimal temperature and pH for the catalytic reaction, as well as the kinetic parameters, were determined. The results showed that MpFNS I exhibited significant differences in affinity and catalytic efficiency for different substrates, with hesperetin exhibiting the highest affinity and catalytic efficiency. Heterologous expression in Arabidopsis thaliana confirmed its in vivo function, demonstrating that UV-B radiation-induced expression of the MpFNS I gene also promoted flavonoid biosynthesis. These findings provide experimental evidence for a comprehensive understanding of the liverwort flavonoid biosynthesis pathway and offer research targets for further investigation into the mechanisms of liverwort flavonoid biosynthesis in response to UV-B radiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The CDS amplification results of MpFNS I; M is a 2000 bp DNA marker, and lanes 1–4 are the target fragments of MpFNS I.

[0026] Figure 2 The PCR results of MpFNS I-pEASY-Blunt colonies are shown; M represents a 2000 bp DNA marker, and lanes 1–5 represent the PCR amplification results of selected clones.

[0027] Figure 3 is the corresponding PCR identification result during the process of MpFNS I E. coli expression vector; Figure 3 A in the middle is the PCR amplification result of the MpFNS I target fragment; Figure 3 Figure B is the colony PCR electrophoresis result of the MpFNS I-pET32a E. coli expression vector; Figure 3 Middle C is the colony PCR identification result of the MpFNS I-pET32a Escherichia coli expression strain.

[0028] Figure 4The SDS-PAGE results of the trial expression of MpFNS I recombinant protein; M is a 10–180 kDa protein marker, lanes 1 to 6 are samples taken before induction of the MpFNS I expression strain, and lanes 7 to 12 are samples taken after induction of the MpFNS I expression strain.

[0029] Figure 5 The results of SDS-PAGE detection of MpFNS I recombinant protein are shown; M is a 10–180 kDa protein marker; lanes 1 and 9 are the supernatant samples after strain disruption and the final eluted protein, respectively.

[0030] Figure 6 This is the in vitro enzymatic activity functional identification of MpFNS I; A–F show the reactions of naringenin, hesperetin, liquiritigenin, chondrostenol, eriodictyol and dihydrokaempferol catalyzed by the recombinant MpFNS I protein.

[0031] Figure 7 is the standard curve of 6 products; the abscissa is the product concentration, and the ordinate is the absorption peak area at the maximum ultraviolet absorption.

[0032] Figure 8 The effect of pH on the activity of MpFNS I recombinant protease; A–F represent naringenin, hesperetin, liquiritigenin, chondrostenol, eriodictyol, and dihydrokaempferol, respectively.

[0033] Figure 9 The effect of temperature on the activity of MpFNS I recombinant protease; A–F represent naringenin, hesperetin, liquiritigenin, chondrostenol, eriodictyol, and dihydrokaempferol, respectively.

[0034] Figure 10 The colony PCR results of the MpFNS I-pCAMBIA1305-35S::myc-GV3101 recombinant strain are shown; M, 2000 bp DNA marker; 1-6 are all positive colonies.

[0035] Figure 11 For the screening and identification of wild-type transgenic Arabidopsis positive plants; Figure 11 Middle A is the screening of 35S::MpFNS I-myc / Clo-0 positive seedlings; Figure 11 Middle B is Western Blot identification of T3 generation MpFNS I gene overexpressing plants; Figure 11 Middle C shows the transcription level analysis of MpFNS I gene in the T3 generation of the overexpression strain.

[0036] Figure 12 To identify the expression levels of genes related to the flavonoid synthesis pathway in the 35S::MpFNS I-myc / Clo-0 strain.

[0037] Figure 13 This is an analysis of the expression levels of genes related to the flavonoid synthesis pathway in the 35S::MpFNS I-myc / Clo-0 strain after UV-B treatment; AE represents MpFNS I, AtC4H, AtPAL, AtCHS and AtCHI genes, respectively.

[0038] Figure 14 HPLC chromatogram of total flavonoids in the 35S::MpFNS I-myc / Clo-0 overexpression strain; Figure 14 Middle A is the HPLC chromatogram of the untreated control group; Figure 14 HPLC chromatogram of the UV-B treatment group treated with medium B; in the HPLC chromatogram of the mixed standard, 1 is quercetin, 2 is apigenin, and 3 is kaempferol.

[0039] Figure 15 Analysis of flavonoid and flavonol contents in 35S::MpFNS I-myc / Clo-0 transgenic Arabidopsis thaliana; A, B, and C are the relative contents of apigenin, quercetin, and kaempferol in the control and UV-B groups, respectively; in group A and B, the control level of Col-0 was set as 1.0; in group C, the control level of Col-51 was set as 1.0; data are expressed as mean ± SD (n = 3).

[0040] Figure 16 Analysis of anthocyanin content in 35S::MpFNS I-myc / Clo-0 transgenic Arabidopsis thaliana; Analysis of anthocyanin content in 35S::MpFNS I-myc / Clo-0 Arabidopsis seedlings treated with UV-B after growth for about 15 days. DETAILED DESCRIPTION

[0041] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0042] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0043] The Takaragaike-1 (Tak-1) strain of liverwort (Marchantia polymorpha.L) used in this invention was kindly donated by Professor Wang Ertao of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. The Takaragaike-1 (Tak-1) strain is described in the literature (Improved G-AgarTrap: A highly efficient transformation method for intact gemmalings of the liverwort Marchantia polymorpha). During cultivation, the relative humidity was controlled at approximately 60%, and the light intensity was 35–40 μmol·m -2 ·s -1 .

[0044] Example 1: Cloning of the MpFNS I gene

[0045] 1. Extraction of Total RNA and Synthesis of cDNA from Marchantia polymorpha

[0046] Prepare 1 / 2 B5 medium (containing 1% sucrose) and pour it into a 10×10 cm sterile plant culture dish after sterilization. Remove the buds from the bud cup on the Marchantia polymorpha gametophyte, place them in sterile water, and evenly spread the buds on the 1 / 2 B5 medium with a pipette tip. At a temperature of 22°C, use 35–40 μmol·m －2 ·s －1 The culture was carried out under a light intensity of 16 h / d.

[0047] Table 1 1 / 2 B5 medium preparation table (400mL)

[0048]

[0049] Note: Adjust the pH to 5.8 with KOH. After preparation, sterilize the mixture in a vertical automatic pressure sterilizer at 121°C for 20 min.

[0050] Total RNA was extracted from Marchantia miliacea leaves cultured in the greenhouse for about 3 weeks using a Broad Spectrum Plant RNA Extraction Kit (DNase I). TM The concentration and purity were determined by NA-NODROP ONE ultra-micro UV spectrophotometer, and cDNA was reverse transcribed according to the instructions of ReScriptTMⅡ RT All-in-One Mix (with dsDNase) cDNA chain synthesis kit.

[0051] 2. Full-length amplification of the MpFNS I gene

[0052] Based on the CDS sequence of MpFNS I in the transcriptome database, primers MpFNS I-CDS-F / R (SEQ ID NO.3-SEQ ID NO.4) were designed to amplify the full-length CDS gene and commissioned to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0053] MpFNS I-CDS-F: 5'-TTTTCTCGTCGAGGCGTTGA-3' (SEQ ID NO. 3);

[0054] MpFNS I-CDS-R: 5'-GACATCGTCCTCATCCGTGA-3' (SEQ ID NO. 4).

[0055] The extracted Marchantia polymorpha cDNA was used as a template to amplify the full-length sequence of the gene using PimeSTAR Max high-fidelity enzyme. The PCR amplification system is shown in Table 2. The above PCR products were subjected to agarose gel electrophoresis, and the full-length CDS of MpFNS I was amplified to be 1069 bp ( Figure 1 After imaging the gel using a gel imager, the target band of the correct size was cut out and the target fragment was recovered according to the instructions of the gel recovery kit. The concentration and purity of the recovered product were measured using an ultra-micro UV spectrophotometer.

[0056] Table 2 MpFNS I gene PCR system (50 μL)

[0057]

[0058] Example 2: Construction of MpFNS I prokaryotic expression vector and expression and purification of MpFNS I protein

[0059] 1. Connecting MpFNS I gene to cloning vector and transformation

[0060] The fragment recovered from the gel in Example 1 was ligated into the pEASY-Blunt cloning vector (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) using a ligation system consisting of 4 μL of recovered PCR product and 1 μL of the pEASY-Blunt cloning vector. The ligation procedure was 22°C for 15 min. The ligation product was transformed into Escherichia coli DH5α via heat stimulation and cultured at 37°C for 14–16 h. Positive clones were identified.

[0061] Pick 8 single colonies and mix them into a 1.5mL centrifuge tube containing 10μL ddH2O. After thorough mixing, take 1μL as a template for colony PCR (system see Table 3). Add 600μL LB medium to the remaining bacterial liquid and continue to culture at 37℃. After the correct band is verified by agarose gel electrophoresis ( Figure 2), and the turbid bacterial solution was sent for sequencing. If the sequencing result was correct, the MpFNS I-pEASY-Blunt vector was successfully constructed.

[0062] Table 3 MpFNS I-pEASY-Blunt-DH5α colony PCR reaction

[0063]

[0064] 2. Construction of prokaryotic expression vector of MpFNS I

[0065] Plasmids MpFNS I-pEASY-Blunt and pET32a were extracted using a plasmid mini kit. The gene fragment was amplified using a high-fidelity enzyme using primers MpFNS I-pET32a-F / MpFNS I-pET32a-R, which carry Kpn I and Xba I recognition sequences, and the MpFNS I-pEASY-Blunt plasmid as a template.

[0066] MpFNS I-pET32a-F:

[0067] 5'-CGCGGATCCATGGCGCCTCCTGCTGCTGAG-3' (SEQ ID NO. 5);

[0068] MpFNS I-pET32a-R:

[0069] 5'-CCGCTCGAGTCAAATGGCAAGAGCGTCGTA-3' (SEQ ID NO. 6).

[0070] The gene fragment obtained by PCR and the pET32a plasmid were double-digested with Kpn I and Xba I, respectively, and digested at 37°C for 3h. Then, the enzymes were detected by agarose gel electrophoresis and the gel was cut and recovered. The recovered target gene fragment and the pET32a expression vector were connected using T4 DNA ligase. The ligation product was then transformed into Escherichia coli DH5α, and a single clone was selected for colony PCR. After successful colony PCR verification, the sample was sent for sequencing verification, and the correctly sequenced plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. The MpFNS I-pET32a-BL21 (DE3) expression strain was obtained by colony PCR verification. The verification results are as follows Figure 3 shown.

[0071] Table 4 MpFNS I gene PCR amplification system (50 μL)

[0072]

[0073] Table 5 Enzyme digestion system (30 μL)

[0074]

[0075] Table 6 MpFNS I-pET32a recombination ligation system (20 μL)

[0076]

[0077] Table 7 MpFNS I-pET32a-DH5α colony PCR reaction system (20 μL)

[0078]

[0079] 3. Trial expression of MpFNS I recombinant protein

[0080] Six monoclonal strains of MpFNS I-pET32a-BL21 (DE3) were selected for the test expression of recombinant proteins. 100 μL of bacterial solution was taken from each strain and 5 mL of C + LB liquid culture medium was placed in a thermostatic shaker at 37°C, 220 rpm, and cultured for 6 hours. 200 μL of the bacterial solution was taken and stored in a 4°C refrigerator before labeling MpFNS I-1 / 2 / 3 / 4 / 5 / 6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to the bacterial solution to a final concentration of 1 mM and cultured in a thermostatic shaker at 37°C, 220 rpm for another 3 hours. 200 μL of the bacterial solution was taken and labeled MpFNS I-1 / 2 / 3 / 4 / 5 / 6. The pre- and post-induction samples were centrifuged at 12,000 rpm for 1 minute, the supernatant discarded, and the pellet was mixed with 20 μL of 1 M Tris-HCl (pH 6.8) and 10 μL of 4× loading buffer using a vortex shaker. The pellet was heated at 100°C in a metal bath for 5 minutes and then stored in a 4°C refrigerator. Then, SDS-PAGE electrophoresis was performed, and white light plates were used for observation and photography to record protein expression.

[0081] SDS-PAGE analysis showed that under 37°C, 3h induction conditions, all six strains tested expressed recombinant protein, with a distinct protein band between 50–70kDa. The theoretical molecular weight of MpFNS I protein is 40.37kDa. When expressed in fusion with the Trx-S-His tag protein, the calculated theoretical molecular weight is approximately 58kDa. Therefore, the protein band obtained by the experimental expression is consistent with the expected molecular weight ( Figure 4 ).

[0082] 4. Large-scale expression and purification of MpFNS I recombinant protein

[0083] The strain MpFNS I-pET32a-BL21 (DE3) with the correct protein size in the test expression was used for large-scale expression and purification of the recombinant protein. 70 μL of bacterial solution was inoculated into 5 mL of C + The cells were cultured in LB liquid medium at 37°C and 220 r / min in a shaker overnight. The next day, 5 mL of the bacterial solution was transferred to 800 mL of C + The culture was continued in liquid LB medium at 37℃ in a constant temperature shaker until OD 600 The RI was approximately 0.6 (measured using a UV-visible spectrophotometer). IPTG was added to a final concentration of 0.5 mM and induced at 16°C for 20 hours. After induction, cells were harvested by centrifugation at 5,000 rpm for 15 minutes using a Beckman Coulbert Avanti J-26S XP high-efficiency centrifuge. The cells were then resuspended in 40 mL of binding buffer. After resuspension, the cells were disrupted three times using a high-pressure homogenizer (1,500 bar). The cell lysate was centrifuged at 12,000 rpm for 30 minutes to obtain the supernatant (containing the recombinant protein).

[0084] Ni Sepharose TM 6Fast Flow (GE healthcare) filler was used for protein affinity chromatography purification. The supernatant was mixed with the filler, incubated at 4°C for 1 hour, and then washed with washing buffer. The concentration of imidazole in the washing buffer was gradually increased, and finally the recombinant protein was eluted with elution buffer. The obtained protein solution was replaced with an ultrafiltration centrifuge tube to remove imidazole, and then the protein solution was preserved for subsequent enzyme activity reaction. The total extract, supernatant and purified protein after cell disruption were used to detect protein expression using SDS-PAGE. The electrophoresis results showed that the size of the expressed recombinant protein of MpFNS I was between 50–70 kDa, which was consistent with the predicted molecular weight of 58 kDa ( Figure 5 The recombinant protein obtained by one-step nickel affinity chromatography has high purity and can be used for subsequent enzyme activity tests.

[0085] Example 3: In vitro enzyme activity assay of MpFNS I

[0086] 1. Enzyme activity reaction system and analysis conditions

[0087] The reaction system contained 50 mM Tris-HCl (pH 7.5), 1 mM α-ketoglutaric acid, 2 mM ascorbic acid, 0.4 mM Fe 2+and 10 μg of purified protein. The final substrate concentration was set to 0.5 mM (see Table 8). Recombinant protein containing the pET32a empty vector was also used as a control. After mixing the above components, the reaction was allowed to proceed at 35°C for 60 min. The reaction was terminated by adding an equal volume of ethyl acetate. The organic phases were combined, evaporated to dryness, and dissolved in 100 μL of chromatography-grade methanol. The mixture was centrifuged at 12,000 rpm for 10 min and analyzed by HPLC.

[0088] HPLC analysis was performed using a 5 μm reversed-phase column (YMC-Triart C18, 4.6 × 150 mm). The analytical conditions were: phase A, water containing 0.1% formic acid; phase B, 100% methanol. The flow rate was 0.8 mL / min. The column and sample tray temperatures were set at 4°C, and the injection volume was 20 μL. Detection wavelengths were set at 268 nm, 310 nm, 336 nm, and 366 nm.

[0089] Table 8 Enzyme activity reaction system (500 μL)

[0090]

[0091] Apigenin, diosmetin 7,4′-dihydroxyflavone, chrysin, luteolin and kaempferol standards were weighed and prepared into standard solutions with concentrations of 1, 5, 10, 20, 40, 80 and 100 μM using chromatography-grade methanol for HPLC detection. Three replicates were set for each concentration, and a peak area-concentration standard curve of the substrate standards was drawn.

[0092] In vitro enzyme activity results showed that the recombinant MpFNS I protein could catalyze the conversion of substrates such as naringenin, hesperetin, liquiritigenin, chondrostenol, and eriodictyol to produce apigenin, diosgenin, 7,4′-dihydroxyflavone, chrysin, and luteolin, respectively, indicating that the recombinant MpFNS I protein has flavonoid synthase function. In addition, MpFNS I can also catalyze the conversion of dihydroflavonols to flavonols, that is, catalyze the conversion of dihydrokaempferol to kaempferol, indicating that it has flavonol synthase (FLS) function. MpFNS I is a bifunctional enzyme of flavonoid synthase and flavonol synthase ( Figure 6 ).

[0093] The concentration-peak area curves of apigenin, diosgenin, 7,4′-dihydroxyflavone, chrysin, luteolin, and kaempferol were drawn respectively. The equation of the chrysin standard curve is y=0.5578*X+0.5776, R 2 =0.998( Figure 7 A); the standard curve equation of luteolin is y=0.5576*X+1.190, R 2 =0.995( Figure 7 B); the standard curve equation for apigenin is y=0.6391*X+0.1401, R2 =0.998( Figure 7 C); the equation of the kaempferol standard curve is y=0.7646*X+0.05715, R 2 =0.999( Figure 7 D); the standard curve equation of lignin is y=0.4539*X-0.03160, R 2 =0.998( Figure 7 E); the standard curve equation of 7,4′-dihydroxyflavone is y=0.9444*X-0.5062, R 2 =0.997( Figure 7 Middle F).

[0094] 2. Analysis of the Enzymatic Properties of MpFNS I

[0095] To explore the catalytic properties of MpFNS I protein, the enzyme activity of MpFNS I protein in the pH range of 6.0–10.5 (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0 and 10.5) was measured at 30°C. The highest enzyme activity was taken as 100% to compare the relative enzyme activities under other pH conditions. 50mM MES-NaOH buffer was used for pH 6.0–7.0, 50mM Tris-HCl buffer was used for pH 7.5–8.5, and 50mM H3BO3 buffer was used for pH 9.0, 10.0 and 10.5. Three sets of parallel experiments were performed to determine the optimal pH for enzyme catalysis. The experimental results showed that MpFNS I protease catalyzed the conversion of naringenin ( Figure 8 A), hesperidin ( Figure 8 B), glycyrrhizin ( Figure 8 Middle C), Qiao Songsu ( Figure 8 D), eriodictyol ( Figure 8 E) and dihydrokaempferol ( Figure 8 The optimum pH values for F) are 7.0, 7.5, 7.0, 8.0, 7.5 and 7.0, respectively.

[0096] Under the standard reaction system, the optimal pH value of the substrate catalyzed by the recombinant protein MpFNS I is mainly distributed in the range of 7.0-7.5. Only the optimal pH value of the chlorpyrifos catalytic system is slightly higher, reaching 8.0. All substrates showed a significant decrease in enzyme activity in extreme pH environments (below 6.5 or above 9.0). pH stability experiments showed that when the reaction environment deviated from the optimal pH by 1.5 units, each catalytic system could still maintain a relative enzyme activity of more than 65%. It is worth noting that the chlorpyrifos catalytic system exhibited the widest pH adaptability range, maintaining a high level of activity in the pH range of 6.5-9.0.

[0097] Further temperature gradient experiments (20–50°C) were conducted under optimal pH conditions. The enzyme activity of MpFNS I recombinant protein against six substrates was measured within the range of 20–50°C (20, 25, 30, 35, 40, 45, and 50°C). The highest enzyme activity measured was taken as 100%, and the relative enzyme activity at other temperatures was compared. Three sets of parallel experiments were performed to determine the optimal reaction temperature. The results showed that there were significant differences in the optimal reaction temperatures of MpFNS I for the six substrates ( Figure 9 Among them, naringenin ( Figure 9 A) and glycyrrhizin ( Figure 9 The optimum reaction temperature of C) is 45℃, which is significantly higher than that of other substrates; Figure 9 Middle B) An obvious activity peak appeared at 35℃, and the enzyme activity was maintained at a high level within the range of 30–45℃, which was about 60–80% of the maximum reaction enzyme activity; while the optimal reaction temperature of chondrostenone, eriodictyol, and dihydrokaempferol was 30℃.

[0098] 3. Enzyme Kinetic Analysis

[0099] At the optimal reaction pH and temperature, reaction systems containing different substrate concentrations (1, 5, 10, 20, 40, 80, 120, 160 μM) were prepared. 10 μg of recombinant protein was added and reacted for 15 min at the optimal temperature and pH conditions for each substrate. The initial reaction rate was measured. K was obtained according to the Michaelis–Menten equation using GraphPad Prism 9 software. m and V max , and calculate k cat / K m value.

[0100] Due to the low catalytic efficiency of MpFNS I recombinant protein for dihydrokaempferol substrate, the reaction time was set to 15 minutes in the initial experiment, and the effective absorption peak of the product could not be measured under HPLC. Therefore, the incubation time was extended to 30 minutes. In addition, when the initial reaction was carried out with glycyrrhizin as the substrate, the active site of the enzyme could not be saturated with the substrate. The measured data showed that the reaction could not reach the maximum reaction rate of enzyme catalysis. When the glycyrrhizin concentration reached 350μM, the fitted Michaelis-Menten curve was still in a rapid growth stage, indicating that the addition of 10μg of recombinant protease was excessive for the reaction. Therefore, in the subsequent reaction with glycyrrhizin, the amount of recombinant protein was reduced to 2μg, and finally a stable and effective Michaelis-Menten curve was fitted ( Figure 10 ).

[0101] As shown in Table 9, the K values of MpFNS I for the six substrates were m The values ranged from 4.28 to 29.66 μM, showing the highest substrate affinity for hesperetin, with K mThe value was 4.28 μM, which was significantly lower than that of other substrates. It had the lowest affinity for dihydrokaempferol, with a K m The value was 29.66 μM; the affinity for liquiritigenin, chondrostenone and eriodictyol was similar, K m The values were 10.39, 10.97, and 10.69 μM, respectively; the K m The value is 25.48μM. MpFNS I has the maximum reaction rate to naringenin, and its V max The value was 70.84 nmol·mg-1·min-1; the V max The lowest value is about 2.46

[0102] nmol·mg-1·min-1; the maximum catalytic rates of the other four substrates were 5.73–32.27nmol·mg-1·min-1. cat / K m The ratio measures the catalytic efficiency of the enzyme. The higher the ratio, the more efficient the enzyme is at low substrate concentrations. MpFNS I showed the highest catalytic efficiency for hesperidin, k cat / K m The value is 5.061; it also has high catalytic efficiency for naringenin, bromocriptine, and liquiritigenin; the catalytic efficiency for eriodictyol is low, only 10% of the catalytic efficiency for hesperetin (k cat / K m In addition, MpFNS I had the lowest catalytic efficiency for dihydrokaempferol, k cat / K m Only 0.083M -1 ·S -1 These results indicate that MpFNS I is a typical flavonoid synthase with high catalytic efficiency for hesperetin, naringenin, naringenin, and liquiritigenin. Although MpFNS I can catalyze the production of kaempferol from dihydrokaempferol and possesses flavonol synthase function, its affinity and catalytic efficiency for dihydrokaempferol are very low. It is speculated that the flavonol synthase function of MpFNS I is not its primary catalytic function.

[0103] Table 9 MpFNS I enzymatic kinetic parameters

[0104]

[0105]

[0106] Example 4: In vivo functional study of MpFNS I

[0107] 1. Construction of MpFNS I gene overexpression vector and screening and identification of positive plants

[0108] Using the MpFNS I-pEASY-Blunt vector plasmid constructed in Example 1 as a template, specific primers for MpFNS I-MYC-F / R containing Kpn I and Xba I restriction sites were designed. High-fidelity enzymes were used for PCR amplification to obtain the MpFNS I gene fragment, which was then recovered from the gel.

[0109] MpFNS I-MYC-F:

[0110] 5'-CGGGGTACCATGGCGCCTCCTGCTGCTGA-3' (SEQ ID NO. 7);

[0111] MpFNS I-MYC-R:

[0112] 5'-TGCTCTAGAAATGGCAAGAGCGTCGTAGT-3' (SEQ ID NO. 8).

[0113] The MpFNS I gene was connected to the pCAMBIA1305-35S::myc vector (pCAMBIA1305-35S::myc vector is described in Chen Qingshuai. Study on the mechanism of rapid response of sugar signal in Arabidopsis [D]. Shandong: Shandong Agricultural University, 2019. DOI:10.7666 / d.D01699098.), and then transformed and positive clones were screened on LB solid medium containing 50 mg / L kanamycin. The positive clones were then verified by bacterial liquid PCR and sequencing to obtain the correct clones. Finally, the correct recombinant plasmid was introduced into GV3101 Agrobacterium competent cells by heat shock transformation. The colony verification results of the MpFNS I-pCAMBIA1305-35S::myc-GV3101 recombinant strain were as follows: Figure 10 shown.

[0114] Wild-type Arabidopsis thaliana Col-0 (Columbia) was infected with Agrobacterium containing the 35S::MpFNS I-myc expression vector. The infection method was as follows: 100 μL of Agrobacterium solution was added to a conical flask containing 50 mL of double-antibody LB liquid, and the culture was shaken at 28°C until the OD 600 The value is approximately 1.5-3.0. Collect the cells by centrifugation at 4000 rpm for 5 minutes at room temperature. Then, gently resuspend the cells in transformation solution (a 5% sucrose solution containing 0.02% Silwet-L 77, prepared the same day) and activate them in a 28°C incubator for approximately 30 minutes. Pre-cut off the opened flowers and pods of the Arabidopsis thaliana, leaving only the flower buds. Immerse all flower buds in the bacterial suspension for 2 minutes, remove them, and incubate them in the dark for 1 day before placing them under normal culture conditions.

[0115] Wild-type Arabidopsis thaliana was infected with Agrobacterium containing the 35S::MpFNS I-myc expression vector and seeds of the T0 generation plants were collected. T1 generation plants were obtained by screening on 1 / 2MS resistance solid medium. The screening results of positive plants are shown in Figure 2. Figure 11 As shown in Figure A. After harvesting individual plants, two generations were screened and the expression of the MpFNS I protein in Arabidopsis was detected by Western blot. The results showed that several homozygous transgenic lines showed brighter bands. When compared with the protein marker band, the obtained protein size ranged from 40 to 50 kDa, which is consistent with the predicted molecular weight of the MpFNS I protein (45.7 kDa). Figure 11 At the same time, RNA was extracted from wild-type Arabidopsis and three overexpression plants to detect the transcription level of MpFNS I gene in different transgenic homozygous lines. Figure 11 Middle C) shows that the expression level of MpFNS I gene in Col-51 plants was the lowest, and the transcription levels of MpFNS I gene in Col-76 and Col-86 plants were 3.3 times and 4.5 times that of Col-51, respectively.

[0116] 2. Gene Expression Analysis in Transgenic Arabidopsis

[0117] Total RNA was extracted from mature leaves / seedlings of wild-type Arabidopsis and various homozygous overexpression lines. cDNA was synthesized by reverse transcription and real-time fluorescence quantitative PCR was performed using the cDNA as a template. The Arabidopsis AtActin 7 gene (AT5G09810) was used as an internal reference gene. qRT-PCR results showed that in the 35S:MpFNS I / Clo-0 overexpression line, the expression of AtPAL (AT2G37040), AtC4H (AT2G30490), AtCHS (AT5G13930), and AtCHI genes (AT2G43570) were significantly upregulated. In particular, the expression level of the AtCHS gene in the three overexpression lines was approximately 12–22 times that of the wild-type ( Figure 12 ).

[0118] As a key intermediate in the flavonoid biosynthesis pathway, naringenin can be converted by various flavonoid biosynthetic enzymes and enter different flavonoid metabolic pathways. In vitro enzyme activity experiments revealed that the MpFNS I protein catalyzes the conversion of naringenin to apigenin. When the MpFNS I gene was heterologously expressed in Arabidopsis thaliana, its expression product catalyzed the conversion of naringenin to apigenin in vivo, resulting in a decrease in naringenin concentration. To meet the Arabidopsis thaliana demand for naringenin, this product activated the expression of genes in the upstream pathway, leading to an overall upregulation of the flavonoid biosynthesis pathway and, in turn, an increase in the flavonoid biosynthesis flux.

[0119]

[0120] 3. Role of MpFNS I in enhancing UV-B tolerance in Arabidopsis

[0121] To further investigate the in vivo function of MpFNS I and analyze its role in enhancing UV-B tolerance in Arabidopsis, Col-0 and three overexpressing strains were treated with UV-B light. UV-B light was applied daily for 12 hours for 7 consecutive days. RNA was extracted from the UV-B-treated and normal light-treated Arabidopsis plants, and expression changes in key flavonoid biosynthesis genes were analyzed using qRT-PCR. Total flavonoids and anthocyanins were also extracted and analyzed for changes in their contents.

[0122] Depend on Figure 13 As shown, under continuous UV-B irradiation, MpFNS I gene expression was significantly upregulated in the overexpression lines Col-76 and Col-86, while no significant change was observed in Col-51. Upstream genes in the Arabidopsis flavonoid pathway exhibited different trends. In wild-type Col-0, expression levels of AtPAL, AtC4H, AtCHS, and AtCHI all decreased significantly after 7 days of UV-B treatment, indicating that long-term UV-B radiation inhibits the expression of genes in the flavonoid biosynthesis pathway. Among the overexpression lines, the expression level of the AtPAL gene did not change significantly in Col-51 and Col-76, but showed a slight increase in the Col-86 line, with the expression level increasing by approximately 20%. The expression level of the AtC4H gene showed an overall downward trend, with a significant downregulation in Col-51, and no significant changes in the other two lines, Col-76 and 86. The expression level of the AtCHI gene also showed a downward trend after UV-B treatment, with a significant downregulation in Col-76 and 86. The expression level of the AtCHS gene showed an upward trend in the overexpression lines, and was significantly upregulated in Col-86, approximately twice that of the control group.

[0123] Total flavonoids were extracted from Arabidopsis wild type Col-0 and MpFNS I gene overexpression lines Col-51 / 76 / 86, and the contents of flavonoids, flavonols and other compounds were determined by HPLC. The results showed that in the control group without UV-B treatment, no apigenin was detected in Col-0 plants, while obvious apigenin chromatographic peaks were detected in the overexpression lines Col-51 / 76 / 86. Among them, the content in Col-51 was the lowest, with a relative amount of 1.0; the relative contents of Col0-76 and Col0-86 were 1.7 and 1.1, respectively. Figure 15 After UV-B treatment, apigenin was still not detected in Col-0 plants, while the apigenin content in the overexpression lines Col-51 / 76 / 86 increased significantly, reaching 3.5, 1.5, and 3.4 times that of the control groups, respectively ( Figure 15The contents of quercetin and kaempferol in each strain were analyzed. The results showed that quercetin and kaempferol were detected in Col-0 and the three overexpression strains in both the UV-B treatment group and the control group ( Figure 14 In the control group, the contents of kaempferol and quercetin in the MpFNS I overexpression line were slightly higher than those in the Col-0 line; however, after UV-B treatment, the contents of kaempferol and quercetin in the overexpression line increased significantly, but were still lower than those in the Col-0 line ( Figure 15 (B, C).

[0124] In the MpFNS I overexpression line, MpFNS I catalyzes flavanones (such as naringenin) to produce flavonoids (such as apigenin), resulting in the accumulation of apigenin in the transgenic line. At the same time, MpFNS I consumes flavanones, weakens the feedback inhibition of flavanones, promotes the upregulation of the expression of upstream genes such as CHS and CHI in flavonoid synthesis, and increases the biosynthesis of flavanones, thereby providing more precursors for the synthesis of downstream flavonoids, flavonols and anthocyanins ( Figure 15 ).

[0125] After UV-B treatment, apigenin content increased significantly in the MpFNS I overexpressing strain, consistent with UV-B signaling activating the expression of key genes in the flavonoid biosynthesis pathway, promoting flavonoid biosynthesis and accumulation. UV-B induction significantly increases the transcriptional levels of key flavonoid pathway genes, driving flavonol synthesis. On the other hand, after UV-B treatment, the flavonol (quercetin and kaempferol) content in the MpFNS I overexpressing strain was significantly lower than that in the Col0 strain, likely due to substrate competition. Flavanone (naringenin) is a common substrate for FNS I and F3H (flavanone 3-hydroxylase). Overexpression of MpFNS I directs a significant amount of naringenin to the flavonoid branch (producing apigenin), reducing the substrate available for AtF3H in Arabidopsis and reducing the synthesis of dihydroflavonols (flavonol precursors).

[0126] In addition, the anthocyanin content in the 35S::MpFNS I-myc / Clo-0 overexpression line was analyzed. The results showed that after UV-B treatment, anthocyanin accumulation in the overexpression line was significantly higher than that in the wild-type Col-0, with a 10–40% increase compared to the control group. It is speculated that because MpFNS I overexpression relieves / weakens the feedback inhibition of flavanones, it increases the concentration of flavanones (naringenin), a common precursor of the downstream branch pathway, thereby enhancing the synthesis and accumulation of anthocyanins. Figure 16 ).

[0127] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent replacements, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A Marchantia polyphylla flavonoid synthase MpFNS I, characterized in that The protein amino acid sequence of the Marchantia polyphylla flavonoid synthase MpFNS I is shown in SEQ ID NO.

1.

2. The gene encoding Marchantia polyphylla flavonoid synthase MpFNS I according to claim 1, characterized in that The CDS nucleotide sequence of the gene encoding Marchantia polyphylla flavonoid synthase MpFNS I is shown in SEQ ID NO.

2.

3. Use of the Marchantia polyphylla flavonoid synthase MpFNS I according to claim 1 in the following (1) or (2): (1) Catalytic substrate; (2) Regulate the expression of flavonoid synthesis genes.

4. The use according to claim 3, characterized in that The optimal pH value of the substrate catalyzed by the Marchantia polyphylla flavonoid synthase MpFNS I is distributed in the range of 7.0-7.

5.

5. The use according to claim 3, characterized in that The Marchantia polyphylla flavonoid synthase MpFNS I catalyzes the K m The values ranged from 4.28–29.66 μM.

6. A recombinant expression vector or genetically engineered bacteria containing the gene encoding the Marchantia polymorpha flavonoid synthase MpFNS I according to claim 2.

7. A method for catalyzing the production of flavonoids from flavanones in a substrate, characterized in that: The method catalyzes flavanone to generate flavonoids by adding Marchantia polyphylla flavonoid synthase MpFNS I to a substrate.

8. The method according to claim 7, characterized in that The flavanone in the substrate is naringenin, hesperetin, liquiritigenin, chondrostenone or eriodictyol, and the generated flavonoids are apigenin, diosmetin, 7,4'-dihydroxyflavone, chrysin or luteolin.

9. A method for catalyzing dihydroflavonol to produce flavonol, characterized in that: The method catalyzes dihydroflavonol (dihydrokaempferol) to generate flavonol (kaempferol) by adding Marchantia polyphylla flavonoid synthase MpFNS I to a substrate.

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