Ginkgo biloba GbF3H gene as well as expression protein and application thereof

Abstract

The invention discloses a gingko GbF3H gene as well as an expression protein and application thereof, and belongs to the field of plant molecular biology. The GbF3H gene is obtained by analyzing and screening on the basis of ginkgo transcriptome data in the earlier stage of a research group, the GbF3H gene is flavanone 3-hydroxylase, the nucleotide sequence of the GbF3H gene is shown as SEQ ID NO.1, and the amino acid sequence of expression protein of the GbF3H gene is shown as SEQ ID NO.2. A GbF3H overexpression vector is constructed and the nicotiana benthamiana is transformed, so that the synthesis of flavonoid substances in a transgenic plant is obviously changed, the difference of 28 flavonoid metabolites is obvious, and the contents of 9 flavonoid substances such as formononin are up-regulated. The invention provides a theoretical basis, a valuable gene resource and a method for analyzing a ginkgo flavone synthesis mechanism and improving the flavone yield through metabolic engineering, and has important significance on development and utilization of ginkgo resources and cultivation of new germplasm with medicinal value.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to a Ginkgo biloba GbF3H gene, its expressed protein, and its applications. Background Technology

[0002] Ginkgo (Ginkgo biloba L.) is the oldest extant relict plant among gymnosperms, and its leaves exhibit high economic value due to their rich content of various active ingredients. The most medicinally active components in ginkgo leaf extract are flavonoids, which can account for up to approximately 24% of the total content. Among these flavonoid glycosides, quercetin, kaempferol, and isorhamnetin account for over 95% of the total flavonoid content, becoming key indicators for evaluating the quality of ginkgo leaf preparations. Ginkgo leaf preparations are made from ginkgo leaf extract as the basic raw material and have broad health and medicinal benefits for humans. These preparations play an important role in the clinical treatment of various diseases such as Alzheimer's disease, myocardial ischemia, and cerebral infarction. The chemical synthesis of ginkgo flavonoids is difficult, and currently, they are mainly extracted from ginkgo leaves. Therefore, increasing the flavonoid content is beneficial to the survival of ginkgo itself and the development of the ginkgo leaf extract pharmaceutical industry, possessing significant practical value and broad application prospects.

[0003] Flavanone 3-hydroxylase (F3H) is a key enzyme in the biosynthesis of flavonols, anthocyanins, and proanthocyanidins. Belonging to the 2-O-ketoglutarate and iron-dependent dioxygenase superfamily (2-ODD), the reaction requires 2-ketoglutarate, oxygen, iron, and ascorbic acid in addition to the substrate naringenin. As a central enzyme in plants entering different flavonoid metabolite branches, F3H separates the F3H branch from flavonoid substances and catalyzes the production of dihydroflavonols, a common precursor in anthocyanin and flavonol synthesis. It plays a crucial regulatory role in the flavonoid synthesis pathway. Therefore, regulating F3H activity or its gene expression can affect the catalytic activity and transcriptional level of flavanones, thereby regulating the flow distribution among different branches of the flavonoid metabolic pathway.

[0004] Currently, the specific molecular characteristics of the F3H gene in Ginkgo biloba and its mechanism for precisely regulating the synthesis of major medicinal flavonoids (such as quercetin) remain unclear. Cloning the Ginkgo biloba F3H gene and elucidating its function is crucial for clarifying the regulatory network of Ginkgo biloba flavonoid biosynthesis and, consequently, guiding genetic improvement strategies to increase flavonoid yield. Summary of the Invention

[0005] To address the problems existing in the prior art, the first technical problem to be solved by this application is to provide a Ginkgo biloba GbDRF2 gene and its expressed protein. The second technical problem to be solved by this application is to provide biological materials containing the aforementioned Ginkgo biloba GbDRF2 gene. The final technical problem to be solved by this application is to provide specific applications of the aforementioned GbDRF2 gene.

[0006] The existing technical problems have not been resolved. The technical solution of this application is as follows:

[0007] A Ginkgo biloba GbF3H gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the Ginkgo GbF3H gene expression protein according to claim 1 is shown in SEQ ID NO.2.

[0009] A biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the Ginkgo GbF3H gene.

[0010] The application of the Ginkgo GbF3H gene or the Ginkgo Gb3GT gene expression protein or the biological material in plant breeding.

[0011] The application of the Ginkgo GbF3H gene or the Ginkgo Gb3GT gene expression protein or the biomaterial described in claim 3 in regulating the synthesis of plant flavonoids.

[0012] A method for increasing the synthesis of flavonoids in plants involves overexpressing the Ginkgo biloba GbF3H gene in plants, wherein the flavonoids are at least one of the following compounds: arbutin, scutellarin, (-)-catechin, scutellarin, isorhamnetin-3-O-neohesperidin, 5-O-demethylhesperidin, isorhamnetin-3-O-glucoside, 2'-hydroxydaidzein, and nepeta glycoside.

[0013] In some embodiments, the method includes the following steps:

[0014] (1) Construct the expression vector of the Ginkgo GbF3H gene;

[0015] (2) Transform the constructed Ginkgo GbF3H gene expression vector into plants or plant tissues;

[0016] (3) Cultivate and screen transgenic plants or plant tissues with increased flavonoid content.

[0017] In some embodiments, the plant is tobacco or ginkgo.

[0018] In some embodiments, the overexpression vector of the Ginkgo GbF3H gene is pCAMBIA1302-Gb3GT.

[0019] In some embodiments, the transformation is mediated by Agrobacterium.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This application identifies a GbF3H gene based on previous analysis of Ginkgo transcriptome data from our research group. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. It is a flavanone 3-hydroxylase. The physicochemical properties, domains, and phylogenetic relationships of the gene were analyzed. Results showed that the Ginkgo GbF3H gene encodes a 369-amino acid hydrophobic acidic protein with DIOX_N and 2OG-FeII_Oxy domains, located in the cytoplasm. Tissue-specific expression analysis indicated that GbF3H expression was higher in immature fruits and young leaves, and its expression was significantly regulated by nitrogen form and abscisic acid induction. Overexpression of F3H in tobacco K326 significantly altered the expression of 28 flavonoid metabolites, with 9 flavonoids, including gentianin, arbutin, and scutellarin, being upregulated. Transcriptome sequencing further revealed that heterologous expression of GbF3H suppressed the expression of key genes Nt4CL, NtFLS1, and NtUFGT, but upregulated NtFLS2, indicating that GbF3H affects the flavonoid metabolism network through multi-level regulation. This application provides a theoretical basis, valuable gene resources, and methods for elucidating the mechanism of Ginkgo flavonoid synthesis and improving flavonoid production through metabolic engineering, which is of great significance for the development and utilization of Ginkgo resources and the cultivation of new germplasm with medicinal value. Attached Figure Description

[0022] Figure 1 Hotspot diagram of Ginkgo F3H gene expression in different tissue sites and under different treatments;

[0023] Figure 2-1 Figure 1 shows the results of structural and phylogenetic analysis of the GbF3H protein from Ginkgo biloba. Figure 2 shows the results of secondary structure analysis of the GbF3H protein, and Figure 3 shows the three-dimensional structural model of the GbF3H protein.

[0024] Figure 2-2 Figure showing the phylogenetic analysis results of the GbF3H protein;

[0025] Figure 3 The diagram shows the results of the genetic transformation of Ginkgo F3H gene into tobacco; where A is a diagram of the growth process of tobacco genetic transformation using the GbF3H leaf disc method; and B is a diagram showing the results of the expression analysis of GbF3H in different transgenic lines.

[0026] Figure 4-1Violin plot of 15 differential metabolites in transgenic tobacco lines, with the horizontal axis representing the group and the vertical axis representing the expression level;

[0027] Figure 4-2 The image shows a violin plot of 13 differentially expressed metabolites in transgenic tobacco lines, with the horizontal axis representing the group and the vertical axis representing the expression level.

[0028] Figure 4-3 The images show bar charts for the fold change of differentially expressed metabolites in transgenic tobacco lines and KEGG enrichment plots for these metabolites. In chart A, the fold change of differentially expressed metabolites is represented by the log2FC (logarithm of the fold change to base 2) on the x-axis and the differentially expressed metabolites on the y-axis. Red indicates upregulated differentially expressed metabolites, and green indicates downregulated differentially expressed metabolites. Chart B shows the enrichment analysis of differentially expressed metabolites. The x-axis represents the rich factor for each pathway, and the y-axis represents the pathway name. The color of the dots represents the P-value; the redder the dot, the more significant the enrichment. The size of the dot represents the number of enriched differentially expressed metabolites.

[0029] Figure 5-1 A diagram showing the expression analysis of the flavonoid synthesis pathway and 10 related transcription factors after Ginkgo F3H transgenic tobacco;

[0030] Figure 5-2 A diagram showing the expression analysis of the flavonoid synthesis pathway and 12 related transcription factors after Ginkgo F3H transgenic tobacco;

[0031] Figure 6 KEGG enrichment map of differentially expressed genes;

[0032] Figure 7-1 This is a diagram of the flavonoid synthesis pathway in F3H transgenic plants; thick orange arrows represent downregulated differential metabolites, red upward arrows represent upregulated genes, and green downward arrows represent downregulated genes.

[0033] Figure 7-2 This diagram illustrates the expression analysis of key differentially expressed genes in the flavonoid synthesis pathway of F3H transgenic plants. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Alternatively, they may be carried out according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0035] Example 1

[0036] 1. Plant materials, gene mining and expression analysis

[0037] This application uses 3-year-old Ginkgo seedlings as experimental material and employs a completely randomized experimental design, setting up different nitrogen forms, namely 5 inorganic nitrogen treatments: NH4 + NO3 - The ratios were 100:0, 75:25, 50:50, 25:75, and 0:100, corresponding to numbers AN0-AN4; two organic nitrogen treatments were used: urea and glycine, numbered Urea and ON, and a blank control (CK). All nitrogen application rates were calculated as pure nitrogen. The experiment was conducted in a greenhouse at the Xiashu Forest Farm of Nanjing Forestry University (32°07′N, 119°13′E). Five pots in six rows were placed on each side of the plants as protective measures to eliminate marginal effects.

[0038] Based on previous transcriptome data of Ginkgo biloba under different nitrogen treatments (SRA: SRP574331), differentially expressed F3H genes were screened. Comparative analysis of transcriptomes of Ginkgo biloba under different nitrogen forms revealed two differentially expressed F3H genes. One of these F3H genes (evm.TU.chr11.1258) has been previously studied (Cloning and characterization of a flavanone 3-hydroxylase gene from Ginkgo biloba). Therefore, this application conducted functional experiments on the other F3H gene (evm.TU.chr8.1972) (hereinafter GbF3H).

[0039] The expression patterns of GbF3H in different tissues (such as male cones, female cones, yellow leaves, and cambium) and different treatment groups of Ginkgo biloba were analyzed by FPKM values ​​and visualized using heatmaps. Further validation was performed using RT-qPCR, with the stably expressed GAPDH gene in Ginkgo biloba as an internal control, and detection was conducted on a qTOWER 2.2 system using SYBR Green Premix (TaKaRa).

[0040] To determine the expression patterns of the GbF3H gene in different tissues and under different treatments in Ginkgo biloba, a heatmap was constructed based on transcriptome data. Figure 1Further investigation revealed that the GbF3H gene exhibited high basal expression levels in various plant tissues. When plants were exposed to different forms of nitrogen treatment, GbF3H expression decreased compared to untreated tissues. GbF3H expression showed more distinct characteristics in specific tissues or developmental stages. For example, GbF3H expression reached its highest level in immature fruit, while its expression was significantly reduced in mature fruit, supporting its specific role at specific stages of fruit development. Relatively high expression levels of F3H were also found in ovulate strobilus and immature leaves. When examining the effects of different forms and concentrations of nitrogen treatment on GbF3H gene expression, corresponding changes in GbF3H expression were observed. Furthermore, GbF3H expression also showed a high level when plants were treated with ABA.

[0041] 2. Cloning of the Ginkgo GbF3H gene

[0042] Total RNA was extracted from Ginkgo biloba leaves using the BioTeKe Universal Plant Total RNA Rapid Extraction Kit. Purity was determined by 1% agarose gel electrophoresis and NanoDrop 2000. =1.8-2.0). cDNA was synthesized using PrimeScript™ RT MasterMix (TaKaRa). Cloning primers were designed based on the GbF3H gene ORF sequence (Table 1), and PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (TaKaRa). Reaction system (50 μL): 2×Premix 25 μL, primers 1 μL each, cDNA template 1 μL. PCR program: 98℃ 3 min; 35 cycles (98℃ 10 s, 55℃ 5 s, 72℃ 15 s); 72℃ 3 min. After gel purification, the amplified product was ligated into the pClone007 Blunt vector (TSINGKE), transformed into Trans1-T1 competent cells, and the sequence correctness was verified by colony PCR and Sanger sequencing. The sequence of Ginkgo biloba GbF3H obtained by cloning is shown in SEQ ID NO.1. It was found that the polypeptide it encodes contains 369 amino acids and its stop codon is TAA. The amino acid sequence is shown in SEQ ID NO.2.

[0043] Table 1 Primer sequence information

[0044]

[0045] 3. Analysis of the physicochemical properties of proteins

[0046] The physicochemical properties of the GbF3H protein were analyzed using ProtParam software, and its total hydrophilicity was predicted using the ProtScale online software. The subcellular localization of the GbF3H protein was predicted using CELLO (subcellular localization predictor). The transmembrane domains of the protein were predicted using Novopro's online tool (https: / / www.novopro.cn / tools / tmhmm.html). The domains of the GbF3H protein were jointly searched and analyzed using NCBI's conserved domain analysis tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow) and the online tool SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?GENOMIC=1). The secondary structure of the GbF3H protein was comprehensively analyzed using NPSA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_seccons.html) combined with three methods: MLRC on GOR4, SIMPA96, SOPMA, DSC, and PHD. A three-dimensional model of the GbF3H protein was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive).

[0047] Physicochemical analysis revealed that the molecular weight of GbF3H protein is 41.88 kDa, its theoretical isoelectric point is 5.73, the total number of positively charged residues (including Arg and Lys) is 41, and the total number of negatively charged residues (including Asp and Glu) is 48. Its adipose coefficient is 87.45, and its instability index is 48.13. Furthermore, the total hydrophilicity of GbF3H protein is -0.277, indicating it is a hydrophobic acidic protein. Subcellular localization prediction of GbF3H showed it to be located in the cytoplasm. Transmembrane domain prediction indicated that GbF3H protein does not possess a transmembrane domain. Interestingly, GbF3H protein possesses two characteristic domains, DIOX_N and 2OG-FeII_Oxy, and also contains two low-complexity domains, belonging to the PLN02515 superfamily. The secondary structure of GbF3H protein mainly consists of α-helices, extended chains, random coils, and some undefined structures. Figure 2-1(A). Meanwhile, a three-dimensional model of the protein was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive). Figure 2-1 In the study (B), the template with the highest homology to GbF3H was found to be A0A0D6QSS1.1.A, which originated from a protein containing the Fe2OG dioxygenase domain in Araucaria cunninghamii (Hoop pine) (Moreton Bay pine). The model coverage was 91%, and the similarity was 53%. Furthermore, phylogenetic analysis revealed that Ginkgo GbF3H clustered with the F3H protein (WIM50945.1) of Larix kaempferi (Pinaceae) and the F3H protein (WFP21701.1) of Psilotum nudum (Pseudomonas nudum) in a single clade. Figure 2-2 This indicates a close kinship, as the number and arrangement of the motifs of these three proteins are also consistent. Figure 2-2 This suggests that the phylogenetic tree results are relatively reliable.

[0048] 4. Construction and genetic transformation of Ginkgo F3H gene vector

[0049] The validated GbF3H gene ORF fragment was cloned downstream of the 35S strong promoter of the plant binary expression vector pCAMBIA1302 using Gateway® technology to construct the recombinant plasmid pCAMBIA1302-F3H. The recombinant plasmid was introduced into Agrobacterium tumefaciens GV3101 via electroporation, and K326 tobacco leaves (K326 tobacco material used for stable genetic transformation was derived from tissue culture seedlings preserved by our research group) were infected using the leaf disc method. After co-culturing for 2 days, the cells were transferred to MS selection medium containing 50 mg / L kanamycin to induce callus and regenerated plants. Positive transgenic lines were screened by PCR and RT-qPCR, with the tobacco Actin gene used as an internal control (Table 1) to verify the stable overexpression of the GbF3H gene.

[0050] GbF3H was overexpressed in K326 tobacco. Using kanamycin as a selection pressure, potential positive transgenic tobacco plants were generated through Agrobacterium-mediated infection of tobacco leaves, callus formation, adventitious bud growth, and root growth after division, via the leaf disc method. Figure 3 As shown in Figure A. Ten tobacco plants were randomly selected for expression level detection. It was found that the Ginkgo GbF3H gene was expressed at a high fold increase of tens of thousands of times in all eight transgenic plants. Three transgenic tobacco lines with relatively high expression levels (F3H3, F3H7, and F3H8) were selected as subjects for further research. The expression levels of genes targeting flavonoids and related to flavonoid synthesis in these transgenic tobacco plants were analyzed. Figure 3 B).

[0051] 5. Flavonoid Analysis of GbF3H Transgenic Tobacco

[0052] (1) Treatment of transgenic K326 tobacco leaves

[0053] Three K326 control samples and three K326 tobacco leaves from transgenic GbF3H overexpression lines were subjected to vacuum freeze-drying. The mixture was then ball-milled at 30 Hz for 1.5 min. 20 mg of powder was accurately weighed and mixed with 10 μL of 4000 nmol / L internal standard working solution and 500 μL of 70% methanol solution. The mixture was then sonicated for 30 min and centrifuged at 12000 r / min for 5 min at 4 °C. Finally, the supernatant was aspirated and filtered through a 0.22 μm filter membrane. The six filtered samples were stored in vials for subsequent LC-MS / MS analysis.

[0054] (2) Chromatographic and mass spectrometric acquisition conditions

[0055] The core components of the data acquisition system include an ultra-high performance liquid chromatograph (UPLC, model ExionLCAD, details available at https: / / sciex.com.cn / ) and a tandem mass spectrometer (MS / MS, model QTRAP 6500+, details available at https: / / sciex.com.cn / ).

[0056] Regarding the configuration of liquid chromatography: (1) Chromatographic separation column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm id); (2) Mobile phase composition: Phase A consists of ultrapure water with 0.05% formic acid added, and Phase B consists of acetonitrile with 0.05% formic acid added; (3) Flow rate and temperature control: The flow rate is set to 0.35 mL / min, the working temperature of the chromatographic column is maintained at 40℃, and the injection volume for each analysis is 2 μL; (4) Gradient elution program: Initially (0 min), the ratio of Phase A to Phase B is 90:10 (V / V), then it is adjusted to 80:20 within 1 min, reaches 30:70 at 9 min, becomes 5:95 at 12.5 min, and is maintained until 13.5 min, then quickly returns to 90:10 at 13.6 min, and ends the analysis at 15 min, still maintaining the 90:10 ratio.

[0057] The conditions for mass spectrometry analysis include: (1) Ionization source settings: The operating temperature of the electrospray ionization source (ESI) is set to 550°C, the mass spectrometry voltage is 5500V in positive ion mode and -4500V in negative ion mode. (2) Ion detection optimization: In the Q-Trap 6500+ mass spectrometer, the detection of each ion pair is based on carefully adjusted declustering potential (DP) and collision energy (CE) for scanning analysis. (3) Curtain gas pressure: The pressure of the CUR (curtain gas) is set to 35 psi.

[0058] (3) Data processing and difference analysis

[0059] Statistical analysis methods for processing flavonoid data can be broadly categorized into univariate and multivariate approaches. Univariate analysis primarily relies on hypothesis testing and fold change (FC) analysis. Multivariate analysis, on the other hand, encompasses advanced techniques such as principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). When using the OPLS-DA model, we utilize the variable importance projection (VIP) index to initially identify metabolite differences between groups; metabolites with a VIP value greater than 1 are considered significantly different. To more accurately screen for differentially expressed metabolites, we further refine the selection by combining the p-value / false discovery rate (FDR) or FC value from the univariate analysis. When comparing two groups of data, the criteria for selecting differentially expressed metabolites are VIP > 1, fold change ≥ 2, and fold change ≤ 0.5.

[0060] (4) Analysis results of flavonoids in transgenic tobacco leaves

[0061] After completing a comprehensive qualitative and quantitative assessment of metabolites in both genetically modified and non-genetically modified tobacco leaves, a total of 52 metabolites were detected, categorized into 9 types. Flavonols were the most abundant category, comprising 15 metabolites. Compared with the CK group, the transgenic GbF3H group showed significant differential expression of 28 flavonoid-related metabolites (9 upregulated and 19 downregulated), mainly distributed in flavonoids and flavonols, each containing 8 differential metabolites. The flavonoid group included 8 differentially expressed metabolites such as baicalin, apigenin, sakuranetin, homoplantaginin, hydroxygenkwanin, tectochrysin, galangin, and 5-O-demethylnobiletin. The flavonol group included astragalin, avicularin, and isorhamnetin-3-O-glucoside. 3-O-glucoside, Baimaside, Kaempferol, Isorhamnetin, Tiliroside, Isorhamnetin-3-O-neohespeidoside Figure 4-1 , 4-2 4-3). Violin plots were created for 28 differentially expressed metabolites. Among the upregulated metabolites, ononin (an isoflavone) was the most abundant, at 4.38 nmol / g in the F3H group and 1.52 nmol / g in the CK group, with a VIP value of 1.11 and an FC value of 2.89. Among the downregulated metabolites, the flavanol catechin gallate (-)-catechin gallate was the most abundant, at 0.66 nmol / g in the F3H group and 2.16 nmol / g in the CK group, with a VIP value of 1.11 and a fold change of 0.31. Figure 4-1 , 4-2 ).

[0062] To further analyze the impact of F3H gene overexpression on changes in flavonoid content, a bar chart was plotted for the top 12 metabolites with the largest fold changes. The metabolite with the largest log2FC value that was upregulated was 2'-hydroxydaidzein (2.28), belonging to the isoflavone category, with a VIP value of 1.07. The metabolite with the largest log2FC value that was downregulated was isorhamnetin, belonging to the flavonol category, with a log2FC of -2.89. Figure 4-3 A). Furthermore, the annotation results of KEGG for significantly differentially expressed metabolites were categorized according to pathway type in KEGG. It was found that the biosynthesis of secondary metabolites contained the most differentially expressed metabolites, with 9 metabolites accounting for 69.2% of the total. Figure 4-3 B). Eight differentially metabolites were identified in flavonoid biosynthesis (61.54% of the total), five in metabolic pathways, four in isoflavone biosynthesis, and four in flavonone and flavonol biosynthesis. The nine upregulated differentially metabolites were: arbutin, engelitin, (-)-catechin, styracin, isorhamnetin-3-O-neohesperidin, 5-O-demethylhesperidin, isorhamnetin-3-O-glucoside, 2'-hydroxydaidzein, and nepeta glycoside.

[0063] 6. Analysis of expression levels of key genes in the flavonoid synthesis pathway after GbF3H transgenic tobacco

[0064] To further understand the specific effects of GbF3H heterologous expression on key enzyme genes and related transcription factors in the flavonoid biosynthesis pathway of K326 tobacco, the expression of 22 key genes in the flavonoid biosynthesis pathway was measured (Table 1). The results showed that the expression levels of key enzyme genes Nt4CL, NtFLS1, and NtUFGT were significantly decreased compared with those in the control group tobacco. Figure 5-1 , 5-2 However, the expression of the NtFLS2 gene in transgenic tobacco was drastically different, showing a significantly higher expression level compared to the control group. Furthermore, the expression levels of NtMYL2b, NtERF4a, and NtERF4b were significantly lower than those in non-transgenic tobacco.

[0065] 7. Transcriptional analysis of the flavonoid synthesis pathway in GbF3H transgenic tobacco and non-transgenic tobacco

[0066] Extraction of RNA from K326 tobacco leaf samples was performed using ethanol precipitation and CTAB-PBIOZOL. The extracted RNA was dissolved in 50 µL of DEPC-treated water. Total RNA was then identified and quantified using a Qubit quantitative PCR instrument and a Qsep400 high-throughput biological fragment analyzer. The raw data was then filtered using FASTP, primarily removing reads with adapters: paired reads were removed if the nitrogen content exceeded 10% of the read's base count; paired reads were also removed if the number of low-quality (Q<=20) bases exceeded 50% of the read's base count. All subsequent analyses were based on clean reads. Sequences were aligned to the tobacco reference genome, new transcripts were predicted using StringTie, gene alignment was calculated using feature counts, and FPKM was calculated for each gene based on gene length. Differential expression analysis was performed between groups using DESeq2, and p-values ​​were corrected using the Benjamini & Hochberg method. The corrected p-value and log2 fold change were used as thresholds for significant differential expression. KEGG enrichment analysis was performed based on hypergeometric tests, and hypergeometric distribution tests were performed on a pathway-by-pathway basis.

[0067] Transcriptome sequencing of F3H transgenic and non-transgenic tobacco leaves yielded 31.21G, successfully annotating 39,247 genes, identifying 4,754 differentially expressed genes (2,901 of which showed significantly upregulated expression). Further investigation into the distribution of these differentially expressed genes in metabolic pathways revealed their involvement in multiple key secondary metabolic pathways. Figure 6 Interestingly, 47 differentially expressed genes were identified in the phenylalanine synthesis pathway; 13 differentially expressed genes were identified in the flavonoid synthesis pathway, a pathway for the synthesis of compounds with broad biological activity; and 4 differentially expressed genes (gene_14759, gene_2555, novel.8734, gene_83065) were identified in the isoflavone and flavonol synthesis pathways, two pathways for the synthesis of compounds with specific biological activities. Figure 7-1 , 7-2The differentially expressed genes found in the flavonoid synthesis pathway directly correspond to key nodes in the synthesis of flavonoid compounds. Downregulation of the expression level of midstream genes in the CHS pathway affects the synthesis of metabolites such as naringenin chalcone, cinnamoyl-CoA, homoeriodictyol, and phloretin, with phloretin showing downregulation. The F3H transgene upregulates the expression of branch point genes, thereby inhibiting the synthesis of dihydrokaempferol, which is a differentially expressed metabolite downregulated. Upregulation of FLS enzyme-encoding genes affects the content levels of kaempferol and galangin.

[0068] The synthesis of flavonoids begins with phenylalanine, which first generates cinnamic acid via PAL, then is converted to p-coumaric acid via C4H, and finally to p-coumaroyl-CoA via C4CL. p-Coumaroyl-CoA is the core intermediate in subsequent metabolism, and through the catalysis of CHS and CHI, it gradually generates naringenin flavanone. Subsequently, dihydrokaempferol is generated through the action of GbF3H. This metabolite is downregulated after GbF3H gene overexpression and enters a branched metabolic pathway: on one hand, it generates flavonols under the action of FLS; on the other hand, it generates dihydroquercetin or dihydromyricetin under the catalysis of F3'H and F3'5'H, and finally generates (-)-epicatechin under the catalysis of FLS. In another branch, phenylalanine generates cinnamyl-CoA under the catalysis of the downregulated gene CHS, which then generates the downregulated metabolite pinocemrin, followed by pinocemrin under the action of F3H, and finally pinocemrin under the action of the upregulated gene FLS. The entire pathway transforms initial substances into various secondary metabolites, such as flavonols and anthocyanins, through a series of enzymatic reactions. These substances play important roles in pigment production and antioxidant processes in plants.

[0069] The Ginkgo biloba GbF3H gene significantly influences the flow allocation of flavonoid metabolic pathways by catalyzing the production of dihydroflavonols. Ginkgo biloba GbF3H gene expression is dynamically regulated by tissue development stage, nitrogen form, and hormone signaling. Genetic transformation revealed that heterologous overexpression of the GbF3H gene alters the composition of flavonoids by inhibiting the expression of Nt4CL, NtFLS1, and NtUFGT genes. In transgenic tobacco K326, overexpression of the Ginkgo biloba GbF3H gene significantly altered 28 flavonoid metabolites, with a significant upregulation of the isoflavone gentianin and a significant downregulation of the flavonol isorhamnetin. Transcriptome analysis further showed that overexpression of the GbF3H gene significantly decreased the expression levels of several key genes in the flavonoid synthesis pathway, including Nt4CL, NtFLS1, and NtUFGT, while significantly increasing the expression of the NtFLS2 gene. Furthermore, the expression levels of some transcription factors, such as NtMYL2b, NtERF4a, and NtERF4b, were also significantly decreased. These changes collectively reveal the mechanism by which the GbF3H gene influences the flavonoid metabolic network through multi-level regulation. This result not only deepens our understanding of the mechanism of Ginkgo flavonoid synthesis but also provides theoretical support for using metabolic engineering to increase the yield of flavonoids in medicinal plants.

Claims

1. A Ginkgo biloba GbF3H gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expression protein of the Ginkgo GbF3H gene according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the Ginkgo GbF3H gene as described in claim 1.

4. The application of the Ginkgo GbF3H gene of claim 1, the Ginkgo Gb3GT gene of claim 2, or the biomaterial of claim 3 in plant breeding.

5. The application of the Ginkgo GbF3H gene of claim 1 or the Ginkgo Gb3GT gene of claim 2, or the biomaterial of claim 3, in regulating the synthesis of plant flavonoids.

6. A method for increasing the synthesis of flavonoids in plants, characterized in that, Overexpression of the Ginkgo GbF3H gene of claim 1 in plants, wherein the flavonoid compound is at least one of the following compounds: arbutin, scutellarin, (-)-catechin, scutellarin, isorhamnetin-3-O-neohesperidin, 5-O-demethylhesperidin, isorhamnetin-3-O-glucoside, 2'-hydroxydaidzein, and euryptin.

7. The method according to claim 6, characterized in that, Includes the following steps: (1) Construct the expression vector of the Ginkgo GbF3H gene as described in claim 1; (2) Transform the constructed Ginkgo GbF3H gene expression vector into plants or plant tissues; (3) Cultivate and screen transgenic plants or plant tissues with increased flavonoid content.

8. The method according to claim 7, characterized in that, The plant in question is either tobacco or ginkgo.

9. The method according to claim 7, characterized in that, The overexpression vector for the Ginkgo GbF3H gene is pCAMBIA1302-Gb3GT.

10. The method according to claim 7, characterized in that, The transformation was mediated by Agrobacterium.

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