Ginkgo biloba GbDRF2 gene as well as expression protein and application thereof

Abstract

The invention discloses a gingko GbDRF2 gene as well as an expression protein and application thereof, and belongs to the field of plant molecular biology. The GbDFR2 gene is obtained by analyzing and screening based on data of a ginkgo transcriptome in the earlier stage of a research group, the nucleotide sequence of the GbDFR2 gene is shown as SEQ ID NO.1, and the amino acid sequence of expression protein of the GbDFR2 gene is shown as SEQ ID NO.2. A GbDFR2 overexpression vector is constructed and nicotiana benthamiana is transformed, so that the synthesis of flavonoid substances in a transgenic plant is obviously changed, the contents of 9 flavonoid substances such as sakuranetin and 3, 7-dimethyl quercetin are increased, and the contents of 10 metabolites such as naringin and chalcone are decreased. The invention discloses a regulation effect of the ginkgo GbDFR2 gene in synthesis of flavonoid compounds, and provides a theoretical basis and a molecular tool for improving plant secondary metabolites through genetic engineering.

Description

Technical Field

[0001] The present invention belongs to the field of plant molecular biology, and in particular relates to a Ginkgo GbDRF2 gene, an expressed protein and an application thereof. Background Art

[0002] Ginkgo biloba L., the only extant species of the Ginkgoaceae, is known as a "living fossil," possessing a unique evolutionary status and high ecological and economic value. Its robust resistance to adversity and widespread distribution, though limited to China after the Ice Age, has now spread globally through human activities to temperate and subtropical regions. Ginkgo leaves and fruits have a millennia-long history of medicinal use, with flavonoids and lactones forming the core active ingredients. The flavonoids possess cardiovascular benefits, antioxidant properties, and anti-cancer properties. These two active ingredients, with their unique chemical and biological properties, have become important raw materials for food and health supplements, providing strong support for the sustainable development of the ginkgo industry.

[0003] Flavonoids are important functional components of Ginkgo biloba leaves and hold great potential for development and utilization. Uncovering the molecular mechanisms of flavonoid biosynthesis is of great significance for Ginkgo biloba's utilization. Anthocyanins are a class of flavonoid compounds and form a branch of the flavonoid biosynthesis pathway. Dihydroflavonol 4-reductase (DFR, EC1.1.1.219), a member of the NADPH-dependent short-chain reductase family, is a key enzyme in the anthocyanin biosynthesis pathway. It catalyzes the first reaction in the anthocyanin biosynthesis pathway, catalyzing three dihydroflavonols and two flavanones to produce five different anthocyanin precursors. These precursors are further converted into various anthocyanins by subsequent enzymes, conferring a rich variety of colors on plant tissues and organs. DFR has potential applications in regulating flower and fruit color. By genetically engineering DFR expression levels or substrate specificity, it is possible to cultivate plant varieties with novel flower and fruit colors. The DFR gene was first isolated from corn and snapdragon in 1985. In recent years, DFR genes have been isolated, identified and studied in many plants. The nucleotide sequences encoding DFR genes in different species are species-specific, but their structural domains and catalytic properties are similar.

[0004] Studies have shown that the regulatory mechanisms of different DFRs genes may differ, and that the patterns of change in individual flavonoid components in ginkgo vary under different environmental inductions. Therefore, obtaining the molecular structure and mechanism of action of ginkgo DFRs is of great significance for understanding how ginkgo DFRs genes regulate the synthesis of ginkgo flavonoids and further exploring the application value of ginkgo flavonoid compounds. Summary of the Invention

[0005] In response to 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 a biological material containing the Ginkgo biloba GbDRF2 gene. The final technical problem to be solved by this application is to provide a specific application of the GbDRF2 gene.

[0006] The existing technical problems are not solved, and the technical solutions of this application are as follows:

[0007] A Ginkgo GbDRF2 gene, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the Ginkgo GbDRF2 gene is shown in SEQ ID NO.2.

[0009] A biological material is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell containing the Ginkgo GbDRF2 gene.

[0010] Application of the Ginkgo GbDRF2 gene or the expression protein of the Ginkgo GbDRF2 gene or the biological material in plant breeding.

[0011] The application of the Ginkgo GbDRF2 gene or the expression protein of the Ginkgo GbDRF2 gene or the biological material in regulating the synthesis of plant flavonoids.

[0012] A method for increasing the synthesis of flavonoids in plants, comprising overexpressing the Ginkgo biloba GbDRF2 gene in the plants, wherein the flavonoids are at least one of the following compounds: cyanidin, kava cyanidin C, diosmin, taxifolin, 3,7-dimethylquercetin, glycyrrhizin isoflavone A, puerarin, sakurain, and genkwain.

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

[0014] (1) constructing an expression vector for the Ginkgo GbDRF2 gene according to claim 1;

[0015] (2) transforming the constructed Ginkgo GbDRF2 gene expression vector into plants or plant tissues;

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

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

[0018] In some embodiments, the Ginkgo GbDRF2 gene overexpression vector is pCAMBIA1302-GbDFR2.

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

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] Based on the research team's previous analysis of Ginkgo biloba transcriptome data, this application identified a gene, GbDFR2. 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 dihydroflavonoid 4-reductase. Its physicochemical properties, domain structure, and phylogenetic relationships were analyzed. Results showed that the GbDFR2 protein is a hydrophobic acidic protein, with highest expression levels in the buds, fruits, and yellow leaves of Ginkgo biloba, respectively. A GbDFR2 overexpression vector was constructed and transformed into Nicotiana benthamiana. Flavonoid biosynthesis was significantly altered in the transgenic plants, with upregulation of nine flavonoids, including sakurain and 3,7-dimethylquercetin, and downregulation of ten metabolites, including naringenin and naringenin chalcone. Further analysis revealed that heterologous expression of GbDFR2 affected the expression of key genes in the flavonoid biosynthesis pathway, such as NbCHI and NbDFR, as well as transcription factors such as NbMYL2b and NbERF4a. This application reveals the key role of the Ginkgo GbDFR2 gene in regulating the synthesis of flavonoids, providing valuable genetic resources and theoretical basis for regulating flower color, fruit color and increasing flavonoid production through genetic engineering technology. It is of great significance to the development and utilization of ginkgo resources and the cultivation of new germplasm with high ornamental and medicinal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A The secondary structure analysis and three-dimensional structure prediction model diagram of Ginkgo DFRs protein;

[0023] Figure 1B This is the result of the evolutionary tree analysis of Ginkgo DFRs protein;

[0024] Figure 1C This is the motif prediction result diagram of DFRs protein;

[0025] Figure 2 This is the result of the expression pattern analysis of Ginkgo DFRs in different tissues and under different treatments;

[0026] Figure 3 Figure 1 shows the expression analysis results of DFR genetically transformed Nicotiana benthamiana and its different strains; A shows the growth process of tobacco genetically transformed with GbDFR2 using the leaf disc method; B shows the expression analysis results of GbDFR2 in different transgenic strains;

[0027] Figure 4A The results of expression pattern analysis of key genes NbPAL1, NbPAL3, NbF3H, NbFLS1, NbANR1, NbANR2, NbbHLH1, and NbbHLH2 of GbDFR2 in different transgenic lines are shown;

[0028] Figure 4B The results of expression pattern analysis of key genes NbC4H, NbHCT, NbFLS2, NbDFR, NbUFGT, NbMYL2a, NbbHLH3, and NbERF4a of GbDFR2 in different transgenic lines are shown;

[0029] Figure 4C The results of expression pattern analysis of key genes NbCHI, NbMYB1, NbMYB2, NbLAR, and NbMYL2b of GbDFR2 in different transgenic lines are shown;

[0030] Figure 5A Violin plots of differential metabolite analysis of GbDFR2 transgenic tobacco plant lines (11 of them);

[0031] Figure 5B Violin plots of differential metabolite analysis of GbDFR2 transgenic tobacco plant lines (8 of them);

[0032] Figure 5C This is a diagram showing the enrichment analysis of differential metabolites in GbDFR2 transgenic tobacco plant lines. DETAILED DESCRIPTION

[0033] To make the objects, technical solutions and advantages of the present invention more clear, the present invention is further described below in conjunction with specific examples. Unless otherwise specified in the following examples, the technical means used are conventional means well known to those skilled in the art, or are carried out according to the kits and product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] Example 1

[0035] 1. Plant materials and mining of Ginkgo DFRs genes

[0036] The material for subcellular localization was Nicotiana benthamiana material stored in the artificial climate chamber of the laboratory of the Department of Economic Forestry at Nanjing Forestry University, while the stably genetically transformed Nicotiana benthamiana tissue culture seedlings were derived from the tissue culture seedlings stored in the tissue culture room of the Ginkgo Project at Nanjing Forestry University.

[0037] In production practice, optimizing nitrogen fertilizer supply levels and forms to improve nitrogen use efficiency (NUE) in ginkgo and achieve stable flavonoid production is of great significance. Nitrogen metabolism provides precursors for flavonoid synthesis in the form of phenylalanine. Based on previous transcriptome sequencing data from ginkgo leaves under different nitrogen forms, the applicants explored and identified key differentially expressed genes (DFRs) involved in the ginkgo flavonoid biosynthesis pathway. The relevant primer sequences designed for these experiments are shown in Table 1 below:

[0038] Table 1 Primer sequence information

[0039]

[0040]

[0041] The applicant screened five DFRs genes from transcriptome data of ginkgo treated with different nitrogen forms. They were differentially expressed between different comparison groups (different ginkgo tissues). Their expression patterns were verified by FPKM and RT-qPCR. Finally, three DFRs genes were identified as differentially expressed under different nitrogen forms (evm.TU.chr12.307, evm.TU.chr12.306, and evm.TU.chr12.287) in ginkgo, respectively. These genes were named DFR1 (GbDFR1), DFR2 (GbDFR2), and DFR3 (GbDFR3) (Table 1). DFR1 and DFR2 encode polypeptides consisting of 337 amino acids, and DFR3 encodes a polypeptide consisting of 333 amino acids.

[0042] Through cloning and sequencing, the open reading frame (ORF) sequence of the Ginkgo DFR2 gene was obtained to be 1014 bp in length, as shown in SEQ ID NO.1, with a stop codon of TGA. The encoded polypeptide consists of 337 amino acids, as shown in SEQ ID NO.2.

[0043] 2. Bioinformatics analysis

[0044] The physicochemical properties of Ginkgo DFR1, DFR2, and DFR3 proteins were analyzed using ProtParam software. The overall hydrophilicity of the proteins was predicted using ProtScale online software. The subcellular localization of the proteins was predicted using CELLO (subcellular localization predictor). Motifs of DFRs proteins were predicted using the MEME online tool (https: / / meme-suite.org / meme / tools / meme). The domain structure of DFRs proteins was analyzed using the NCBI 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 protein secondary structure was comprehensively analyzed using NPSA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_seccons.html) in combination with MLRC, GOR4, SIMPA96, SOPMA, DSC, and PHD methods. A three-dimensional protein model was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive).

[0045] The results of the physicochemical property analysis of the three DFRs proteins are shown in Table 1. The molecular weight of DFRs is approximately 37 kDa, the theoretical isoelectric point is 5.58 (DFR3)-6.45 (DFR2), the total number of negatively charged residues (Asp+Glu) in the protein is the highest in DFR3, 39, the total number of positively charged residues (Arg+Lys) is the highest in DFR2 (32), the instability index is 38.18 (DFR1)-41.14 (DFR3), and the fat coefficient is the highest in DFR3, 89.55. Further study of the characteristics of DFRs proteins revealed that the total hydrophilicity of DFRs proteins is less than 0, and thus it is speculated that they are hydrophobic acidic proteins. Subcellular localization prediction results showed that DFR1 is located in the cytoplasm, while DFR2 and DFR3 are mainly located extracellularly. The results of conserved domain analysis showed that the three DFRs proteins all belonged to the NADB_Rossmann superfamily, and DFRs was classified as an atypical member FR of the SDR family, which plays an important role in the reduction of NADP-dependent flavonoids (ketone-containing plant secondary metabolites).

[0046] Table 2 Physicochemical properties and subcellular localization information of proteins encoded by three DFR structural genes

[0047]

[0048] A comprehensive analysis of the secondary structures of the three DFRs proteins showed that they are mainly composed of α-helices, extended chains, random coils and some unclear structures. In addition, the three-dimensional model of the protein was constructed and it was found that the template with the highest homology to DFR1 was numbered 8fem.1.A, which is the DFR protein and NADP complex of Patchouli (Panicum vigratum), with a model coverage rate of 94% and a model similarity of 42%; the template with the highest homology to DFR2 and DFR3 was numbered 2nnl.1.A, which is a DFR protein, but the binding of two substrate analog molecules to DFR changes the functional geometry of the catalytic site. The model coverage rate of DFR2 is 94%, and the model similarity is 41%; the model coverage rate of DFR3 is 97%, and the model similarity is 42% ( Figure 1A ).

[0049] Phylogenetic tree analysis showed that the three DFR proteins clustered together with the dihydroflavonol 4-reductase (XP_059069589.1) from Cryptomeria japonica of the Cupressaceae family, and were closely related. They were also more distantly related to the dihydroflavonol 4-reductases (XP_058725319.1, XP_059461396.1, XP_062165496.1) from Viciavillosa of the Leguminosae family, Corylus avellana of the Betulaceae family, and Alnus glutinosa of the European Alnus family. Figure 1B Motif prediction of the three DFRs proteins in Ginkgo biloba showed that the protein motifs of DFR1 and DFR2 were similar, while DFR3 had lost motif9 ( Figure 1C In summary, DFR1 and DFR2 have the same 9 motifs, and DFR3 lacks motif9, which is consistent with the phylogenetic tree results.

[0050] 3. Expression pattern analysis

[0051] To investigate the expression of the GbDFR gene in Ginkgo biloba, we obtained publicly available raw RNA sequencing (RNA-seq) data from the European Nucleotide Sequence Database (ENA) (https: / / www.ebi.ac.uk / ena / browser / view / ). Fastq RNA-seq files for different tissues, including buds, male flowers, and megasporophyll (PRJNA289172); roots (PRJNA373812); stems (PRJNA473396); cambium (PRJNA488475); and developing leaves (PRJNA473396, PRJNA517218, and PRJNA578374), were downloaded from the EMBL-EBI database (https: / / www.ebi.ac.uk / ena / browser / home) by searching accession numbers. After assessing the quality of raw fastq data using FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc), low-quality reads and adapter sequences were removed using Trimmatic (Bolger et al., 2014). Clean reads were mapped to the latest Ginkgo genome using STAR (Dobin et al., 2013), and FPKM (fragments per kilobase per million mapped reads) was calculated using RSEM (Raghupathy et al., 2018). Finally, the transcript abundance of GbDFRs was converted to log2(FPKM+1) values. A heat map of the transcript abundance of GbDFRs genes in different tissues was generated using the Cloudtutu (http: / / cloudtutu.com.cn / ) online website.

[0052] Based on the existing Ginkgo biloba transcriptome data, the expression patterns of three DFRs genes under different treatments and tissues were analyzed ( Figure 2The results showed that compared to other genes, the expression level of the DFR3 gene showed the most significant peak in the yellow leaf tissue of Ginkgo biloba, while its expression level dropped to the lowest level in immature fruit. Meanwhile, the expression level of the DFR1 gene in reproductive organs such as female buds, ovulate strobilus, and male buds showed a significant advantage compared to other genes. As for the DFR2 gene, its expression level was relatively high in immature fruit. Under treatment with different concentrations of brassinosteroids (BR), the DFR3 gene responded particularly positively to BR1, BR3, and BR4, with its expression level showing a significant upward trend. Under ABA treatment, the expression level of the DFR3 gene was higher at the ABA1 concentration than that of other genes. On the other hand, the expression level of the DFR1 gene under ABA treatment was also significantly higher than that of other genes, further emphasizing the important role of the DFR1 gene in the ABA signal transduction pathway.

[0053] 4. Vector Construction and Genetic Transformation of Nicotiana benthamiana

[0054] Homologous recombination and golden gate seamless cloning were used to construct the overexpression vector pCAMBIA1302-GbDFR2, which contains a strong 35S promoter. Sequencing was used to verify the presence of the target fragment in the overexpression vector. Plasmids were extracted from bacterial cultures containing the target fragment bands. 1 μL of the plasmid was added to 50 μL of GV3101 competent Agrobacterium cells, mixed thoroughly, and then transferred to an electroporation cuvette. After electroporation, 1 mL of LB liquid medium was added, mixed thoroughly, and then transferred to a 1.5 mL centrifuge tube. Cultured on a shaker at 28°C and 180 rpm for 30 minutes. 50 μL of the activated Agrobacterium culture was inoculated onto LB solid medium and incubated in the dark at 28°C for 48 hours. Detection primers were synthesized, and samples showing clear electrophoretic bands of the correct size were selected for genetic transformation of Nicotiana benthamiana.

[0055] Cut the sterile tobacco leaves into small pieces with a scalpel and inoculate them on the pre-culture medium. Pick up Agrobacterium in the infection solution and prepare the OD 600= 0.2 Agrobacterium suspension. Inoculate tobacco leaves pre-cultured for 2-3 days with the Agrobacterium suspension for infection for 10-15 minutes. The infected leaves are then plated onto filter paper, air-dried, and inoculated onto co-culture medium for incubation in the dark for 48-72 hours. After 2 days of co-culture, transfer the leaves to induction medium for callus induction and wait for approximately 10 days for callus to form. Select well-growing calli and inoculate them onto kan-resistant screening medium for 15-30 days at 23±2°C. Actively growing positive calli from the second screening are inoculated onto differentiation medium, with 4-5 calli per dish, incubated at 23°C with a 16h / 8h light / dark cycle for 15-30 days. If seedlings form from the calli during differentiation, inoculate them onto seedling growth medium and grow them for 7-10 days. Tobacco genomic DNA was extracted using the CTAB method and then tested by PCR. RNA from potential positive seedlings was extracted and reverse transcribed into cDNA for RT-qPCR expression level detection of different strains.

[0056] To understand the biological function of GbDFR2, a stable overexpression vector of GbDFR2 gene was constructed and genetically transformed into Nicotiana benthamiana by leaf disc method. After induction, screening, differentiation and rooting, transgenic plants were obtained ( Figure 3 The expression levels of 10 transgenic Nicotiana benthamiana strains were measured, and it was observed that DFR was highly expressed in multiple transgenic plants. The three Nicotiana benthamiana strains with the highest expression levels, DFR1, DFR8, and DFR9, were selected as the subjects of subsequent research ( Figure 3 (B) They were subjected to targeted flavonoid determination and subsequent expression level analysis of key genes and transcription factors in the flavonoid biosynthesis pathway.

[0057] 5. Expression analysis of flavonoid synthesis pathway in transgenic plants

[0058] To further investigate the specific effects of heterologous expression of GbDFR2 on key enzyme genes in the flavonoid biosynthesis pathway in Nicotiana benthamiana, the expression of 13 key genes in the flavonoid biosynthesis pathway was measured: NbPAL1, NbPAL3, NbC4H, NbHCT, NbCHI, NbF3H, NbFLS1, NbFLS2, NbDFR, NbLAR, NbANR1, NbANR2, and NbUFGT. The results showed that compared with the control, the expression levels of NbCHI and NbDFR genes were significantly increased in transgenic Nicotiana benthamiana. However, the expression patterns of several genes in transgenic Nicotiana benthamiana were significantly different. The expression levels of the key enzyme genes NbPAL3, NbFLS1, NbANR1, and NbUFGT were all significantly decreased compared with the control Nicotiana benthamiana. In addition, the expression levels of eight transcription factors (NbMYL2a, NbMYL2b, NbbHLH1, NbbHLH2, NbbHLH3, NbERF4a, NbMYB1, and NbMYB2) that may affect flavonoid biosynthesis in transgenic tobacco were studied. Compared with the control group, the expression levels of NbMYL2b, NbERF4a, and NbERF4b in the transgenic lines were significantly decreased ( Figure 4A 、 Figure 4B and Figure 4C )

[0059] 6. Analysis of flavonoids in DFR2 transgenic tobacco

[0060] (1) Sample pretreatment method for flavonoid content determination:

[0061] First, each sample was vacuum freeze-dried to ensure its stability and facilitate subsequent operation. Subsequently, each sample was ground for 1.5 minutes using a ball mill at a frequency of 30 Hz until it was in a fine powder. Accurately weigh 20 mg of powder for each sample, and add 10 μL of 4000 nmol / L internal standard mixed working solution and 500 μL of 70% methanol solution to dissolve and extract the sample. The sample was then ultrasonically treated for 30 minutes and then centrifuged at 12000 r / min for 5 minutes at 4°C. After centrifugation, the supernatant was aspirated and filtered through a 0.22 μm filter membrane. Finally, the treated sample was stored in an injection bottle for subsequent LC-MS / MS analysis.

[0062] (2) Chromatographic mass spectrometry acquisition conditions for flavonoid content determination:

[0063] The data acquisition instrument system only needs to include ultra-high performance liquid chromatography (UPLC) (ExionLC AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP 6500+, https: / / sciex.com.cn / ).

[0064] Liquid phase conditions included the following: 1) Column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm id); 2) Mobile phase: Phase A: ultrapure water (with 0.05% formic acid), Phase B: acetonitrile (with 0.05% formic acid); 3) Flow rate: 0.35 mL / min; Column temperature: 40°C; Injection volume: 2 μL. 4) Elution gradient: A / B: 90:10 (V / V) at 0 min, 80:20 (V / V) at 1 min, 30:70 (V / V) at 9 min, 5:95 (V / V) at 12.5 min, 5:95 (V / V) at 13.5 min, 90:10 (V / V) at 13.6 min, and 90:10 (V / V) at 15 min.

[0065] For mass spectrometry, an electrospray ionization (ESI) source was selected, with a temperature set to 550°C. In positive ion mode, the mass spectrometer voltage was 5500 V; in negative ion mode, the mass spectrometer voltage was -4500 V. The curtain gas (CUR) pressure was set to 35 psi. In a Q-Trap 6500+ mass spectrometer, each ion pair was scanned and detected based on optimized declustering potential (DP) and collision energy (CE) to ensure analytical accuracy and sensitivity.

[0066] (3) Differential metabolite analysis method

[0067] To further explore the differences in metabolites between groups, an OPLS-DA model was first used for analysis. This model generates variable importance projection (VIP) values, which serve as a key indicator for assessing the degree of metabolite differences between groups. By setting a threshold of VIP values ​​greater than 1, significantly different metabolites were initially screened. Furthermore, the fold change (FC) metric was introduced. By comparing the fold change in metabolite concentrations between groups, metabolites with a fold change ≥ 2 or fold change ≤ 0.5 were selected to further narrow the range of differential metabolites and ensure that the selected metabolites were significantly different and biologically significant. Furthermore, to further understand the biological functions and mechanisms of action of these differentially expressed metabolites, the KEGG database (http: / / www.kegg.jp / kegg / compound / ) was used to annotate the selected metabolites in detail. The rich information in the KEGG database provides key information about each metabolite, including its chemical structure, biosynthetic pathway, and its function in vivo. Finally, the KEGG annotation results of the significantly different metabolites were classified and organized according to the type of KEGG pathway (http: / / www.kegg.jp / kegg / pathway.html).

[0068] (4) Expression analysis results of flavonoid synthesis pathway in transgenic plants

[0069] By overexpressing the DFR2 gene in Nicotiana benthamiana, and completing comprehensive qualitative and quantitative metabolite analysis in the collected samples, a total of 54 flavonoids were identified. These substances were classified into 10 categories: dihydroflavones, dihydroflavonoid glycosides, dihydroflavonols, biflavones, isoflavones, chalcones, phenolic acids, flavanols, flavones, and flavonols. Among them, chalcones include 4 metabolites, accounting for 7.41% of the total, dihydroflavonoids have 6 metabolites, accounting for 31.48%, dihydroflavonols have 2 metabolites, accounting for 3.7% of the total, dihydroflavonoid glycosides have 1 metabolite, accounting for 1.85%, phenolic acids contain 2 metabolites, accounting for 3.7% of the total substances, flavonoids contain 17 metabolites, accounting for 31.48%, which is the most metabolites among all categories, flavonols contain 14 metabolites, accounting for 25.93% of the total, flavanols include 3 metabolites, accounting for 5.56%, biflavonoids contain 1 metabolite, accounting for 1.85%, and isoflavones contain 4 metabolites, accounting for 7.41% of the total substances.

[0070] In order to further compare the quantitative information of metabolites between different groups, 19 metabolites with significant differences were screened out by applying the OPLS-DA model and combining comprehensive evaluation of key indicators such as VIP value and ploidy change (10 metabolites showed a down-regulation trend in DFR2 transgenic plants, and 9 differential metabolites showed an up-regulation trend). The differential metabolites can be divided into 9 categories. Chalcones have three differential metabolites, namely naringenin chalcone, phloretin and benzylideneacetophenone, accounting for 15% of the total. Flavonoids have two metabolites, namely eriocitrin and liquiritin, accounting for 10%. The metabolite of flavonols is taxifolin, the metabolite of flavonoid glycosides is naringenin-7-O-glucoside, and the metabolite of phenolic acids is flavokawain C. C), flavonoids include narcissin, sakuranetin, genkwanin, jaceosidin, and diosmin, accounting for 25% of the total substances; flavonols include isorhamnetin and 3,7-methylquercetin; flavanols include catechin gallate and epicatechin; isoflavones include puerarin and licoisoflavone A, both accounting for 10% of the total.

[0071] The 19 differential metabolites were displayed on violins, and it was found that among the up-regulated differential metabolites cyanidin, kava piperine C, diosmin, taxifolin, 3,7-dimethylquercetin, glycyrrhizin A, puerarin, sakuratin, and genkwain, the flavonoid sakuratin had the highest expression level, with the content in the DFR group being 0.23 nmol / g, which was 2.07 times that of the CK group. Among the down-regulated differential metabolites, narcissin had the highest expression level, with the content in the DFR group being 4.71 nmol / g, which was 5.42 nmol / g lower than that of the CK group. It is a type of flavonoid. In addition, compared with the CK group (NbCK group, non-transgenic tobacco group), the flavonoid genkwain was the substance with the largest up-regulated differential expression fold, followed by the flavonol 3,7-dimethylquercetin. The chalcone substance naringenin chalcone ( Figure 5A 、 Figure 5B). In order to gain a deeper understanding of the biological functions and mechanisms of action of these differential metabolites, the KEGG database (http: / / www.kegg.jp / kegg / compound / ) was used to annotate and classify the screened metabolites in detail. The results showed that there were 5 differential metabolites in metabolic pathways, 6 differential metabolites in flavonoid biosynthesis, namely, naringenin chalcone, eriodictyol, isorhamnetin, sakura sakura, flavigny tretinoin, (-)-epicatechin, 1 differential metabolite 3,7-dimethylquercetin in flavonoid and flavonol biosynthesis, and 7 differential metabolites in the biosynthesis of secondary metabolites, which was the largest number and accounted for 100% of the total ( Figure 5C ).

[0072] These experiments demonstrate that DFR2 regulates the synthesis and accumulation of flavonoids through a multi-layered mechanism. First, DFR directly catalyzes the conversion of dihydroflavonols to anthocyanin precursors (such as leucoanthocyanidins), driving metabolic flux toward the anthocyanin branch. This results in a significant upregulation of flavonoids in transgenic tobacco, while chalcone production is significantly downregulated due to enhanced activity of the upstream enzyme CHI. Second, DFR influences metabolic flux by regulating transcription factor networks. For example, heterologous expression of GbDFR2 inhibits the expression of transcription factors such as NbMYL2b and NbERF4a, potentially coordinating resource allocation between metabolic branches by repressing genes in the flavonol branch or activating genes involved in anthocyanin synthesis. Furthermore, DFR expression may trigger negative feedback regulation of phenylpropanoid metabolism, such as the inhibition of NbPAL3 and NbFLS1, suggesting that it maintains metabolic homeostasis by balancing precursor supply. The high expression and extracellular localization of GbDFR3 in yellow leaves of Ginkgo biloba suggest that it may participate in environmental stress responses, alleviating oxidative damage by enhancing anthocyanin synthesis.

[0073] In summary, this application reveals the key role of the Ginkgo DFR2 gene in regulating the synthesis of flavonoids, providing valuable genetic resources and theoretical basis for regulating flower color, fruit color and increasing flavonoid production through genetic engineering technology, which is of great significance for the development and utilization of Ginkgo resources and the cultivation of new germplasm with high ornamental and medicinal value.

Claims

1. A Ginkgo biloba GbDRF2 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the Ginkgo biloba GbDRF2 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that The biological material is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell containing the Ginkgo GbDRF2 gene according to claim 1.

4. Use of the Ginkgo GbDRF2 gene according to claim 1, the expressed protein of the Ginkgo GbDRF2 gene according to claim 2, or the biomaterial according to claim 3 in plant breeding.

5. Use of the Ginkgo GbDRF2 gene according to claim 1, the expressed protein of the Ginkgo GbDRF2 gene according to claim 2, or the biomaterial according to claim 3 in regulating the synthesis of plant flavonoids.

6. A method for increasing the synthesis of flavonoids in plants, characterized in that: The Ginkgo GbDRF2 gene of claim 1 is overexpressed in a plant, wherein the flavonoid compound is at least one of the following compounds: cyanidin, kava cyanidin C, diosmin, taxifolin, 3,7-dimethylquercetin, glycyrrhizin isoflavone A, puerarin, sakurain, and genkwain.

7. The method according to claim 6, characterized in that The following steps are involved: (1) constructing an expression vector for the Ginkgo GbDRF2 gene according to claim 1; (2) transforming the constructed Ginkgo GbDRF2 gene expression vector into plants or plant tissues; (3) Cultivating and screening transgenic plants or plant tissues with increased flavonoid content.

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

9. The method according to claim 7, characterized in that The overexpression vector of the Ginkgo GbDRF2 gene is pCAMBIA1302-GbDFR2.

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

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