Ginkgo biloba Gb3GT gene as well as expression protein and application thereof

Abstract

The invention discloses a gingko Gb3GT gene as well as an expression protein and application thereof, and belongs to the field of plant molecular biology. The nucleotide sequence of the ginkgo Gb3GT gene provided by the invention is as shown in SEQ ID NO.1, the amino acid sequence of the encoded protein of the ginkgo Gb3GT gene is as shown in SEQ ID NO.2, and the ginkgo Gb3GT gene belongs to flavonoid 3-O-glycosyltransferase and is a key enzyme for glycosylation modification of ginkgo flavonoid compounds. Genetic transformation experiments of Benjiang tobacco show that the overexpression of Gb3GT significantly up-regulates the expression quantity of key genes NtPAL1, NtCHS1, NtCHI and NtANR1 for flavone synthesis, promotes the accumulation of 19 beneficial flavone metabolites and down-regulates 7 metabolites at the same time, and has the function of regulating and controlling the biosynthesis of flavone compounds. The invention provides an effective theoretical basis and a molecular tool for molecular breeding of ginkgo biloba with high flavone content and utilization of plant flavone components, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant molecular biology, and particularly relates to a function and identification of a Ginkgo biloba L. Gb3GT gene. BACKGROUND

[0002] Ginkgo biloba L., as the oldest gymnosperm existing, originated in the Carboniferous period and survived the Quaternary glaciation in some "refuges" along the Yangtze River in China. It is the oldest plant existing at the same time as dinosaurs among the existing gymnosperms. The rich active ingredients contained in the leaves of Ginkgo biloba L. have extremely high economic value. Flavonoids are one of the most active ingredients for medicinal use, including flavonols, biflavonoids and flavonoid glycosides. The three types of substances with a high proportion are quercetin, kaempferol and isorhamnetin. Ginkgo biloba L. flavonols have multiple biological activities such as antioxidant, anti-inflammatory and anti-tumor activities. Studies have shown that flavonoids can enhance the tolerance of plants to high temperature, pathogens and other stresses by maintaining the active oxygen steady state and inducing disease resistance, and the content of flavonoids is related to the stress resistance of Ginkgo biloba L.

[0003] The biosynthesis of flavonoids begins with the phenylalanine metabolic pathway, and glycosylation modification catalyzed by glycosyltransferase (GT, EC 2.4.x.y) is a common post-processing step. Glycosyltransferase forms glycoside derivatives by transferring sugar groups to flavonoid skeletons, which significantly improves the water solubility and biological activity of the compounds. According to the catalytic mechanism, the glycosyltransferase superfamily can be divided into retention type (R) and inversion type (I), and according to the three-dimensional folding pattern, most glycosyltransferases are mainly classified into GT-A and GT-B families. Existing studies have shown that 3-O-glycosyltransferase plays an important role in plant anthocyanin synthesis and stress response, for example, the expression of heterologous 3GT gene in transgenic petunia can change the flower color, and the accumulation of flavonol glycosides mediated by 3GT in Arabidopsis thaliana can improve the resistance to ultraviolet stress.

[0004] Although the biosynthesis of ginkgo flavonoids is clearly regulated by genes such as chalcone synthase (CHS) and flavonol synthase (FLS), the functional understanding of key glycosylation enzymes—particularly flavonoid 3-O-glycosyltransferase (3GT)—remains largely unexplored. The lack of systematic analysis of the gene function and metabolic regulatory network of ginkgo glycosyltransferases in existing technologies has hindered the development of diverse ginkgo flavonoids and the targeted enhancement of their medicinal activity. Therefore, understanding the molecular structure and mechanism of action of ginkgo glycosyltransferases is crucial for fully exploring the application value of plant flavonoids. 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 Gb3GT gene that expresses flavonoid 3-O-glucosyltransferase (3GT). The second technical problem to be solved by this application is to provide a biomaterial containing the Ginkgo biloba Gb3GT gene. The final technical problem to be solved by this application is to provide a specific application of the Gb3GT gene.

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

[0007] A Ginkgo Gb3GT 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 Gb3GT gene is shown in SEQ ID NO.2.

[0009] A biomaterial, which is an expression cassette, recombinant vector, recombinant bacteria or recombinant cell containing the Ginkgo Gb3GT gene according to claim 1.

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

[0011] In some embodiments, the plant breeding is specifically ginkgo breeding.

[0012] The application of the Ginkgo Gb3GT gene or the expression protein of the Ginkgo Gb3GT gene or the biological material in regulating the synthesis of plant flavonoids.

[0013] A method for increasing the synthesis of flavonoids in plants comprises overexpressing the Ginkgo biloba Gb3GT gene in the plants, wherein the flavonoids are at least one of the following compounds: astragaloside, rutin, narcissin, sakuraside, puerarin, dihydrokaempferol, kaempferol, isosakuraside, 3,7-di-O-methylquercetin, hesperetin, robinin, phloridzin, dihydroquercetin, glycyrrhizin isoflavone A, baicalin, quercetin-7-O-glucoside, (E)-cardamine, formononetin, and dihydromyricetin.

[0014] In some embodiments, the method comprises the following steps:

[0015] (1) constructing an expression vector of the Ginkgo Gb3GT gene;

[0016] (2) transforming the constructed Ginkgo biloba Gb3GT gene expression vector into plants or plant tissues;

[0017] (3) Cultivating and screening transgenic plants or plant tissues with increased flavonoid content.

[0018] In some embodiments, the plant is tobacco or ginkgo.

[0019] In some embodiments, the Ginkgo Gb3GT gene overexpression vector is pCAMBIA1302-Gb3GT.

[0020] In some embodiments, the transformation is mediated by Agrobacterium.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Based on the previous Ginkgo transcriptome data, the applicant screened and cloned the differentially expressed Gb3GT gene (encoding 224 amino acids), and conducted bioinformatics analysis, expression pattern analysis and genetic transformation functional verification of Nicotiana benthamiana. The results showed that the protein encoded by Gb3GT is a hydrophobic alkaline protein, belongs to the GTB superfamily of glycosyltransferases, and contains a UDPGT conserved domain. Phylogenetic analysis showed that Gb3GT is most closely related to the UGT proteins of Siraitia grosvenorii and Nymphaea thermarum. Subcellular localization showed that Gb3GT is localized in the chloroplast. Through genetic transformation of Nicotiana benthamiana, it was found that overexpression of Gb3GT significantly upregulated the expression levels of key flavonoid biosynthesis genes NtPAL1, NtCHS1, NtCHI, and NtANR1, and promoted the accumulation of 19 flavonoid metabolites such as astragalin, rutin and formononetin (most of which have wide application value in the fields of medicine, food and health products), while downregulating 7 metabolites such as cynaroside and quercitrin. This study, published for the first time, discloses the Gb3GT gene and its expressed protein from Ginkgo biloba and reveals its regulatory role in the biosynthesis of Ginkgo flavonoids. This discovery has important technical significance for enhancing the industrial utilization value of flavonoid compounds and provides a theoretical basis and molecular tools for molecular breeding of high-flavonoid varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A This is the phylogenetic tree diagram of Gb3GT;

[0024] Figure 1B This is a comparison diagram of the motif analysis of Gb3GT protein;

[0025] Figure 2 Figure 3 is a heat map of 3GT gene expression in different tissues and under different treatments and its subcellular localization analysis. A is a heat map of 3GT gene expression in different tissues and under different treatments. B is a subcellular localization analysis of 3GT gene, including fluorescence channel, chloroplast channel, bright field, and overlay from left to right. The scale bar in the figure is 10 μm.

[0026] Figure 3 Figure 1 is a diagram showing the growth process of Nicotiana benthamiana genetically transformed with Gb3GT and an analysis diagram of the expression pattern of Gb3GT (B), wherein A is a diagram showing the growth process of Nicotiana benthamiana genetically transformed with Gb3GT, and B is an analysis diagram of the expression pattern of Gb3GT in Nicotiana benthamiana genetically transformed with Gb3GT;

[0027] Figure 4AThis is the expression analysis diagram of flavonoid synthesis-related genes NbPAL1, NbPAL3, NbCHI, NbFLS1, NbANR1, NbANR2, NbbHLH1, and NbbHLH2 in Nicotiana benthamiana leaves after Gb3GT genetic transformation;

[0028] Figure 4B This is the expression analysis diagram of flavonoid synthesis-related genes NbC4H, NbHCT, NbFLS2, NbDFR, NbUFGT, NbMYL2a, NbbHLH3, and NbERF4a in leaves of Nicotiana benthamiana after genetic transformation with Gb3GT;

[0029] Figure 4C This is the expression analysis diagram of flavonoid biosynthesis-related genes NbCHS1, NbMYB1, NbLAR, NbMYB4, NbMYL2b, and NbERF4b in Nicotiana benthamiana leaves after Gb3GT genetic transformation;

[0030] Figure 5A This is a violin plot of differential metabolites (14 of them) in leaves of Nicotiana benthamiana after genetic transformation with Gb3GT;

[0031] Figure 5B This is a violin plot of differential metabolites (12 of which) in leaves of Nicotiana benthamiana after genetic transformation with Gb3GT;

[0032] Figure 5C This is a bar graph showing the top 12 differential metabolites between leaves of Gb3GT-transformed Nicotiana benthamiana and the control. The horizontal axis represents the log2FC of the differential metabolites, i.e., the logarithm of the fold difference of the differential metabolites with base 2, and the vertical axis represents the differential metabolites. Red represents up-regulated differentially expressed metabolites, and green represents down-regulated differentially expressed metabolites.

[0033] Figure 5D This is a KEGG enrichment analysis diagram of differential metabolites in leaves of Nicotiana benthamiana after Gb3GT genetic transformation; the horizontal axis in the figure represents the richness factor (Rich factor) corresponding to each pathway, the vertical axis is the pathway name, the color of the point is the P-Value, the redder the better the enrichment, and the size of the point represents the number of enriched differential metabolites. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0035] Example 1: Identification, cloning and expression analysis of the Ginkgo biloba Gb3GT gene

[0036] Based on the existing transcriptome data of Ginkgo biloba leaves, the sequence of the significantly differentially expressed 3GT gene (evm.TU.chr7.1785) was screened out, and its open reading frame (ORF) was obtained by BioXM 2.6 software analysis. The primers were designed using Oligo 6.0 software (Table 1), and the total RNA was extracted from Ginkgo biloba leaves (from Ginkgo biloba trees in the campus of Nanjing Forestry University). The ORF sequence was cloned using high-fidelity PCR enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, Dalian, China). The 50 μL PCR reaction system was as follows: Primer Star Max 25 μL, 1 μL of forward and reverse primers, 1 μL of cDNA template, and 22 μL of ddH2O. The PCR reaction program was as follows: 98 ℃ for 3 min; 98 ℃ for 10 s, 55 ℃ for 5 s, and 72 ℃ for 15 s, for 35 cycles; 72 ℃ for 3 min, and 4 ℃ for permanent preservation. The amplified product was connected with the vector according to the requirements of pClone007 Blunt Vector Kit (TSINGKE, Nanjing, China), and was transformed into E. coli competent cells. After 6-8 h of shaking culture at 250 rpm and 37 ℃, the white bacterial solution was verified by bacterial solution PCR using 2×T5 Super PCR Mix (Colony) (TSINGKE, Nanjing, China). The positive bacterial solution was sent to Nanjing Tsingke Biological Technology Co., Ltd. for first-generation Sanger sequencing verification.

[0037] Table 1 Primer sequence information

[0038]

[0039] Through cloning and sequencing, the open reading frame (ORF) sequence of the Ginkgo biloba 3GT gene (Gb3GT) was obtained, which was 675 bp in length, as shown in SEQ ID NO. 1. The termination codon was TGA, and the encoded polypeptide consisted of 224 amino acids, as shown in SEQ ID NO. 2.

[0040] The physicochemical properties of the protein were analyzed using ProtParam software, the transmembrane domain of the protein was predicted using the online tool of NovoPro (https: / / www.novopro.cn / tools / tmhmm.html), the protein domain was analyzed using the conserved domain analysis tool of NCBI (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow) and the online network tool SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?GENOMIC=1). The secondary structure of the protein was analyzed using NPSA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_seccons.html) in combination with MLRCon GOR4, SIMPA96 and SOPMA, DSC and PHD, the three-dimensional model of the protein was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive), the protein sequence alignment was performed using MEGAX software and the phylogenetic tree was constructed using the Neighbour-joining Bootstrap resampling method with 1000 times.

[0041] The physicochemical property analysis of the protein showed that the molecular weight of Gb3GT was 25.35551 kDa, and the theoretical isoelectric point was 9.08. Among them, the total number of negatively charged residues (Asp+Glu) in the protein was 15, the total number of positively charged residues (Arg+Lys) was 21, the fatty coefficient was 84.42, and the instability index was 40.57. The overall hydrophilicity value of Gb3GT protein was -0.102, it was speculated that the protein was a hydrophobic basic protein, the transmembrane domain prediction result of the protein showed that 3GT did not have a transmembrane domain. The domain analysis showed that Gb3GT belonged to Glycosyltransferase_GTB-type superfamily and contained the specific UDPGT domain of the family. The main components of Gb3GT folded protein were α-helix, extended strand and random coil structure. The three-dimensional model of the protein was constructed using SWISS-MODEL, and it was found that the homology of Gb3GT was the highest, and the template number was 6jem.1.A. The template corresponded to UGT2 protein of Phytolacca americana, the model coverage reached 65%, and the model similarity was 37%.

[0042] As Figure 1AAs shown in the figure, the phylogenetic tree analysis of the protein showed that the Ginkgo 3GT protein was closely related to the UDP-glucosyltransferase protein (AEM43002.1) of Siraitia grosvenorii of the Cucurbitaceae family and the β-D-glucosylcrocin β-1-6-glucosyltransferase (KAF3795419.1) of Nymphaea thermarum of the Nymphaeaceae family. It was also closely related to the UDP-glucosyltransferases (KAK4425798.1, XP_009609569.1, XP_059669660.1) of Sesamum alatum of the Sesamaceae family, Nicotiana atomtomentosiformis of the Solanaceae family, Cornus florida of the Cornaceae family, and Olea europaea of ​​the Oleaceae family. europaeasubsp.europaea) is more distantly related to the β-D-glucosylcrocin β-1-6-glucosyltransferase (CAA3031259.1). Motif analysis showed that the Gb3GT protein had a partial domain deletion and contained only two conserved motifs ( Figure 1B ).

[0043] Example 2: Vector Construction of Ginkgo Gb3GT Gene, Genetic Transformation and Detection of Nicotiana benthamiana

[0044] Transient expression vectors Pan580-Enhancer esp3l(D)-Gb3GT (the target gene is fused to the N-terminal of the fluorescent protein) and overexpression vector pCAMBIA1302 (pCAMBIA1302-Gb3GT) containing a strong 35S promoter were constructed by homologous recombination and golden gate seamless cloning methods. The plasmid DNA was transformed into tobacco protoplasts using PEG-mediated method, and the protoplast transformation step was referred to the method of Yoo et al. (2007). The Gb3GT localization results were observed under the laser confocal microscope Nikon C2-ER instrument, and the chlorophyll fluorescence parameters were: chloroplast excitation light 640 nm, emission light 510 nm; the green fluorescent protein GFP parameters were: excitation light 488, emission light 510. The overexpression vector was verified by sequencing whether it contained the target fragment, and GV3101 Agrobacterium strain was used for transformation by heat shock method, and leaf disc method was used to infect N. benthamiana, and the transgenic lines were identified by PCR and the expression level of RNA was detected (the method was referred to: Yoo Sang-Dong, Cho Young-Hee & Sheen Jen. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nature Protocols 2, 1565-1572 (2007).).

[0045] (1) Expression level of Gb3GT in different tissue parts and under different treatments and subcellular localization analysis

[0046] Based on the obtained transcriptome data, the expression pattern of Gb3GT gene in different tissue parts was explored Figure 2 A), and the results showed that the expression amount of Gb3GT gene in the cambium part was the highest, followed by the ovulate strobilus part. In addition, the effect of different treatments on the expression of Gb3GT gene was investigated, and the results showed that the expression amount of Gb3GT was higher when using SA treatment, on the contrary, the expression amount of 3GT gene was relatively lower when using ABA treatment. Different concentrations of BR treatment showed great difference in the expression amount of Gb3GT gene. By constructing transient expression vector to explore the subcellular localization of Ginkgo biloba Gb3GT gene, based on the observation and analysis of tobacco protoplasts by laser confocal microscope, it was found that Ginkgo biloba Gb3GT might be located in chloroplast Figure 2 B).

[0047] (2) Detection of the expression amount of Ginkgo 3GT in genetically transformed N. benthamiana

[0048] A plant overexpression vector was constructed and genetic engineering technology was used to further stably transform the Ginkgo 3GT gene into Nicotiana benthamiana. A group of potential positive transgenic tobacco plants were obtained by leaf disc method with kanamycin as the selection pressure ( Figure 3 By randomly testing six robust transgenic Nicotiana benthamiana strains, it was found that 3GT was efficiently overexpressed in all four strains ( Figure 3 (B) Three transgenic Nicotiana benthamiana lines, 3GT2, 3GT3, and 3GT5, with expression levels exceeding 2000-fold, were selected as the focus of subsequent experiments to test their targeted flavonoids and expression levels.

[0049] (3) Analysis of expression levels of key genes for flavonoid synthesis in 3GT-genetic Nicotiana benthamiana

[0050] NbCHS1, NbMYB1, NbLAR, NbMYB4, NbMYL2b, NbERF4b

[0051] In order to further understand the effect of Gb3GT heterologous overexpression on the expression levels of key enzyme genes (NbPAL1, NbPAL3, NbC4H, NbHCT, NbCHS1, NbCHI, NbFLS1, NbFLS2, NbDFR, NbLAR, NbANR1, NbANR2 and NbUFGT) in the Nicotiana benthamiana flavonoid biosynthesis pathway, real-time fluorescence quantitative PCR amplification of the target genes was performed using the relevant primer sequences in Table 1. The results are shown in Figure 1. Figure 4A 、 Figure 4B 、 Figure 4C As shown, compared with the control group, the expression levels of the NbPAL1, NbCHS1, NbCHI, and NbANR1 genes in transgenic Nicotiana benthamiana plants were significantly increased, by 3.5-fold, 4.1-fold, 2.8-fold, and 2.3-fold, respectively. In contrast, the expression levels of the key enzyme genes NbHCT and NbFLS2 were significantly decreased compared with the control group, falling to 40% and 55% of the control group, respectively. Analysis of the expression of related transcription factors in tobacco revealed that the expression level of NbERF4a was significantly decreased compared with the control group, while the expression levels of NbMYL2a, NbbHLH2, and NbERF4b were significantly increased.

[0052] Example 3: Detection of flavonoid content in tobacco transformed with the Ginkgo Gb3GT gene

[0053] (1) Sample processing

[0054] Transgenic 3GT tobacco leaves and control Nicotiana benthamiana leaves of the same period were freeze-dried and ground into powder using a ball mill (30 Hz, 1.5 min). 20 mg of the powder was weighed and added with 10 μL of 4000 nmol / L internal standard mixed working solution and 500 μL of 70% methanol solution. The mixture was sonicated for 30 min and centrifuged at 4°C (12,000 rpm for 5 min). The supernatant was aspirated and the sample was filtered through a 0.22 μm filter membrane and stored in an injection vial for LC-MS / MS analysis.

[0055] (2) Chromatographic mass spectrometry acquisition conditions

[0056] The data acquisition instrument system mainly includes ultra-high performance liquid chromatography (UPLC) (ExionLC AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (MS / MS) (QTRAP 6500+, https: / / sciex.com.cn / ).

[0057] Liquid chromatography conditions included the following: 1) Chromatographic 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.

[0058] Mass spectrometry conditions included an electrospray ionization (ESI) source temperature of 550°C, a mass spectrometer voltage of 5500 V in positive ion mode, a mass spectrometer voltage of -4500 V in negative ion mode, and a curtain gas (CUR) pressure of 35 psi. In a Q-Trap 6500+, each ion transition was scanned based on optimized declustering potential (DP) and collision energy (CE).

[0059] (3) Differential metabolite screening and KEGG enrichment analysis of differential metabolites

[0060] Based on the variable importance projection (VIP) obtained from the OPLS-DA model, metabolites with a VIP > 1 were selected to initially identify metabolites with significant differences between groups. Metabolites with a fold change ≥ 2 and a fold change ≤ 0.5 were also selected to further identify significantly different metabolites. Metabolites were annotated using the KEGG database (http: / / www.kegg.jp / kegg / compound / ), and the KEGG annotation results for significantly different metabolites were classified according to the KEGG pathway type (http: / / www.kegg.jp / kegg / pathway.html).

[0061] (4) Analysis results of flavonoid content in transgenic plants

[0062] After the Gb3GT gene was genetically transformed into tobacco, the metabolites in its leaves were detected. After qualitative and quantitative evaluation, a total of 60 flavonoid metabolites were detected, belonging to ten categories. Flavonols contained three metabolites, namely dihydrokaempferol, dihydroquercetin, and dihydromyricetin. Flavonols contained astragalin, rutin, quercitrin, baimaside, kaempferol, 3,7-di-O-methylquercetin (3, The flavonoids contain seven metabolites, namely cynaroside, narcissin, sakuranetin, jaceosidin, scutellarin, and wogonoside. The isoflavonols contain four metabolites, namely puerarin, 6-O-acetylglycitin, licoisoflavone A, and formononetin. The chalcones contain two metabolites, namely xanthohumol and phlorizin. The phenonic acids contain one metabolite, namely (E)-Cardamonin. Among them, there are 26 differential metabolites ( Figure 5A ), in order to intuitively show the distribution of data and its probability density characteristics, a violin plot was drawn to show that compared with the CK group, the specific up-regulated differential metabolites were astragaloside, rutin, narcissin, sakuratin, puerarin, dihydrokaempferol, kaempferol, isosakuratin, 3,7-di-O-methylquercetin, hesperetin, acaciatin, phloridzin, dihydroquercetin, glycyrrhizin A, baicalin, quercetin-7-O-glucoside, (E)-cardamomine, formononetin and dihydromyricetin, these 19 were found. The seven down-regulated metabolites were luteolin, quercetin, cyperibotrysin, xanthohumol, 6"-O-acetyl glycitin, baicalin and (-)-epicatechin. Among the down-regulated flavonoids, luteolin had the most significant differential expression, with a variable importance projection value of 1.08 and a difference fold value of 0.23 ( Figure 5A 、 Figure 5B ).

[0063] In order to further compare the fold changes of metabolite quantitative information in the transgenic and non-transgenic groups, a bar chart was made showing the fold changes of the top 12 metabolites. It was found that 3,7-di-O-methylquercetin had the largest fold change among the up-regulated metabolites, with a VIP value of 1.09 and a log2Fold Change value of 1.96. The second largest flavonol substance was astragalin, with a VIP value of 1.04 and a log2Fold Change of 1.66 ( Figure 5C Further KEGG enrichment analysis based on the results of differential metabolites revealed that the most significantly enriched 12 differential metabolites were found in the biosynthesis of secondary metabolites (the largest number, accounting for 80% of the total), followed by 9 differential metabolites in the flavonoid biosynthesis pathway, namely sakurain, xanthohumol, dihydrokaempferol, isosakurain, phloridzin, dihydromyricetin, quercetin, (-)-epicatechin, and kaempferol. There are 6 differential metabolites in the flavonoid and flavonol biosynthesis pathway, including kaempferol, astragaloside, rutin, quercetin, cyperiside, and 3,7-di-O-methylquercetin. There is 1 differential metabolite, formononetin, in the isoflavonoid biosynthesis. The content of 3GT in this sample is 0.03 nmol / g, while that in the CK group is 0 nmol / g. Figure 5D ).

[0064] The results of the above examples indicate that Gb3GT encodes an enzyme belonging to the GTB-type glycosyltransferase superfamily, localized to the chloroplast, and may be involved in the regulation of chloroplast-related secondary metabolism. This enzyme specifically promotes the accumulation of specific flavonoid glycosides by positively regulating upstream genes for flavonoid synthesis and inhibiting competing genes in branched pathways, thus regulating metabolic flux toward specific flavonoid derivatives. This application not only elucidates the molecular mechanism of Gb3GT-mediated flavonoid diversification in Ginkgo biloba but also provides a powerful molecular tool for metabolic engineering of high-flavonoid plants.

Claims

1. A Ginkgo biloba Gb3GT gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the Ginkgo biloba Gb3GT 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 Gb3GT gene according to claim 1.

4. Use of the Ginkgo Gb3GT gene according to claim 1, the expressed protein of the Ginkgo Gb3GT gene according to claim 2, or the biomaterial according to claim 3 in plant breeding.

5. Use of the Ginkgo Gb3GT gene according to claim 1, the expressed protein of the Ginkgo Gb3GT 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 Gb3GT gene according to claim 1 is overexpressed in a plant, wherein the flavonoid compound is at least one of the following compounds: astragaloside, rutin, narcissin, sakuraside, puerarin, dihydrokaempferol, kaempferol, isosakuraside, 3,7-di-O-methylquercetin, hesperetin, robinin, phloridzin, dihydroquercetin, glycyrrhizin isoflavone A, baicalin, quercetin-7-O-glucoside, (E)-cardamine, formononetin and dihydromyricetin.

7. The method according to claim 6, characterized in that The following steps are involved: (1) constructing an expression vector for the Ginkgo Gb3GT gene according to claim 1; (2) transforming the constructed Ginkgo biloba Gb3GT 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 Gb3GT gene is pCAMBIA1302-Gb3GT.

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

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