Ginkgo biloba gbC4H1 gene, its encoded protein and application

Abstract

The application discloses a ginkgo biloba L. GbC4H1 gene, a coding protein and application thereof, and relates to the technical field of plant genetic engineering.The ginkgo biloba L. GbC4H1 gene disclosed by the application has a nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the coding protein is as shown in SEQ ID NO.2.The expression vector of the ginkgo biloba L. GbC4H1 gene is constructed and transformed into Nicotiana benthamiana; and a transgenic plant with increased flavonoid metabolite content is obtained through cultivation and screening.Compared with a control group, the expression level of a transcription factor of a flavone synthesis pathway related gene and a flavone synthesis pathway structural gene in the transgenic strain is significantly increased.The flavonoid metabolite content in the transgenic strain is increased, and compared with the control group, 14 kinds of differential metabolites are up-regulated and 20 kinds of differential metabolites are down-regulated.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the Ginkgo GbC4H1 gene and its encoded protein and its applications. Background Technology

[0002] Ginkgo biloba is the only extant species in the Ginkgoaceae family, a world-renowned relict gymnosperm, and is hailed as a "living fossil" with significant scientific research value. Ginkgo leaves are rich in flavonoids, ginkgolides, and other compounds, possessing multiple effects including antioxidant activity, anti-platelet-activating factor activity, regulation of vasoactive activity, and protection of the nervous system. Ginkgo leaf extract has been clinically used to treat various diseases, including cardiovascular diseases, nervous system disorders, and hyperlipidemia, achieving significant therapeutic effects.

[0003] Cinnamate 4-hydroxylase (C4H) belongs to the cytochrome P450 enzyme family and affects the synthesis and metabolism of substances such as flavonoids, lignin, and alkaloids. In plant secondary metabolism, the phenylpropanoid pathway plays a crucial role in plant growth, development, and the synthesis of secondary metabolites. This pathway is generally believed to promote the formation of flavonols and anthocyanins through a series of biological reactions. This process requires the synergistic catalysis of multiple enzymes, including phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL). C4H, as one of the key enzymes in this pathway, catalyzes the hydroxylation of cinnamate to produce 4-coumarate, and is the second key enzyme in the phenylpropanoid pathway after PAL. Studies have found that the expression levels of phenylpropane metabolites and their associated C4H genes play a crucial role in the formation of plant color, aroma, and fruit texture. Furthermore, increasing the expression levels of C4H-related genes can effectively enhance plant resistance. In research on black tea processing, the expression level of the C4H gene is also closely related to tea quality.

[0004] Flavonoids are an important class of plant secondary metabolites, possessing various pharmacological functions such as antioxidation, anticancer and antitumor activity, anti-aging, and protection of the cardiovascular system. C4H, as a key enzyme in the phenylpropane metabolic pathway, also participates in the synthesis of flavonoids. By catalyzing the conversion of cinnamic acid to p-coumaric acid, C4H provides precursors for the synthesis of flavonoids, thus affecting the biosynthesis and accumulation of flavonoids (Liang Liang, Han Xiaomin, Zhang Zheng, et al. Cloning and expression analysis of cinnamic acid-4-hydroxylase (C4H) gene in Aucklandia lappa [J]. Chinese Journal of Traditional Chinese Medicine, 2014, 39(10): 1767-1771.). When plants encounter external stimuli such as ultraviolet radiation, physical mechanical damage, or fungal infection and induction, their defense mechanisms are activated, which in turn induces the expression of the C4H gene, increasing its expression level and promoting the production of secondary metabolites such as flavonoids. The accumulation of these secondary metabolites is crucial for plants to defend against damage from adverse external factors (Lee SS, Lee EM, An BC, et al. Molecular Cloning and Characterization of Cinnamate-4Hydroxylase Gene from Rubus coreanus[J].The Open Plant ScienceJournal, 2008, 2(1):31-36.DOI:10.2174 / 1874294700802010031.). Furthermore, in Arabidopsis mutants, inactivation of the protein encoded by the C4H gene leads to growth restriction and abnormal stamen function, as well as a significant decrease in flavonoid levels (Schilmiller AL, Stout J, Weng JK, et al. Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis.[J]. Plant Journal, 2010, 60(5):771-782.DOI:10.1111 / j.1365-313X.2009.03996.x.). This indicates that the C4H gene has a certain influence on flavonoid synthesis and accumulation under different environmental conditions. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the Ginkgo GbC4H1 gene. Another technical problem this invention aims to solve is to provide the protein encoded by the Ginkgo GbC4H1 gene. A further technical problem this invention aims to solve is to provide applications for the Ginkgo GbC4H1 gene in regulating the synthesis and accumulation of flavonoids in plants under different nitrogen forms.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A Ginkgo biloba GbC4H1 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0008] The amino acid sequence of the protein encoded by the Ginkgo GbC4H1 gene is shown in SEQ ID NO. 2.

[0009] Vectors and recombinant bacteria containing the Ginkgo GbC4H1 gene.

[0010] Application of Ginkgo GbC4H1 gene in regulating the expression of genes related to the flavonoid synthesis pathway.

[0011] Specifically, the expression levels of genes related to the flavonoid synthesis pathway, namely NtF3H, NtFLS1, NtFLS2, NtPAL1, NtCHI, NtCHS1, NtLAR, and NtDFR, were significantly increased.

[0012] Application of Ginkgo GbC4H1 gene in regulating the expression of transcription factors that regulate structural genes in the flavonoid synthesis pathway.

[0013] Specifically, the expression levels of transcription factors NtMYL2a, NtMYL2b, NtbHLH3, and NtERF4b, which regulate the structural genes of the flavonoid synthesis pathway, were significantly increased.

[0014] Application of the Ginkgo GbC4H1 gene in regulating the content of flavonoid metabolites in plants.

[0015] Specifically, it promotes an increase in the content of flavonoid metabolites in plants.

[0016] Application of the Ginkgo biloba GbC4H1 gene in regulating differential flavonoid metabolites in plants.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1) The present invention cloned the Ginkgo GbC4H1 gene from Ginkgo leaves, the nucleotide sequence of which is shown in SEQ ID NO. 1 and the amino acid sequence of which is shown in SEQ ID NO. 2.

[0019] 2) In this invention, the GbC4H1 tobacco Benedict line was constructed. Compared with the control group, the expression levels of NtCHI, NtCHS1 and NtDFR genes in the transgenic line were significantly increased, while the expression levels of NtMYL2a and NtMYL2b genes among transcription factors were significantly increased; the expression level of the key enzyme gene NtANR1 was significantly decreased.

[0020] 3) This invention constructed the GbC4H1 tobacco Benedict strain and qualitatively and quantitatively analyzed its flavonoid metabolites. The results showed that, compared with the control group, 14 differentially expressed metabolites were upregulated and 20 differentially expressed metabolites were downregulated. Overexpression of GbC4H1 significantly increased the content of syringaldehyde and baimaside in transgenic plants. Attached Figure Description

[0021] Figure 1 The diagram shows the protein properties of five Ginkgo C4H genes (A represents the transmembrane domain of the encoded protein, B and C represent conserved domains, D represents the predicted secondary structure of the encoded protein, and E represents the predicted tertiary structure of the encoded protein).

[0022] Figure 2 Phylogenetic diagram (A) and motif analysis diagram (B) of five Ginkgo C4Hs genes;

[0023] Figure 3 A diagram showing the expression patterns of five C4Hs genes in different tissue sites and under different nitrogen species.

[0024] Figure 4 The process of stable overexpression of GbC4H1 in tobacco and the analysis of expression levels (A is the process of genetic transformation of GbC4H1 gene into tobacco, B is the expression level of GbC4H1 gene in tobacco).

[0025] Figure 5 This is a diagram showing the expression levels of key enzyme genes and important transcription factors in the flavonoid synthesis pathway in transgenic Nicotiana benthamiana.

[0026] Figure 6 The diagram shows a violin plot of differential metabolites (A, where the horizontal axis represents the group and the vertical axis represents the expression level) and a KEGG classification plot of differential metabolites (B, where the vertical axis indicates the name of the KEGG metabolic pathway, the number represents the number of differential metabolites annotated in that pathway, and the number in parentheses shows the proportion of differential metabolites annotated in that pathway to the total number of differential metabolites annotated in KEGG). Detailed Implementation

[0027] 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. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0028] The plant material used in this application was 3-year-old bare-root Ginkgo seedlings, planted at the Xiashu Forest Farm of Nanjing Forestry University. The experiment was conducted in July, when Ginkgo is in its vigorous growth phase, using a completely randomized experimental design with 7 different nitrogen forms (5 inorganic nitrogen treatments, NH4+, and 44+). + NO3 - =100:0, 75:25, 50:50, 25:75 and 0:100, NO3 - Provided by potassium nitrate, NH4 + Ammonium sulfate was used as the nitrogen source; two organic nitrogen treatments (urea and glycine) and a blank control were included. The application rate was based on the nitrogen application rate, with a total of eight treatments (15 plants per treatment, 5 plants per biological replicate). The nitrogen application rate was 5.55 g / plant. All treatments received nitrogen fertilizer in three applications, 10 days apart. Each application consisted of 1 / 3 of the fertilizer dissolved in 200 mL of tap water and poured into the pot. The control group received 200 mL of tap water. The fertilizer solution was kept from overflowing. Simultaneously, a nitrification inhibitor, DCD (7% of the nitrogen application rate, 0.3885 g / plant), was applied to reduce the loss of nitrate nitrogen. To ensure K... + The supply was consistent, and all treatments were supplemented with KCl, which was applied together with nitrogen fertilizer. The date of completion of application was the start date of the treatment. The specific experimental design is shown in Table 1.

[0029] Table 1 Experimental Design

[0030]

[0031] Note: The table shows the total amount of 15 seedlings used for each treatment, in grams.

[0032] Example 1

[0033] 1. Cloning of the Ginkgo biloba GbC4H1 gene

[0034] Total RNA was extracted from seven groups of nitrogen-treated Ginkgo biloba leaves using a universal plant total RNA rapid extraction kit (BioTeKe). The specific method was performed according to the manufacturer's instructions. Then, TaKaRa's PrimeScript was used to extract the RNA. TMcDNA was synthesized via reverse transcription using the RT MasterMix (Perfect Real Time) kit. Based on differentially expressed C4H1 sequences screened from Ginkgo transcriptome data, open reading frame (ORF) primers were designed using Oligo 6.0 software (Table 2). The high-fidelity PCR enzyme PrimeSTAR from Takara was then used. ® Max DNA Polymerase clones its ORF sequence.

[0035] 50 μL PCR reaction system: Primer Star Max 25 μL, front and rear primers 1 μL each, cDNA template 1 μL, ddH2O 22 μL.

[0036] PCR reaction program: 98 ℃ for 3 min; 98 ℃ for 10 s, 55 ℃ for 5 s, 72 ℃ for 15 s, 35 cycles; 72 ℃ for 3 min, then incubate at 8 ℃.

[0037] Table 2 Primer information for Ginkgo C4Hs gene

[0038]

[0039] The PCR product of the C4H1 gene was detected by 1% agarose gel electrophoresis. The band that matched the predicted length of the target gene amplification product was excised and purified using the BioTeKe Rapid Agarose Gel DNA Recovery Kit. The purified product was ligated to a vector according to the instructions of the pClone007 Blunt Vector Kit (TSINGKE), and transformed into competent E. coli cells. After a short recovery period, the cells were plated on ampicillin-resistant medium and cultured overnight. Single colonies were randomly selected and cultured using 2×T5 Super PCR Mix (Colony) (TSINGKE) for PCR verification. Positive colonies were sent to CT Biotech Co., Ltd. for sequencing verification.

[0040] 20 μL bacterial culture PCR system: 10 μL 2×T5 Super PCR Mix, 0.75 μL universal primers M13-F, 0.75 μL M13-R, 1.5 μL bacterial culture, and 6.5 μL ddH2O.

[0041] PCR reaction program: 98 ℃ for 2 min; 98 ℃ for 10 s, 55 ℃ for 10 s, 72 ℃ for 15 s, 35 cycles; 72 ℃ for 2 min, then incubate at 8 ℃.

[0042] Positive clones were sent to Qingke Biotechnology for sequencing. The final sequencing yielded the ORF nucleotide sequences of the GbC4H1, GbC4H2, GbC4H3, GbC4H4, and GbC4H5 genes, as shown in SEQ ID NO. 1-5, and the amino acid sequences of their encoded proteins, as shown in SEQ ID NO. 6-10, respectively.

[0043] 2. Identification and bioinformatics analysis of differentially expressed genes C4Hs in Ginkgo biloba

[0044] The physicochemical properties of C4H proteins were analyzed using the ProtParam online tool. The online tool ProtScale (https: / / web.expasy.org / protscale / ) was used to predict the hydrophilicity and hydrophobicity of Ginkgo C4H proteins and to plot their hydrophilicity and hydrophobicity maps. Novopro online tools (https: / / www.novopro.cn / tools / tmhmm.html), SMART online tool (http: / / smart.embl-heidelberg.de / ), and the NCBI online conserved domain retrieval tool CDART (Conserved Domain Architecture Retrieval) were used. The protein's domains, including transmembrane domains, functional domains, and conserved domains, were analyzed using the Softberry Tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow). Subcellular localization of the C4H protein was predicted using the Softberry online website (http: / / www.softberry.com / ). Its secondary structure was analyzed using the SOPMA online tool (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html). A three-dimensional model was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / ) for validation. A phylogenetic tree was constructed using MEGAX software with a 1000-run neighbor-joining bootstrap test.

[0045] The results are shown in Table 3. Based on the transcriptome data of Ginkgo biloba leaves under different nitrogen forms, key enzyme genes in the phenylalanine synthesis pathway were identified and extracted. For the C4H enzyme gene, which had the largest number of differentially expressed genes, differentially expressed genes were extracted from all comparison groups except the control (CK) group under different nitrogen forms. A total of five genes were obtained and named C4H1, C4H2, C4H3, C4H4, and C4H5 according to their chromosomal order. All five proteins are hydrophobic basic proteins. The amino acid sequences of C4H2, C4H3, C4H4, and C4H5 proteins are identical, and their encoding genes are multi-copy genes. The similarity in physicochemical properties indicates that a few mutations occurred during evolution, but the structure remained unchanged. Subcellular localization prediction results showed that C4H2, C4H3, and C4H5 may be located in both the cytoplasm and mitochondria, C4H1 is located only in the mitochondria, and C4H4 is located only in the cytoplasm.

[0046] Table 3 Physicochemical properties and subcellular localization prediction of proteins encoded by structural genes

[0047]

[0048] The results are as follows Figure 1 As shown, C4H1, C4H2, C4H3, C4H4, and C4H5 all possess transmembrane domains, and each has only one such domain. A small number of amino acids are located intracellularly, while the majority are located extracellularly. Figure 1 A). The conserved domain analysis tool from NCBI and the online tool SMART were used to identify protein domains. C4H1, C4H2, C4H3, C4H4, and C4H5 all belong to the CYP450 family and possess a transmembrane region and an NmrA domain. Figure 1 B and C). Using NPSA, a combined analysis of MLRC on GOR4, SIMPA96 and SOPMA, DSC, and PHD methods revealed that the protein's secondary structure was mainly composed of α-helices, extended strands, random coils, and some temporarily unidentified structures. Figure 1 D). Among the five C4H proteins, α-helices were the most prevalent, while random coils were more common in the remaining proteins. Using SWISS-MODEL to construct 3D models of the proteins, the template number with the highest homology for C4H1 was Q9XEH8.1.A, which is the TC4H protein from *Pinus taeda* (Loblolly pine), with a model coverage of 95% and a model similarity of 52%. The template number with the highest homology for C4H2, C4H3, C4H4, and C4H5 was 6vby.1.A, which is the SbC4H1 from *Sorghum bicolor*, with a model coverage of 90% and a model similarity of 51% for all of them. Figure 1E).

[0049] The results are as follows Figure 2 As shown, Ginkgo C4H2 and C4H5 clustered in one branch, while C4H3 and C4H4 clustered in another. These proteins are closely related to the CYP72A171 protein (ATG29969.1) of *Taxus chinensis* (Taxus chinensis), the C4H protein (USH99603.1) of *Cephalotaxus hainanensis* (Cephalotaxus hainanensis), and the C4H2 protein (XP_057839147.2) of *Cryptomeria japonica* (Cryptomeria japonica). Figure 2 A). C4H1 was separated into its own branch. Motif analysis showed that C4H1 had severe sequence deletions, losing most of its motif, indicating that its driving function is somewhat different. C4H1 was also the gene with the most differential expression pattern among different nitrogen species comparison groups, suggesting that it may play a role in regulating flavonoid synthesis and accumulation. Figure 2 B).

[0050] 2. Expression patterns of five C4Hs genes in different tissue sites and under different nitrogen forms

[0051] The results are as follows Figure 3 As shown, all five C4Hs were expressed at the highest levels in AN2 or AN3. Among different tissues, C4H1 was expressed at the highest level in male cones, C4H2 at the highest level in stems, and C4H3, C4H4, and C4H5 were expressed at the highest levels in yellow leaves.

[0052] Example 2

[0053] 1. Constructing a stable overexpression vector and stably genetically transforming Nicotiana benthamiana.

[0054] Add 1 µL of plasmid to 50 µL of GV3101 Agrobacterium competent cells, mix thoroughly, and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 28°C and 180 rpm for 30 min on a shaker. Inoculate 50 µL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 28°C for 48 h. Synthesize the corresponding detection primers and select samples with clear and correctly sized electrophoretic bands from the PCR amplification results for genetic transformation of Nicotiana benthamiana.

[0055] Sterile tobacco leaves were cut into small pieces with a scalpel and inoculated onto a pre-culture medium. Agrobacterium was picked and placed in the infection solution to prepare OD. 600Inoculate tobacco leaves pre-cultured for 2-3 days with a 0.2% Agrobacterium suspension for 10-15 minutes. After inoculation, transfer the infected tobacco leaves onto filter paper, air-dry them, and then onto a co-culture medium. Incubate in the dark for 48-72 hours. After 2 days of co-culture, transfer the leaves to an induction medium to induce callus growth. After approximately 10 days, callus tissue will develop. Select well-growing callus tissue and inoculate it onto a KAN-resistant selection medium. Culture for 15-30 days at 23±2℃. Inoculate 4-5 vigorous positive callus tissues per plate onto a differentiation medium at 23℃ under 16h / 8h light / dark conditions for 15-30 days. If seedlings form during differentiation, inoculate them onto a seedling growth medium and allow them to grow for 7-10 days. Tobacco genomic DNA was extracted using the CTAB method and detected by PCR. At the same time, RNA was extracted from potential positive seedlings, reverse transcribed into cDNA, and its expression level in different strains was detected by RT-qPCR.

[0056] 2. Real-time quantitative PCR analysis

[0057] Total RNA was extracted from leaves using an RNA extraction kit (Beijing Biotech Biotechnology Co., Ltd.), and then transcribed using a cDNA synthesis kit (Shanghai Pudi Biotechnology Co., Ltd.). RT-qPCR reactions were performed using a qTOWER 2.2 qPCR instrument. The program was as follows: starting with a denaturation step at 95℃ for 3 min, followed by 40 cycles of 95℃ for 10 s, 60℃ for 10 s, and 72℃ for 15 s. The derivation steps for the melting curve are as follows: 95℃ for 15 s, 60℃ for 30 s, and 95℃ for 15 s. The internal control gene was GAPDH, which has been reported to be stably expressed in Ginkgo biloba. The internal control gene for Nicotiana benthamiana was NbActin. The quantitative primer sequences for genes related to tobacco flavonoid synthesis are shown in Table 2.

[0058] The results are as follows Figure 4 As shown, after tobacco co-culture, induction, screening, differentiation, and rooting ( Figure 4 A) Six robust transgenic lines of *Nicotiana benthamiana* were randomly tested, and the results showed that GbC4H1 overexpression achieved efficient expression in multiple transgenic lines. Therefore, three *Nicotiana benthamiana* lines with high GbC4H1 expression levels were selected as subjects for subsequent experiments.

[0059] 3. Expression of genes and key transcription factors related to the flavonoid synthesis pathway

[0060] The expression levels of related genes and key transcription factors were analyzed in three lines with high CK and GbC4H1 expression levels, including genes of the flavonoid synthesis pathway: NtPAL1, C4H, NtHCT, NtCHI, NtCHS1, NtF3H, NtF3H, NtFLS1, NtFLS2, NtDFR, NtLAR, NtANR1, NtANR2, and NtUFGT, as well as transcription factors regulating structural genes of the flavonoid synthesis pathway, specifically NtMYL2a, NtMYL2b, NtbHLH1, NtbHLH2, NtbHLH3, NtERF4a, and NtERF4b.

[0061] The results are as follows Figure 5 As shown, compared with the control group, the expression levels of NtCHI, NtCHS1 and NtDFR genes in transgenic Nicotiana benthamiana lines were significantly increased, while the expression levels of NtMYL2a and NtMYL2b genes among transcription factors were significantly increased; the expression level of the key enzyme gene NtANR1 was significantly decreased.

[0062] Example 3

[0063] 1. Targeted flavonoid metabolite assay

[0064] Leaf samples of transgenic *Nicotiana benthamiana* and control *Nicotiana benthamiana* (CK) were collected and freeze-dried. Each sample was ground separately using a ball mill at 30 Hz for 1.5 min until it became powder. 20 mg of the powder was weighed and divided into two portions. One portion was added to 10 μL of 4000 nmol / L internal standard working solution, and the other portion was added to 500 μL of 70% methanol solution. The mixtures were then sonicated for 30 min. The samples were centrifuged at 12000 r / min for 5 min at 4°C. The supernatant of each sample was collected and filtered through a 0.22 μm filter membrane. The samples were then stored in sample vials for LC-MS / MS analysis.

[0065] The main liquid phase conditions include: (1) chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm id); (2) mobile phase: phase A is ultrapure water (with 0.05% formic acid added), phase B is acetonitrile (with 0.05% formic acid added); (3) flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL; (4) elution gradient: 0 min A / B is 90:10 (V / V), 1 min A / B is 80:20 (V / V), 9 min is 30:70 (V / V), 12.5 min A / B is 5:95 (V / V), 13.5 min A / B is 5:95 (V / V), 13.6 min is 90:10 (V / V), 15 min is 90:10 (V / V).

[0066] The main mass spectrometry conditions include: (1) Electrospray ionization (ESI) temperature 550°C; (2) Mass spectrometry voltage 5500 V in positive ion mode, mass spectrometry voltage -4500 V in negative ion mode, and Curtain Gas (CUR) 35 psi; (3) In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0067] The data acquisition instrument system mainly includes: Ultra Performance Liquid Chromatography (UPLC) (ExionLC AD, https: / / sciex.com.cn / ) and Tandem Mass Spectrometry (MS / MS) (QTRAP 6500+, https: / / sciex.com.cn / ).

[0068] 2. Screening of differentially metabolized flavonoids

[0069] Variable Importance in Projection (VIP) obtained from an Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model can initially screen metabolites between different groups. Generally, a VIP value greater than 1 indicates a significant difference in the metabolite. Further screening of differentially expressed metabolites is then conducted using univariate analysis with p-value / FDR or FC values. Metabolites with fold change ≥ 2 and fold change ≤ 0.5 are selected; a difference of more than 2-fold or less than 0.5-fold between the control and experimental groups is considered significant. The selected metabolites are then annotated in the KEGG database. The KEGG database (http: / / www.kegg.jp / kegg / compound / ) is used to identify significantly different metabolites, and the annotation results are categorized according to pathway types in KEGG (http: / / www.kegg.jp / kegg / pathway.html).

[0070] The mean was compared using one-way ANOVA with SPSS 22.0 software, and Duncan calculated the significance of differences in flavonoid synthesis-related genes among different comparison groups (P < 0.05).

[0071] The results are as follows Figure 6 As shown, after qualitative and quantitative evaluation of metabolites in tobacco leaves, the results showed that a total of 56 flavonoid metabolites were detected in the overexpressed transgenic lines and the CK group. Compared with the control group, 14 differentially expressed metabolites were upregulated and 20 differentially expressed metabolites were downregulated. A violin plot was used to display the 34 significantly different metabolites. Among the upregulated differentially expressed metabolites, the phenolic acid syringaldehyde showed the highest fold change (VIP) at 1.07 and a Fold Change (FC) of 7.20; the flavonoid baimaside was second, with a Fold Change (Fold Change) of 4.63 and a VIP of 1.07. Correspondingly, among the downregulated differentially expressed metabolites, narcissin showed the largest fold change, followed by rutin. Figure 6 A). Classification of significantly differentially metabolites within the KEGG pathway revealed that the most numerous differentially metabolites were found in flavonoid biosynthesis and secondary metabolite biosynthesis, with eight in each, accounting for 61.54% of the total. Figure 6 B). In addition, there are 6 differential metabolites in flavonone and flavonol biosynthesis, 5 differential metabolites in metabolic pathways, and 1 differential metabolite in isoflavonoid biosynthesis, namely Apogenin 7-glucoside, which belongs to the flavonoid class.

[0072] In summary, the GbC4H1 gene was expressed at the highest levels in the AN3 treatment group and male cones. Overexpression of the Ginkgo GbC4H1 gene promoted the expression levels of NtCHI, NtCHS1, NtDFR, NtMYL2a, and NtMYL2b genes in transgenic plants, while reducing the expression level of the key enzyme gene NtANR1. Overexpression of GbC4H1 significantly increased the content of syringaldehyde and baimaside in transgenic plants.

[0073] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A Ginkgo biloba GbC4H1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the Ginkgo GbC4H1 gene of claim 1, the amino acid sequence of which is shown in SEQ ID NO.

6.

3. A vector or recombinant bacteria containing the Ginkgo GbC4H1 gene as described in claim 1.

4. The application of the Ginkgo GbC4H1 gene as described in claim 1 in increasing the content of syringaldehyde and ephedrine, metabolites of Tobacco Benzoinus.

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