Ginkgo biloba GbC4H1 gene as well as encoding protein and application thereof

Abstract

The invention discloses a gingko GbC4H1 gene as well as an encoding protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the gingko GbC4H1 gene disclosed by the invention is as shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the gingko GbC4H1 gene is as shown in SEQ ID NO. 2. The method comprises the following steps: constructing an expression vector of the gingko GbC4H1 gene, and transforming the expression vector into the nicotiana benthamiana; and cultivating and screening to obtain a transgenic plant with increased flavonoid metabolic substance content. Compared with a control group, the expression level of regulation and control transcription factors of flavone synthesis pathway related genes and flavone synthesis pathway structural genes in a transgenic line is remarkably improved. The content of flavonoid metabolites in a transgenic strain is increased, and compared with a control group, 14 kinds of differential metabolites are up-regulated and 20 kinds of differential metabolites are down-regulated.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to the Ginkgo biloba GbC4H1 gene, its encoded protein, and applications thereof. Background Art

[0002] Ginkgo biloba is the only extant species of Ginkgoopsida and is a well-known relict gymnosperm in the world, known as a "living fossil" and having important scientific research value. Ginkgo leaves are rich in compounds such as flavonoid glycosides and ginkgolides, and have multiple effects such as antioxidant, anti-platelet activating factor, regulating vascular activity, and protecting the nervous system. Ginkgo leaf extract has been clinically used to treat various diseases such as cardiovascular diseases, nervous system diseases, and hyperlipidemia, and has achieved remarkable curative effects.

[0003] Cinnamate 4-hydroxylase (C4H) belongs to the cytochrome P450 enzyme family and can affect the synthesis and metabolism of substances such as flavonoids, lignins, and alkaloids. In plant secondary metabolism, the phenylpropanoid metabolic pathway plays an important role in plant growth and development and the synthesis of secondary metabolites. Generally, it is believed that this pathway promotes the formation of flavonols and anthocyanins through a series of biological reactions, and this process requires the synergistic catalysis of multiple enzymes such as phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL). Among them, C4H, as one of the key enzymes in this pathway, catalyzes the hydroxylation of cinnamic acid to produce 4-coumarate, and is the second key enzyme after L-phenylalanine ammonia-lyase (PAL) in the phenylpropanoid pathway. Research has found that phenylpropanoid metabolites and the expression levels of related C4H genes play a crucial role in the formation of plant color, aroma, and pulp texture, and further increasing the expression levels of C4H-related phenylpropanoid metabolic genes can effectively enhance plant resistance. During the study of black tea processing, the expression level of the C4H gene is also closely related to the quality of tea.

[0004] Flavonoids are an important class of plant secondary metabolites, with multiple pharmacological functions such as anti-oxidation, anti-cancer and anti-tumor, anti-aging, and cardiovascular protection. C4H, as a key enzyme in the phenylpropanoid metabolic pathway, is also involved in the synthesis of flavonoids. By catalyzing the conversion of cinnamic acid to p-coumaric acid, C4H provides precursors for the synthesis of flavonoids, thereby affecting the biosynthesis and accumulation of flavonoids (Liang Liang, Han Xiaomin, Zhang Zheng, et al. Cloning and expression analysis of the gene of cinnamic acid-4-hydroxylase (C4H) from Aquilaria sinensis[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 will be activated, thereby inducing the expression of the C4H gene, increasing its expression level, and promoting the increase in the content 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 Science Journal, 2008, 2(1):31-36. DOI:10.2174 / 1874294700802010031.). In addition, in Arabidopsis mutants, the inactivation of the protein encoded by the C4H gene causes Arabidopsis to exhibit growth restriction, stamen dysfunction and other characteristics, and also leads to a significant decrease in its 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 shows that the C4H gene has a certain effect on the synthesis and accumulation of flavonoids under different environments. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide the Ginkgo biloba GbC4H1 gene. Another technical problem to be solved by the present invention is to provide the encoded protein of the Ginkgo biloba GbC4H1 gene. The technical problem to be solved by the present invention is also to provide the application of the Ginkgo biloba GbC4H1 gene in regulating the synthesis and accumulation of flavonoids in plants under different nitrogen forms.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

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

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

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

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

[0011] Specifically, it promotes a significant increase in the expression levels of genes related to the flavonoid synthesis pathway, such as NtF3H, NtFLS1, NtFLS2, NtPAL1, NtCHI, NtCHS1, NtLAR, and NtDFR genes.

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

[0013] Specifically, it promotes a significant increase in the expression levels of transcription factors NtMYL2a, NtMYL2b, NtbHLH3, and NtERF4b that regulate structural genes in the flavonoid synthesis pathway.

[0014] Application of the Ginkgo biloba 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 flavonoid differential metabolites in plants.

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

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

[0019] 2) The present invention constructs the GbC4H1 Nicotiana benthamiana strain. Compared with the control group, the expression levels of the NtCHI, NtCHS1, and NtDFR genes in the transgenic strains are significantly increased, while the expression levels of the NtMYL2a and NtMYL2b genes among the transcription factors are significantly increased; the expression level of the key enzyme gene NtANR1 is significantly decreased.

[0020] 3) The present invention constructs the GbC4H1 Nicotiana benthamiana strain and qualitatively and quantitatively analyzes the flavonoid metabolites in it. The results show that, compared with the control group, 14 differential metabolites are up-regulated and 20 differential metabolites are down-regulated. Overexpression of GbC4H1 significantly increases the contents of syringaldehyde and baimaside in the transgenic plants. Description of the Drawings

[0021] Figure 1 It is a protein property analysis diagram of 5 Ginkgo biloba C4H genes (A is the transmembrane domain of the encoded protein, B and C are conserved domains, D is the prediction of the secondary structure of the encoded protein, and E is the prediction of the tertiary structure of the encoded protein);

[0022] Figure 2 It is a phylogenetic tree diagram (A) and motif analysis diagram (B) of 5 Ginkgo biloba C4Hs genes;

[0023] Figure 3 It is an analysis diagram of the expression patterns of 5 C4Hs genes in different tissue parts and under different nitrogen forms;

[0024] Figure 4 It is a process and expression level analysis diagram of stable overexpression of GbC4H1 in tobacco (A is the process of genetic transformation of tobacco with the GbC4H1 gene, and B is the expression level of the GbC4H1 gene in tobacco);

[0025] Figure 5 It is an analysis diagram of the expression levels of key enzyme genes and important transcription factors in the flavonoid synthesis pathway in transgenic Nicotiana benthamiana;

[0026] Figure 6 It is a violin diagram of differential metabolites (A, the abscissa is the group, and the ordinate is the expression level) and a KEGG classification diagram of differential metabolites (B, the ordinate is marked with the name of the KEGG metabolic pathway, the number represents the number of differential metabolites annotated in this pathway, and the number in the parentheses shows the proportion of the differential metabolites annotated in this pathway to the total number of differential metabolites annotated on KEGG). Detailed Embodiments

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are all conventional means well-known to those skilled in the art. For molecular biology experimental methods without specific descriptions, they can be referred to the methods listed in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product manuals.

[0028] The plant material used in this application is 3-year-old ginkgo seedlings with bare roots, planted in Xiashu Forest Farm of Nanjing Forestry University. The experiment was carried out in July when ginkgo was growing vigorously, and a completely randomized experimental design was adopted. Seven different nitrogen form treatments were set (5 inorganic nitrogen treatments, NH4 + :NO3 - = 100:0, 75:25, 50:50, 25:75, and 0:100, NO3 - was provided by potassium nitrate, and NH4 + was provided by ammonium sulfate; 2 organic nitrogen treatments, urea and glycine), and a blank control. The application ratio was based on the nitrogen application rate, with a total of 8 treatments (15 plants for each treatment, and every 5 plants were taken as 1 biological replicate). The nitrogen application rate was 5.55 g / plant. The nitrogen fertilizers for all treatments were applied in 3 equal amounts, with a 10-day interval between each application. Each time, 1 / 3 of the fertilizer was dissolved in 200 mL of tap water and poured into the pots. The control group was watered with 200 mL of tap water to ensure that the fertilizer solution would not overflow. At the same time, the nitrification inhibitor DCD was applied (the dosage was 7% of the nitrogen application rate, 0.3885 g / plant) to reduce the loss in the form of nitrate nitrogen. To ensure the consistency of K + supply, each treatment was supplemented with KCl and applied together with the 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 values in the table are the total amounts for 15 plants in each treatment, with the unit of g.

[0032] Example 1

[0033] 1. Cloning of Ginkgo biloba GbC4H1 Gene

[0034] The total RNA of ginkgo leaves after 7 nitrogen treatments was extracted using a universal plant total RNA rapid extraction kit (BioTeKe Corporation). The specific method was referred to the instruction manual. Then, PrimeScript of TaKaRa was used. TMThe RT MasterMix (Perfect Real Time) kit was used for reverse transcription to synthesize cDNA. Based on the differentially expressed C4H1 sequences screened from Ginkgo biloba transcriptome data, primers for its open reading frame (ORF) were designed using Oligo 6.0 software (Table 2), and the PrimeSTAR ® Max DNA Polymerase from Takara was used to clone its ORF sequence.

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

[0036] PCR reaction program: 98 °C for 3 min; 98 °C for 10 s, 55 °C for 5 s, 72 °C for 15 s, for 35 cycles; 72 °C for 3 min, hold at 8 °C indefinitely.

[0037] Table 2 Primer information for Ginkgo biloba C4Hs genes

[0038]

[0039] The PCR products of the C4H1 gene were detected by 1% agarose gel electrophoresis. The bands corresponding to the predicted length of the target gene amplification products were excised, recovered and purified using the BioTeKe Quick Agarose Gel DNA Recovery Kit. According to the requirements of the pClone007 Blunt Vector Kit (TSINGKE), the purified products were ligated with the vector, transferred into competent Escherichia coli, briefly revived and then spread on ampicillin-resistant medium. After overnight culture, single colonies were randomly selected and verified by colony PCR using 2×T5 Super PCR Mix (Colony) (TSINGKE). The positive bacterial solutions were sent to Tsingke Biotechnology Co., Ltd. for sequencing verification.

[0040] 20 μL colony PCR system: 10 μL of 2×T5 Super PCR Mix, 0.75 μL of universal primer M13-F, 0.75 μL of M13-R, 1.5 μL of bacterial solution, 6.5 μL of ddH2O.

[0041] PCR reaction program: 98 °C for 2 min; 98 °C for 10 s, 55 °C for 10 s, 72 °C for 15 s, for 35 cycles; 72 °C for 2 min, hold at 8 °C indefinitely.

[0042] The positive clones were sent to Tsingke Biological for sequencing after detection. The ORF nucleotide sequences of the GbC4H1, GbC4H2, GbC4H3, GbC4H4, and GbC4H5 genes obtained by final sequencing are shown in SEQ ID NO.1-5, and the amino acid sequences of their encoded proteins are 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 C4Hs proteins were analyzed using the ProtParam online tool. The hydrophilicity and hydrophobicity of Ginkgo biloba C4H proteins were predicted using the online tool ProtScale (https: / / web.expasy.org / protscale / ) and the protein hydrophilicity-hydrophobicity map was drawn; the online tools of Novopro Biosciences (https: / / www.novopro.cn / tools / tmhmm.html), SMART (http: / / smart.embl-heidelberg.de / ), and the NCBI online conserved domain retrieval tool CDART (Conserved Domain Architecture Retrieval Tool) (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow) were used to analyze the protein domains, including transmembrane domains, functional domains, and conserved domains; the subcellular localization of C4H proteins was predicted using the Softberry online website (http: / / www.softberry.com / ); the secondary structure was analyzed using the SOPMA online tool (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa_sopma.html); the three-dimensional model was constructed and verified using SWISS-MODEL (https: / / swissmodel.expasy.org / ). A phylogenetic tree was constructed using MEGAX software with the neighbor-joining method and 1000 bootstrap replicates.

[0045] The results are shown in Table 3. Based on the transcriptome data of Ginkgo biloba leaves under different nitrogen form treatments, the key enzyme genes in the phenylalanine synthesis pathway were mined and identified. For the C4H enzyme gene with the largest number of differential genes, in addition to the comparison groups of different nitrogen form treatments and CK, the differential genes between all other comparison groups were also extracted, and a total of 5 genes were obtained. They were named C4H1, C4H2, C4H3, C4H4, and C4H5 according to the arrangement order of chromosomes. All 5 proteins are hydrophobic basic proteins. The amino acid sequences of C4H2, C4H3, C4H4, and C4H5 are identical, and their encoding genes are multi-copy genes. The similar physicochemical properties indicate that there have been a few mutations during evolution, but the structure has not changed. The results of subcellular localization prediction show that C4H2, C4H3, and C4H5 may be simultaneously localized in the cytoplasm and mitochondria, C4H1 is only localized in the mitochondria, and C4H4 is only localized in the cytoplasm.

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

[0047]

[0048] The results are as Figure 1 shown. C4H1, C4H2, C4H3, C4H4, and C4H5 all have a transmembrane domain, and there is only one. A small number of amino acids are located inside the cell, and most amino acids are located outside the cell ( Figure 1 A). The conserved domain analysis tool of NCBI and the online tool SMART were used together to search for the protein domains. C4H1, C4H2, C4H3, C4H4, and C4H5 all belong to the CYP450 family and all have a transmembrane region and an NmrA domain ( Figure 1 B and C). NPSA was used to comprehensively analyze the secondary structure of the protein in three ways: MLRC on GOR4, SIMPA96 and SOPMA, DSC and PHD. The results show that the folded protein is mainly composed of α-helices, extended strands, random coils, and some temporarily unknown structures ( Figure 1 D). Among the 5 C4H proteins, the proportion of α-helices is the largest, and the proportion of random coils is larger in the other proteins. The three-dimensional model of the protein was constructed using SWISS-MODEL. It was found that the template with the highest homology for C4H1 is Q9XEH8.1.A, which is the TC4H protein of Pinus taeda (Loblolly pine), with a model coverage rate of 95% and a model similarity of 52%; the templates with the highest homology for C4H2, C4H3, C4H4, and C4H5 are all 6vby.1.A, which is the SbC4H1 of Sorghum bicolor, with a model coverage rate of 90% and a model similarity of 51% for all. Figure 1E).

[0049] The results are as follows Figure 2 As shown in the figure, Ginkgo C4H2 and C4H5 are clustered into one branch, and C4H3 and C4H4 are clustered into one branch, which are closely related to the CYP72A171 protein (ATG29969.1) of Taxus chinensis, the C4H protein (USH99603.1) of Cephalotaxushainanensis, and the C4H2 protein (XP_057839147.2) of Cryptomeria japonica. Figure 2 A). C4H1 was divided into a separate branch. The results of motif analysis showed that the C4H1 sequence was seriously missing and most of the motifs were lost, indicating that there were certain differences in its driving functions. C4H1 was also the gene with the largest difference in differential expression patterns among the different nitrogen form comparison groups, suggesting that it may play a certain role in regulating flavonoid synthesis and accumulation ( Figure 2 B).

[0050] 2. Analysis of expression patterns of five C4Hs genes in different tissues and under different nitrogen forms

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

[0052] Example 2

[0053] 1. Construction of stable overexpression vector and stable genetic transformation of Nicotiana benthamiana

[0054] Take 1µL plasmid and add it to 50µL GV3101 Agrobacterium competent cells, mix thoroughly and then transfer to the electroporation cup, add 1mL LB liquid medium after electroporation, mix thoroughly and then transfer to a 1.5mL centrifuge tube, shake and culture in a shaker at 28℃ and 180rpm for 30min, take 50µL of the activated Agrobacterium liquid and inoculate it on LB solid medium, and culture it in the dark at 28℃ for 48h. Synthesize the corresponding detection primers, and select the samples with clear electrophoresis bands and correct sizes in the PCR amplification results 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 Agrobacterium into the infection solution and prepare the OD 600Agrobacterium resuspension solution with an OD value of 0.2. Inoculate tobacco leaves pre-cultured for 2 - 3 days into the Agrobacterium suspension and infect for 10 - 15 minutes. Place the infected tobacco leaves on filter paper, air-dry them, and then inoculate them onto the co-culture medium. Incubate in the dark for 48 - 72 hours. Transfer the leaves after 2 days of co-culture to the induction medium to induce callus for about 10 days until callus tissue grows. Screen the callus with better growth and inoculate it onto the screening medium with kanamycin resistance for 15 - 30 days at a culture temperature of 23 ± 2°C. Inoculate the vigorously growing positive callus after the second screening onto the differentiation medium, 4 - 5 callus per dish, and culture at 23°C with a 16h / 8h light / dark cycle for 15 - 30 days. During the differentiation process, if seedlings form from the callus, inoculate them onto the seedling strengthening medium to grow for 7 - 10 days. Extract tobacco genomic DNA using the CTAB method for PCR detection. At the same time, extract the RNA of potential positive seedlings, reverse transcribe it into cDNA, and perform RT-qPCR to detect the expression levels of different strains.

[0056] 2. Real-time fluorescence quantitative PCR analysis

[0057] Extract total RNA from leaves using an RNA extraction kit (Beijing Bioteke Corporation), and then perform transcription using a cDNA synthesis kit (Shanghai Pudi Biotechnology Co., Ltd.). Use a qTOWER 2.2 qPCR instrument for RT-qPCR reactions. The program starts with a denaturation step at 95°C for 3 minutes, followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s. The steps for deriving the melting curve are as follows: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s. The internal reference gene is the gene GAPDH that has been reported to be stably expressed in Ginkgo biloba, and the internal reference for Nicotiana benthamiana is NbActin. The quantitative primer sequences for genes related to tobacco flavonoid synthesis are shown in Table 2.

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

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

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

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

[0062] Example 3

[0063] 1. Determination of targeted flavonoid metabolites

[0064] Samples of transgenic Nicotiana benthamiana leaves and CK leaves of Nicotiana benthamiana as the control at the same period were collected and freeze-dried; each sample was ground separately using a ball mill and ground for 1.5 min at 30 Hz until the sample became powdery; 20 mg of the powder was weighed and divided into two parts. One part was added with 10 μL of an internal standard mixed working solution with a concentration of 4000 nmol / L, and the other part was added with 500 μL of 70% methanol solution, and then ultrasonicated for 30 min; centrifuged at 12000 r / min for 5 min at 4°C, the supernatant of each sample was aspirated, and finally the sample was filtered through a 0.22 μm filter membrane and stored in a sample vial for LC-MS / MS analysis.

[0065] The liquid phase conditions mainly include: (1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm i.d.); (2) Mobile phase: Phase A is ultrapure water (added with 0.05% formic acid), and Phase B is acetonitrile (added with 0.05% formic acid); (3) Flow rate: 0.35 mL / min; Column temperature: 40 °C; Injection volume: 2 μL; (4) Elution gradient: At 0 min, A / B is 90:10 (V / V), at 1 min, A / B is 80:20 (V / V), at 9 min, it is 30:70 (V / V), at 12.5 min, A / B is 5:95 (V / V), at 13.5 min, A / B is 5:95 (V / V), at 13.6 min, it is 90:10 (V / V), and at 15 min, it is 90:10 (V / V).

[0066] The mass spectrometry conditions mainly include: (1) Temperature of the electrospray ionization (ESI) source: 550 °C; (2) Mass spectrometry voltage in positive ion mode: 5500 V, mass spectrometry voltage in negative ion mode: -4500 V, 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 flavonoid differential metabolites

[0069] The Variable Importance in Projection (VIP) obtained from the Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model can preliminarily screen out metabolites between different groups. Generally speaking, when the VIP value is greater than 1, the difference in this metabolite is considered significant. At the same time, combined with the P-value / FDR or FC value of univariate analysis, differential metabolites are further screened out. Metabolites with fold change ≥ 2 and fold change ≤ 0.5 are selected. If the difference in metabolites between the control group and the experimental group is more than 2-fold or less than 0.5-fold, it is considered significantly different. And the screened metabolites are 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 classified according to the pathway types in KEGG (http: / / www.kegg.jp / kegg / pathway.html).

[0070] One-way ANOVA of SPSS 22.0 software was used to compare the means, and Duncan's post hoc multiple comparisons were used to calculate the significance of differences in flavonoid synthesis-related genes between different comparison groups (P < 0.05).

[0071] The results are as Figure 6 shown. After qualitative and quantitative evaluation of the metabolites detected in tobacco leaves, the results showed that a total of 56 flavonoid metabolites were detected in the overexpressed transgenic line group and the CK group. Among them, compared with the control group, 14 differential metabolites were up-regulated and 20 differential metabolites were down-regulated. Violin Plot diagrams were shown for 34 significantly different metabolites. Among the up-regulated differential metabolites, the differential multiple of the phenolic acid syringaldehyde was the highest, with a VIP value of 1.07 and a Fold Change (FC) of 7.20; the flavonoid baimaside was the second, with a Fold Change value of 4.63 and a VIP value of 1.07; correspondingly, among the down-regulated differential metabolites, the differential expression multiple of Narcissin was the largest, and rutin was the second ( Figure 6 A). Classification of significantly different metabolites in the KEGG pathway showed that there were the most differential metabolites in Flavonoid biosynthesis and Biosynthesis of secondary metabolites, with 8 each, accounting for 61.54% of the total number of differential metabolites (Figure 6 B). In addition, there are 6 differential metabolites in flavone and flavonol biosynthesis, 5 differential metabolites in metabolic pathways, and 1 differential metabolite in isoflavonoid biosynthesis, which is Apigenin 7-glucoside and belongs to flavonoids.

[0072] In summary, the expression level of GbC4H1 gene is the highest in the AN3 treatment group and male cones. Overexpression of the Ginkgo biloba GbC4H1 gene promoted the increased expression levels of NtCHI, NtCHS1, NtDFR, NtMYL2a, and NtMYL2b genes in transgenic plants; it decreased the expression level of the key enzyme gene NtANR1. Overexpression of GbC4H1 significantly increased the contents of syringaldehyde and baimaside in transgenic plants.

[0073] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them 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, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector or recombinant bacterium containing the Ginkgo GbC4H1 gene according to claim 1.

4. Use of the Ginkgo GbC4H1 gene according to claim 1 in regulating the expression of genes related to the flavonoid biosynthesis pathway.

5. The use according to claim 4, characterized in that: Specifically, the expression levels of genes NtF3H, NtFLS1, NtFLS2, NtPAL1, NtCHI, NtCHS1, NtLAR and NtDFR related to the flavonoid synthesis pathway were significantly increased.

6. Use of the Ginkgo GbC4H1 gene of claim 1 in regulating the expression of transcription factors of structural genes in the flavonoid biosynthesis pathway.

7. The use according to claim 6, characterized in that: Specifically, the expression levels of regulatory transcription factors NtMYL2a, NtMYL2b, NtbHLH3 and NtERF4b, which promote the structural genes of the flavonoid synthesis pathway, were significantly increased.

8. Use of the Ginkgo GbC4H1 gene of claim 1 in regulating the content of flavonoid metabolites in plants.

9. The use according to claim 6, characterized in that: Specifically, it promotes the increase of flavonoid metabolites in plants.

10. Use of the Ginkgo GbC4H1 gene of claim 1 in regulating differential flavonoid metabolites in plants.

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