Ginkgo biloba GbHCT1 gene and expression protein and application thereof

Abstract

The invention discloses a gingko GbHCT1 gene as well as an expression protein and application thereof, and belongs to the field of plant molecular biology. The GbHCT1 gene is obtained by analyzing and screening based on data of a ginkgo transcriptome in the earlier stage of a research group, the nucleotide sequence of the GbHCT1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of expression protein of the GbHCT1 gene is as shown in SEQ ID NO.2. A GbHCT1 overexpression vector is constructed and the nicotiana benthamiana is transformed, so that the content of ten flavonoid substances such as luteolin and isorhamnetin-3-O-glucoside in the transgenic GbHCT1 tobacco is remarkably increased. KEGG enrichment shows that differential metabolites are mainly enriched in a flavone synthesis pathway. The application reveals that the ginkgo GbHCT1 gene has dual effects on synthesis of flavonoid compounds by regulating carbon source distribution of a phenylpropane metabolic pathway, and a theoretical basis, a gene resource and a method are provided for secondary metabolism regulation and genetic improvement of ginkgo.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, specifically relating to a Ginkgo biloba GbHCT1 gene, its expressed protein, and its applications. Background Technology

[0002] Ginkgo biloba is a precious "living fossil" tree species, not only highly adaptable and resistant to pests and diseases, but its leaf extract (GBE) is also an important medicinal resource. The core active components of GBE include flavonoid glycosides and terpenoids, which have pharmacological effects such as scavenging free radicals, protecting cells, and anti-fibrosis.

[0003] Hydroxycinnamyltransferase (HCT) is a key acyltransferase (BAHD family member) in the phenylpropanoid metabolic pathway in plants, catalyzing the reaction of acyl-CoA with a hydroxyl acceptor. HCT possesses dual activity in catalyzing the hydroxycinnamylation of shikimic acid / quinic acid, playing a central role in lignin biosynthesis: it controls the direction of carbon flow, catalyzes key steps (such as p-coumaroyl-CoA → p-coumaroylshikimic acid → caffeoyl ester), and regulates lignin content and structure to improve plant traits (such as stress resistance and processing ability). Studies have also shown that HCT (such as blackberry RuHCT1) may affect anthocyanin synthesis and inhibit lignan production. Although HCT catalyzes a similar reaction to its family-related HQT enzymes (generating shikimic acid / quinic acid esters, which are then converted into lignin precursors or chlorogenic acid CGA), HCT significantly favors shikimic acid substrates, and its downregulation primarily affects lignin rather than CGA. Furthermore, the HCT-catalyzed steps (generating p-coumaroyl shikimic acid, caffeoyl ester, etc.) are important precursor reactions in the biosynthesis of flavonoids, suggesting that they may indirectly regulate flavonoid synthesis.

[0004] Currently, research on HCT function mainly focuses on lignin synthesis, and how it affects the biosynthesis of key medicinal components (especially flavonoids) in Ginkgo biloba remains unclear. Discovering the Ginkgo biloba HCT gene is of great significance for elucidating its secondary metabolic regulatory mechanism and guiding genetic improvement of Ginkgo biloba. Summary of the Invention

[0005] To address the problems existing in the prior art, the first technical problem to be solved in this application is to provide a Ginkgo biloba GbHCT1 gene and its expressed protein. The second technical problem to be solved in this application is to provide biological materials containing the aforementioned Ginkgo biloba GbHCT1 gene. The final technical problem to be solved in this application is to provide specific applications of the aforementioned GbHCT1 gene.

[0006] To address the problems in the existing technology, the technical solution of this application is as follows:

[0007] A Ginkgo biloba GbHCT1 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The protein encoded by the Ginkgo GbHCT1 gene has the amino acid sequence shown in SEQ ID NO.2.

[0009] A biological material containing an expression cassette, recombinant vector, recombinant bacteria, or recombinant cells of the Ginkgo GbHCT1 gene.

[0010] The application of the Ginkgo GbHCT1 gene expression protein or the biological material in plant breeding.

[0011] The application of the Ginkgo GbHCT1 gene, the expressed protein of the Ginkgo GbHCT1 gene, or the biological material in regulating the synthesis of plant flavonoids.

[0012] A method for increasing the synthesis of flavonoids in plants: overexpressing the Ginkgo GbHCT1 gene in plants, wherein the flavonoids are at least one of the following compounds: isomangiin, baicalin, mangiferin, 6-hydroxyflavone, crotonin, cyanidin, luteolin, romaine, isorhamnetin-3-O-glucoside, and naringin.

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

[0014] (1) Construct the expression vector of the Ginkgo GbHCT1 gene;

[0015] (2) Transform the constructed Ginkgo GbHCT1 gene expression vector into plants or plant tissues;

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

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

[0018] In some embodiments, the overexpression vector of the Ginkgo GbHCT1 gene is pCAMBIA1302-GbHCT1.

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

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

[0021] Based on previous analysis of Ginkgo transcriptome data, this application identified a GbHCT1 gene, whose nucleotide sequence is shown in SEQ ID NO.1 and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. It is a hydroxycinnamoyl transferase. Its physicochemical properties, domains, and phylogenetic relationships were analyzed. The results showed that the GbHCT1 protein is a hydrophobic acidic protein, with the highest expression levels observed in the buds, fruits, and yellow leaves of Ginkgo. A GbHCT1 overexpression vector was constructed and transformed into Nicotiana benthamiana. The levels of flavonoids, such as luteolin and isorhamnetin-3-O-glucoside, were significantly upregulated in Ginkgo biloba transformed with the GbHCT1 gene, while the levels of flavanols, such as catechin gallate, were significantly downregulated. KEGG enrichment indicated that the differentially expressed metabolites were mainly enriched in the flavonoid synthesis pathway. This application reveals that the Ginkgo biloba GbHCT1 gene plays a dual role in the synthesis of flavonoids by regulating carbon source allocation in the phenylpropane metabolic pathway, providing a theoretical basis, gene resources, and methods for the regulation and genetic improvement of Ginkgo biloba secondary metabolism. Attached Figure Description

[0022] Figure 1-1 The images show the results of the GbHCT1 protein structure analysis, where A represents the secondary structure analysis results of the GbHCT1 protein; and B represents the three-dimensional structure model of the GbHCT1 protein.

[0023] Figure 1-2 Figure showing the phylogenetic analysis results of the GbHCT1 protein;

[0024] Figure 1-3 The image shows the motif prediction results for the GbHCT1 protein.

[0025] Figure 2 This is a diagram illustrating the expression pattern of Ginkgo HCTs genes.

[0026] Figure 3 This is a subcellular localization analysis diagram of GbHCT1; from left to right, the empty vector (35S::GFP) control shows the fluorescence channel, chloroplast channel, bright field, and overlay diagram; the target gene (35S::HCT1) shows the fluorescence channel, chloroplast channel, bright field, and overlay diagram from left to right.

[0027] Figure 4-1 The diagram shows the process and expression level of stable overexpression of GbHCT1 in tobacco; A represents the process of genetic transformation of the GbHCT1 gene into tobacco; B represents the expression level of the GbHCT1 gene in tobacco.

[0028] Figure 4-2 This is a diagram showing the expression levels of eight key enzyme genes and important transcription factors in the flavonoid synthesis pathway in GbHCT1 transgenic tobacco.

[0029] Figure 4-3This is a diagram showing the expression levels of 12 key enzyme genes and important transcription factors in the flavonoid synthesis pathway in GbHCT1 transgenic tobacco.

[0030] Figure 5-1 A violin plot showing the analysis of 14 differential metabolites in GbHCT1 transgenic tobacco plants, with the horizontal axis representing the group and the vertical axis representing the expression level.

[0031] Figure 5-2 A violin plot showing the analysis of 12 differential metabolites in GbHCT1 transgenic tobacco plants, with the horizontal axis representing the group and the vertical axis representing the expression level.

[0032] Figure 5-3 A graph showing the differential KEGG enrichment of the GbHCT1 transgenic tobacco plant line;

[0033] Figure 6 This diagram illustrates the flavonoid biosynthesis metabolic pathway and expression analysis in transgenic plants. Green arrows indicate downregulation, red arrows indicate upregulation, green text represents downregulated genes, and red text represents upregulated genes. Detailed Implementation

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

[0035] Example 1

[0036] 1. Plant materials

[0037] This application uses 3-year-old Ginkgo seedlings grown in the greenhouse of Xiashu Forest Farm, Nanjing Forestry University (32°07′N, 119°13′E) as material for gene cloning. Subcellular localization experiments were conducted using Nicotiana benthamiana material cultured in an artificial climate chamber by the Ginkgo biloba research group of Nanjing Forestry University, while the stably genetically transformed Nicotiana benthamiana K326 plants were obtained from tissue culture seedlings preserved by the research group.

[0038] 2. This application, through analysis of transcriptome data of different nitrogen forms in Ginkgo biloba, screened and identified two differentially expressed HCT genes (evm.TU.chr4.150 and evm.TU.chr12.1826), named HCT1 (encoding 494 amino acids) and HCT2 (encoding 419 amino acids), respectively. The online tool Expasy-protfaram (https: / / web.expasy.org / protparam / ) was used to predict the molecular weight, isoelectric point, total number of charged residues, instability coefficient, adipose coefficient, and hydrophobicity of the proteins encoded by these structural genes. The characteristics of the HCT protein were analyzed using the ProtScale online software. Subcellular localization was predicted using CELLO (subcellular localization predictor). Motif prediction of the HCT protein was performed using the MEME online tool (https: / / meme-suite.org / meme / tools / meme). The transmembrane structure of the HCT protein was predicted using Novopro's online tool (https: / / www.novopro.cn / tools / tmhmm.html). Joint analysis of the protein's domains was conducted using NCBI's conserved domain analysis tool (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi#opennewwindow) and the online tool SMART (http: / / smart.embl-heidelberg.de / smart / set_mode.cgi?GENOMIC=1). In-depth analysis of the secondary structure of the HCT protein was conducted using NPSA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_seccons.html), combining MLRC on GOR4, SIMPA96 and SOPMA, DSC and PHD. A three-dimensional model of the HCT protein was constructed using SWISS-MODEL (https: / / swissmodel.expasy.org / interactive).

[0039] Predicted using Expasy-Protfaram, the molecular weights of HCT1 and HCT2 are 55.93496 and 47.51469 kDa, respectively; their theoretical isoelectric points are 6.29 and 6.28, respectively; their instability indices are 41.24 and 48.9, respectively; and their adiposeity coefficients are 83.3 and 80.21, respectively. Predicted using the online ProtScale software, their total hydrophilicity is -0.238 and -0.118, respectively, suggesting they are hydrophobic acidic proteins. Bioinformatics analysis revealed that HCTs proteins lack transmembrane domains. Subcellular localization prediction showed that HCTs proteins are located in the cytoplasm. Joint analysis of the HCT protein domains using NCBI's conserved domain analysis tool and the online tool SMART revealed that both HCT proteins belong to the transferase superfamily; specifically, HCT1 has only one transferase domain, while HCT2 has two transferase domains. Secondary structure analysis of HCT proteins revealed that HCT1 protein is mainly composed of α-helices, extended chains, random coils, and some structures yet to be identified. In HCT2 protein, the proportion of unidentified structures is significant, reaching 29.36%. Figure 1-1 A). By constructing three-dimensional models of HCT proteins using SWISS-MODEL, the highest homologous template for HCT1 was found to be 4g0b.1.A, derived from the HCT protein in Coffea canephora, with a model coverage of 86% and a similarity of 34%. For HCT2, the highest homologous template was 5kjt.1.A, derived from the crystal structure of the HCT complex with p-coumaroyl-CoA in Arabidopsis thaliana, with a model coverage of 91% and a similarity of 36%. Figure 1-1 B). Furthermore, phylogenetic tree construction revealed that Ginkgo HCT1 and the hydroxycinnamoyltransferase (XP_057833148.2) of Cryptomeria japonica clustered together, indicating a closer phylogenetic relationship. In contrast, Ginkgo HCT2 was slightly less related. Figure 1-2 Motif analysis showed that Ginkgo HCT1 and XP_057833148.2 had the highest motif similarity, with HCT2 lacking only motif 7 compared to XP_057833148.2. Compared to HCT proteins from other species, HCT2 lacks motif 8, and compared to HCT1 protein, it has two more motifs (motif 11 and motif 14), but also lacks motif 9. Figure 1-3 ).

[0040] 2. Expression pattern of Ginkgo HCTs genes

[0041] To determine the expression levels of Ginkgo HCTs genes in different tissues and under different treatments, RNA-seq data from different Ginkgo tissues were obtained from a public transcriptome database. The sequencing data were aligned to the Ginkgo reference genome using STAR software, and gene expression levels were calculated using RSEM and normalized to log2(FPKM+1). Heatmap analysis revealed tissue-specific expression patterns among different gene families.

[0042] The expression patterns of two HCT genes in different tissues and treatment groups of Ginkgo biloba were analyzed using FPKM values, and visualized using heatmaps. Figure 2 As shown in the heatmap, the expression levels of both HCT genes are generally higher in female buds, indicating their important roles in this specific tissue. In contrast, HCT gene expression levels are relatively lower in the cambium, mature leaf, mature fruit, and kernel. The expression pattern of the HCT2 gene also exhibits some tissue specificity, with higher expression levels in ovulate strobilus, male buds, and immature leaf, while expression levels are relatively lower in the root. The expression levels of HCT1 and HCT2 genes also differ across tissues, with HCT1 expression being higher in immature leaf tissue compared to other tissues. In addition to tissue specificity, the effects of BR, ABA, and SA treatments on HCT1 and HCT2 gene expression were also investigated. The results showed that under different concentrations of BR treatment, the expression level of HCT2 was higher than that of HCT1, indicating that BR tends to promote the expression of HCT2. However, under different concentrations of ABA treatment, the opposite trend was observed, that is, the expression level of gene HCT1 was higher than that of HCT2. Under SA treatment, the expression level of HCT2 was higher than that of HCT1. Figure 2 ).

[0043] 3. Cloning of the GbHCT1 (i.e., HCT1) gene, vector construction, and detection of transgenic plants.

[0044] Total RNA was extracted from Ginkgo biloba leaves using the BioTeKe Universal Plant Total RNA Rapid Extraction Kit, following the manufacturer's instructions. RNA integrity was verified by 1% agarose gel electrophoresis, and purity was measured using a NanoDrop 2000 spectrophotometer. To determine the biological function of GbHCT1, specific primers were designed using Oligo 6.0 software, and PCR amplification was performed using TaKaRaPrimeSTAR Max DNA Polymerase. The open reading frame sequence of the Ginkgo biloba GbHCT1 gene was obtained using molecular cloning technology (Table 1). The cloned sequence of Ginkgo biloba GbHCT1 is shown in SEQ ID NO.1, and it was found that the encoded polypeptide contains 494 amino acids, with the stop codon being TAA. The amino acid sequence is shown in SEQ ID NO.2. The ORF plasmid obtained after first-generation Sanger sequencing was then ligated into the multiple cloning site of the plant binary expression vector pCAMBIA1302, which contains a 35S strong promoter and a GUS reporter gene. After amplification of the recombinant plasmid in E. coli, the correctness of the inserted sequence was verified by first-generation Sanger sequencing (Qingke Biotechnology). Positive vectors were introduced into Agrobacterium tumefaciens GV3101 via electroporation. Subsequently, the overexpression vector pCAMBIA1302 of the target gene was transformed into tobacco using the Agrobacterium-mediated leaf disc method. After kanamycin selection, PCR, and RT-qPCR verification, high-expression transgenic lines were selected. Total RNA was extracted from transgenic tobacco leaf tissue using the VazymeFastPure Universal Plant Total RNA Isolation Kit (RC411). Residual genomic DNA was digested with DNase I, followed by reverse transcription to synthesize cDNA. RT-qPCR analysis was performed using SYBR Green Premix (TaKaRa, China), with the tobacco Actin gene (Table 1) as an internal control. -ΔΔCt The relative expression levels of the target gene were calculated using a method, and the data were statistically analyzed using GraphPad Prism 9.0.

[0045] Table 1 Primer sequence information

[0046]

[0047] To further clarify the functional location of the protein encoded by the Ginkgo biloba GbHCT1 gene within cells and its mechanism of action in the flavonoid synthesis pathway, based on tobacco protoplast results, HCT1 may be located in the nucleus and cytoplasm. Figure 3 ).

[0048] 4. Identification of expression level of Ginkgo GbHCT1 gene in tobacco and analysis of related gene expression patterns

[0049] To elucidate the biological function of HCT, GbHCT1 was overexpressed in K326 tobacco, and the cells were screened using kanamycin. Figure 4-1 Following A), 10 transgenic tobacco lines were randomly selected for expression level detection. The results showed that GbHCT was highly expressed in multiple transgenic lines. The three transgenic K326 tobacco lines with the highest expression levels (HCT1, HCT4, and HCT9) were selected for expression level detection of structural genes and key transcription factors in the flavonoid synthesis pathway. Figure 4-1 B). To further understand the effects of GbHCT1 heterologous expression on the expression of key enzyme genes and transcription factors in the tobacco flavonoid biosynthesis pathway ( Figure 4-2 , 4-3 The study found that, compared to the control group, the expression levels of key enzyme genes NtHCT, NtPAL1, and NtDFR were significantly increased in transgenic tobacco. However, the expression level of NtFLS1 was significantly decreased in transgenic tobacco. Furthermore, analysis of the expression levels of different transcription factors in transgenic tobacco revealed that, compared to the control group, the expression levels of NtbHLH1, NtERF4a, and NtERF4b were significantly decreased in the three transgenic lines, while the expression levels of NtMYL2a and NtMYB4 were significantly increased in transgenic tobacco. Figure 4-2 , 4-3 ).

[0050] 5. Flavonoid Analysis of GbHCT1 Transgenic Tobacco

[0051] (1) Sample pretreatment for the determination of flavonoid metabolites

[0052] Six samples of K326 tobacco leaves were freeze-dried under vacuum, then ground in a ball mill at a vibration frequency of 30 Hz for 1.5 min. 20 mg of the powder sample was then taken, and 10 μL of a 4000 nmol / L internal standard working solution and 500 μL of 70% methanol solution were added to prepare extracts for each sample. After sonication for 30 min, the extracts were centrifuged at 4°C for 5 min at 12000 rpm. The supernatant was collected after centrifugation, filtered through a 0.22 μm filter membrane, and stored in sample vials for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0053] (2) Chromatographic and mass spectrometric acquisition conditions

[0054] 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 / ).

[0055] The mass spectrometry system configuration includes: an electrospray ionization (ESI) source set at 550 °C. The mass spectrometry voltage is set to 5500 V in positive ion mode and -4500 V in negative ion mode; the curtain gas (CUR) is 35 psi. In the Q-Trap 6500+, each ion pair is scanned and detected based on optimized declustering potential (DP) and collision energy (CE).

[0056] The liquid chromatography system configuration includes: (1) Column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 100 mm × 2.1 mm id). (2) Mobile phase composition: Mobile phase A is ultrapure water with 0.05% formic acid added; Mobile phase B is acetonitrile with 0.05% formic acid added. (3) Operating parameters: The flow rate is set to 0.35 mL / min, the column temperature is maintained at 40 ℃, and the sample injection volume is 2 μL. (4) Elution program: At the start of elution (0 min), the ratio of phase A to phase B is 90:10; after 1 min, the ratio is adjusted to 80:20; at 9 min, the ratio becomes 30:70; at 12.5 min, the ratio is further adjusted to 5:95 and maintained until 13.5 min; after 13.6 min, the ratio quickly returns to 90:10 and continues until 15 min.

[0057] (3) Screening and enrichment analysis of differential metabolites

[0058] Metabolites were identified using the OPLS-DA analysis model and variable importance projection (VIP) scores. Metabolites with a VIP score greater than 1 were defined as metabolites with preliminary significant differences between groups. Then, metabolites with fold change ≥ 2 and fold change ≤ 0.5 were selected to further screen for metabolites with more pronounced differences. Furthermore, differentially expressed metabolites were annotated and visualized using the KEGG database.

[0059] Qualitative and quantitative data analysis of flavonoids in transgenic K326 tobacco leaves revealed 56 metabolites. Further screening identified 26 differentially regulated metabolites (16 downregulated and 10 upregulated). Figure 5-1 , 5-2This includes eight categories: dihydroflavonoids, dihydroflavonols, succinones, isoflavones, chalcones, flavanols, flavones, and flavonols. Flavonoids contain the most differentially metabolized substances, including 11 substances such as luteolin, apigenin, diosmetin, jaceosidin, scutellarin, homoplantaginin, hydroxygenkwanin, 6-hydroxyflavone, tectochrysin, hispidulin, and galangin. Flavonols include isorhamnetin-3-O-glucoside. Four differentially metabolized compounds were identified: 3-O-glucoside, kaempferol, isorhamnetin, and tiliroside; four differentially metabolized compounds were identified in dihydroflavonoids: pinocembrin, eriodictyol, narirutin, and poncirin; the differentially metabolized compounds of xanthones were mangiferin and isomangiferin; two differentially metabolized compounds of flavanols were identified: (-)-catechin gallate and (-)-epicatechin; the differentially metabolized compound of dihydroflavonols was dihydrokaempferol; the differentially metabolized compound of isoflavones was glycitin; and the differentially metabolized compound of chalcones was phloretin. Among the metabolites, luteolin was the most significantly upregulated metabolite compared to the control group, with an upregulation of 3.3-fold. This was followed by isorhamnetin 3-O-glucoside, a flavonol, which was upregulated 2.56-fold compared to the control group. Among the downregulated metabolites, (-)-catechin gallate was the most significantly downregulated among flavanols, with an average content of approximately 0.26 nmol / g in the HCT group, a downregulation of approximately 8.16-fold. This was followed by linalool, a flavonoid, with an average content of approximately 0.07 nmol / g in the HCT group and approximately 0.35 nmol / g in the control group, a downregulation of 4.21-fold.The 10 upregulated differential metabolites were: isomangiin, baicalin, mangiferin, 6-hydroxyflavone, cynomolgin, cyanidin, luteolin, romaine, isorhamnetin-3-O-glucoside, and naringin.

[0060] Furthermore, further classification of differentially metabolized flavonoids in transgenic and non-transgenic plants according to pathway types in KEGG revealed nine differentially metabolites in flavonoid biosynthesis, including sakuranetin, pinocembrin, phlorizin, xanthohumol, naringenin chalcone, quercetin, kaempferol, apigenin, and luteolin, which were the most numerous, accounting for 90% of the significantly differentially metabolites. Three differentially metabolites were found in flavonoid and flavonol biosynthesis: luteolin, apigenin, and kaempferol. One differentially metabolite was found in isoflavone biosynthesis: daidzein. There are seven metabolic differentials in both metabolic pathways and the biosynthesis of secondary metabolites, accounting for 70% of the total. Figure 5-3 ).

[0061] 6. Transcriptome sequencing analysis of transgenic tobacco

[0062] (1) Transcriptome sequencing

[0063] RNA was extracted from tobacco leaves of the CK group and the transgenic K326 group. RNA concentration was first measured using a Qubit 4.0 fluorometer, and then RNA integrity was precisely checked using a Qsep400 bioanalyzer. After passing the tests, library construction was performed. mRNA was then isolated, cDNA was synthesized, and the entire library preparation process involved end repair, A-tailing, sequencing adapter addition, purification, PCR amplification, denaturation, and circularization. After library construction, preliminary quantification was performed using Qubit, followed by insertion fragment detection using an Agilent 2100 to ensure library quality. Following these steps, library quality control was performed. Preliminary quantification was performed using Qubit 2.0, and the insert size was checked using a fragment analyzer. If the insert size met expectations, the effective concentration of the library was accurately quantified using Q-PCR (effective concentration > 2 nM). Only after the library quality control results met the requirements could sequencing proceed. After passing the library quality control, DNA Nano Balls (DNBs) were prepared, loaded onto sequencing chips, and sequenced using a high-throughput sequencer.

[0064] (2) Transcriptome analysis

[0065] HISAT was used to build an index, and clean reads were aligned to a reference tobacco genome. StringTie was used for new gene prediction, employing a network flow algorithm and optional de novo assembly to assemble transcripts. Gene expression levels were quantified using the most commonly used FPKM method. Differential expression analysis between the two groups was performed using DESeq2, with p-values ​​corrected using the Benjamini & Hochberg method. The corrected p-value and log2foldchangel were used as thresholds for significant differential expression. Enrichment analysis was performed based on hypergeometric tests; for KEGG, hypergeometric distribution tests were performed on a pathway-by-pathway basis; for GO, GO term-based enrichment was used.

[0066] (3) Differential gene analysis

[0067] Differential expression analysis was performed using DESeq2, with corrected P-value (FDR < 0.05) and |log2(FoldChange)| ≥ 1 as screening thresholds. Differential genes were functionally enriched using the KEGG and GO databases.

[0068] Transcriptome sequencing analysis of HCT transgenic and non-transgenic tobacco leaves yielded 27.86 G data records (Table 2). 39,134 genes were annotated, of which 3,617 were differentially expressed (2,517 genes were upregulated). Further investigation into the functional distribution of these differentially expressed genes revealed their important roles in several key secondary metabolic pathways, including phenylpropanoid biosynthesis, flavonoid biosynthesis, flavonone and flavonol biosynthesis, and isoflavone biosynthesis. Particularly in the phenylalanine biosynthesis pathway, 35 differentially expressed genes were identified, indicating that the transgenic HCT gene may significantly affect the biosynthesis of phenylalanine and its downstream products (Figure 5). In the flavonoid biosynthesis pathway, 12 differentially expressed genes were annotated. Two differentially expressed genes exist in the isoflavone and flavonol synthesis pathways: flavonol-3-O-glucoside L-rhamnosyltransferase and flavonoid 3'-monooxygenase.

[0069] The biosynthetic pathway of flavonoids begins with phenylalanine, which is converted to cinnamic acid by PAL, followed by C4H hydroxylation to p-coumaric acid, and then to p-coumaroyl-CoA via 4CL. This substance then proceeds through multiple branching pathways, undergoing HCT and C3′H reactions to convert to caffeoyl-CoA, providing a precursor for subsequent flavonoid skeletal synthesis. Caffeoyl-CoA, under CHS catalysis, generates the downregulated differential metabolite sennarol, which is further converted to (-)-Epicatechin by enzymes such as F3H. Meanwhile, naringenin and other enzymes can also be catalyzed by other enzymes such as CYP75B1 to form various flavonoids. Some flavonoids can also be converted to (-)-epicatechin by ANR. Figure 5-1 , 5-2Coumarin-CoA, under the catalysis of CHS, enters different branch pathways, generating naringenin, phloretin, and pinocembrin-, respectively. Phloretin and pinocembrin showed downregulation after HCT overexpression. Pinocembrin, under the action of F3H and FLS, ultimately generates galangin. Naringenin, under the catalysis of FLS, generates the downregulated differential metabolite apigenin, which is then catalyzed by CYP75B1 to form luteolin. Overexpression of HCT upregulates this metabolite, which is then converted to (-)-epigallocatechin by ANR. Further metabolomics data revealed a high correlation between differentially expressed genes and the accumulation of flavonoid metabolites. Figure 6 The shunting trend of caffeoyl-CoA in the phenylpropane pathway was consistent with the expression changes of most genes. Integrated transcriptomic and metabolomic analysis showed that GbHCT1 overexpression activates upstream genes and inhibits downstream flavanol synthesis-related genes through positive feedback, ultimately driving the massive accumulation of flavonoids. These results elucidate the dual regulatory role of Ginkgo GbHCT1 in secondary metabolism from a multi-omics perspective.

[0070] Table 2

[0071]

[0072] In summary, Ginkgo GbHCT1 belongs to the Transferase superfamily. GbHCT1 is a hydrophobic acidic protein containing a single domain, lacking a transmembrane domain, and subcellularly located in the cytoplasm. GbHCT1 is highly expressed in female buds and under ABA treatment. Overexpression of the Ginkgo GbHCT1 gene significantly affects the synthesis and accumulation of flavonoids by dynamically regulating carbon source allocation in the phenylpropanoid metabolic pathway. Heterologous expression of GbHCT1 promotes the accumulation of flavonoids in tobacco while inhibiting the production of flavanol metabolites. At the gene expression level, GbHCT1 overexpression significantly upregulates key genes in the phenylpropanoid pathway, forming a positive feedback loop to enhance flavonoid synthesis; simultaneously, it inhibits flavonol synthase genes and transcription factors, leading to reduced flavanol pathway activity. KEGG enrichment analysis further confirmed that differentially expressed metabolites are mainly enriched in the flavonoid biosynthesis pathway, indicating that GbHCT1 reshapes the synthetic pattern of secondary metabolites by coordinating carbon source allocation and transcriptional regulatory networks. In summary, this application reveals for the first time the dual regulatory mechanism of the Ginkgo biloba GbHCT1 gene in the biosynthesis of flavonoids. It upregulates key genes such as NtPAL1, NtHCT, and NtDFR to promote the synthesis and accumulation of flavonoids, such as luteolin, while inhibiting flavanol branch gene NtFLS1 and transcription factors such as NtbHLH1 and NtERF4a / b, resulting in a significant reduction in flavanol metabolites.

[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 GbHCT1 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the Ginkgo GbHCT1 gene according to claim 1, the amino acid sequence of which is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the Ginkgo GbHCT1 gene as described in claim 1.

4. The application of the Ginkgo GbHCT1 gene of claim 1, the expression protein of the Ginkgo GbHCT1 gene of claim 2, or the biomaterial of claim 3 in plant breeding.

5. The application of the Ginkgo GbHCT1 gene of claim 1, the expression protein of the Ginkgo GbHCT1 gene of claim 2, or the biomaterial of claim 3 in regulating the synthesis of plant flavonoids.

6. A method for increasing the synthesis of flavonoids in plants, characterized in that, Overexpression of the Ginkgo GbHCT1 gene of claim 1 in plants, wherein the flavonoid compound is at least one of the following compounds: isomangiin, baicalin, mangiferin, 6-hydroxyflavone, crotonin, cyanidin, luteolin, romaine, isorhamnetin-3-O-glucoside, and naringin.

7. The method according to claim 6, characterized in that, Includes the following steps: (1) Construct the expression vector of the Ginkgo GbHCT1 gene as described in claim 1; (2) Transform the constructed Ginkgo GbHCT1 gene expression vector into plants or plant tissues; (3) Cultivate and screen transgenic plants or plant tissues with increased flavonoid content.

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

9. The method according to claim 7, characterized in that, The overexpression vector for the Ginkgo GbHCT1 gene is pCAMBIA1302-GbHCT1.

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

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