Strawberry flavonoid biosynthesis regulation gene and application thereof

Abstract

The application discloses a strawberry flavonoid biosynthesis regulation gene and application thereof, and relates to the field of plant genetic engineering. Specifically, a gene closely related to the regulation of strawberry flavonoid biosynthesis is identified FaGeBP3 , and the influence of the gene on flavonoid biosynthesis is verified by constructing overexpression and interference strains through agrobacterium transformation, and finally, the molecular mechanism of the regulation of metabolism is tried to be researched. The gene can be used in the regulation of plant flavonoid biosynthesis, and helps to cultivate plants with more flavors and nutrients, especially new strawberry varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to flavonoid regulatory genes and their application in regulating plant flavonoid synthesis. Background Technology

[0002] Strawberries are favored by consumers worldwide for their unique flavor and rich nutrients (especially polyphenols). Among them, flavonoids are an important class of secondary metabolites that not only affect fruit color and antioxidant capacity, but also influence plant development, reproduction, and adaptation to adversity.

[0003] Flavonoid biosynthesis is a complex network originating from the phenylpropane pathway, in which chalcone isomerase (CHI) acts as a key rate-limiting enzyme, catalyzing the formation of flavanones, which are common precursors to various flavonoid subclasses. Due to their important biological significance, the transcriptional regulation of flavonoid biosynthesis by environmental factors and hormonal signals has become a research hotspot.

[0004] The GLABROUS1 enhancer-binding protein (GeBP) family is a plant-specific transcription factor family, first identified in Arabidopsis thaliana in 2003. Arabidopsis thaliana GeBPs were identified in the GLABROUS1 gene enhancer due to their strong binding affinity. The GeBP family differs from typical leucine zipper transcription factor families in the number of leucine residues within their domains and the distance between these residues. This unique structural configuration allows GeBPs to form both homodimers and heterodimers, thus enabling more flexible regulation of target genes. Studies in multiple species have shown that GeBPs are involved in developmental processes and stress responses. For example, AtGPL in Arabidopsis controls leaf senescence, and in apples… Malus domestica MdGeBP3 in *Solanum lysate* regulates root formation and flowering. Other homologs in Chinese cabbage and rice respond to drought and heavy metal stress, while SlGeBP in Solanaceae responds to viral infection. Although already described... GeBP Genes respond to biotic and abiotic stresses, but little is known about the molecular mechanisms underlying their biological effects. Furthermore, the role of GeBP transcription factors in strawberries has not been reported; in particular, their regulatory role in secondary metabolism remains to be explored. Summary of the Invention

[0005] To address the aforementioned technical issues, this invention identified a novel gene regulating flavonoid synthesis, verified its function, and revealed its molecular mechanism.

[0006] On the one hand, the present invention provides a gene that regulates the biosynthesis of plant flavonoids.

[0007] In some embodiments, the gene encodes a protein with an amino acid sequence as shown in SEQ ID NO:2.

[0008] In some implementations, the gene is FaGeBP3 In some specific implementations, the aforementioned FaGeBP3 The gene comprises a nucleotide sequence as shown in SEQ ID NO:1. In some specific embodiments, the... FaGeBP3 The gene has a nucleotide sequence as shown in SEQ ID NO:1.

[0009] In some embodiments, the gene comprises a nucleotide sequence as shown in SEQ ID NO:1.

[0010] In some embodiments, the gene has a nucleotide sequence as shown in SEQ ID NO:1.

[0011] In some embodiments, the plant includes strawberries, peaches, pears, and apples. In some specific embodiments, the plant is a strawberry.

[0012] On the other hand, the present invention provides FaGeBP3 Application of genes in regulating the biosynthesis of flavonoids in plants.

[0013] In some implementations, the FaGeBP3 Genes regulate the biosynthesis of flavonoids by interacting with the FaCHI promoter.

[0014] In some embodiments, the gene encodes a protein with an amino acid sequence as shown in SEQ ID NO:2.

[0015] In some embodiments, the FaGeBP3 gene comprises the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the FaGeBP3 gene has the nucleotide sequence shown in SEQ ID NO:1. In some embodiments, the FaGeBP3 gene has the nucleotide sequence shown in SEQ ID NO:1.

[0016] In some implementations, the FaGeBP3 gene is normally expressed or overexpressed, negatively regulating the biosynthesis of flavonoids.

[0017] In some implementations, the FaGeBP3 gene is downexpressed or not expressed, positively regulating and enhancing the biosynthesis of flavonoids.

[0018] In some embodiments, the plant includes strawberries, peaches, pears, and apples. In some specific embodiments, the plant is a strawberry.

[0019] On the one hand, the present invention provides a method for improving the biosynthesis of plant flavonoids.

[0020] In some implementations, the biosynthesis of plant flavonoids is enhanced by inhibiting or knocking out the expression of the FaGeBP3 gene.

[0021] In some embodiments, the FaGeBP3 gene has a nucleotide sequence as shown in SEQ ID NO:1.

[0022] In some embodiments, the plant includes strawberries, peaches, pears, and apples. In some specific embodiments, the plant is a strawberry. Attached Figure Description

[0023] Figure 1 This section presents the distribution of GeBP genes in different species and the results of phylogenetic analysis. (a) is a bar chart showing the number of GeBP genes in different species. Colors represent different taxa and ploidy groups: orange for diploid strawberries, yellow for tetraploid strawberries, green for octoploid strawberries, blue for other Rosaceae species, and gray for representative species from more distant families. The numbers within the red circles above the bars indicate the number of unique GeBP gene loci identified in each strawberry germplasm. (b) is a phylogenetic tree of GeBP proteins from 20 strawberry germplasms, 13 Rosaceae species, and 6 more distant families. Labels beginning with "FvRg" represent 'Ruegen' forest strawberries. Fragaria vesca ), 'FvYW' stands for 'Yellow Miracle' ( Fragaria vesca 'Yellow Wonder'), 'FvH4' represents 'Hawaii No. 4' forest strawberry ( Fragaria vesca 'Hawaii4', 'Fii' represents Northeast strawberries ( Fragaria iinumae 'Fnip' stands for Japanese strawberry ( Fragaria nipponica 'Fviri' stands for green strawberry ( Fragaria viridis 'Fnil' represents yellow-haired strawberry ( Fragaria nilgerrensis 'Fda' represents Tibetan strawberry ( Fragaria daltoniana 'Fma' represents Northeast strawberries ( Fragaria mandschurica 'Fnu' represents the split-calyx strawberry ( Fragaria nubicola 'Fpe' stands for five-leaf strawberry ( Fragaria pentaphylla 'Fmo' represents Southwest strawberry ( Fragaria moupinensis 'For' represents Oriental Strawberry ( Fragaria orientalis 'Fchi' stands for Chilean strawberry ( Fragaria chiloensis 'Fvirg' stands for Virginia strawberry ( Fragaria virginiana'FaFB' stands for 'Florida Glorious' pineapple strawberry. Fragaria × ananassa 'Florida Brilliance', 'FaCL' represents 'first love' pineapple strawberry ( Fragaria × ananassa 'Chulian', 'FaYL' represents 'vibrant' pineapple strawberry ( Fragaria × ananassa 'Yanli'), 'FaFVC' represents 'FVC' pineapple strawberry ( Fragaria × ananassa 'FVC', 'FaBe' represents 'Red Beauty' pineapple strawberry ( Fragaria × ananassa (Benihoppe). The pie chart on the branches shows the proportion of strawberry GeBP genes in each branch relative to the total number of strawberry GeBP genes (n=138). The colored circles at the nodes represent the self-support values: dark red (>90%), light red (70%-90%), and gray (<70%) represent high, medium, and low confidence levels, respectively.

[0024] Figure 2 This represents the orthologous genomes of the GeBP family in 20 strawberry varieties. Specifically: (a) Homologous gene cluster (OGG) identification and clustering analysis: Homologous genes from different materials were clustered through sequence alignment, and the number of genes contained in each homologous group (OGG1-OGG7) was counted. (b) Gene presence / deletion variation (gPAV) analysis: The distribution of each homologous genome in the 20 strawberry materials was detected and visualized. (c)-(d) Pan-genome component classification analysis: Based on the frequency of gene occurrence in the population, genes were classified into functional categories such as core genes, softcore genes, and shell genes.

[0025] Figure 3 This represents a phylogenetic tree of GeBP proteins based on 20 strawberry germplasms.

[0026] Figure 4 This diagram illustrates the conserved motifs and gene structures of the GeBP gene family in strawberry. (a) shows a phylogenetic tree displaying OGG (homologous group) clustering. (b) A schematic diagram of the 10 most conserved protein motifs. The scale bar indicates amino acid sequence length. (c) The gene structure of GeBPs, showing the UTR, CDS, and introns. The scale bar indicates gene sequence length.

[0027] Figure 5This section presents an analysis of replication events and selection pressures in the strawberry GeBP gene. (a) Distribution of gene replication types within each OGG. Stacked bar charts show the number of each replication type identified within each OGG, while a proportionate dot plot shows the proportion of each replication type. (b) Comparison of Ka values ​​between core GeBP and non-essential GeBP. (c) Changes in the Ka / Ks ratio among OGGs.

[0028] Figure 6 The values ​​represent the expression patterns of the GeBP gene in different tissues as detected by RT-qPCR. (a)-(c) correspond to *Strawberry 'First Love'*, *Strawberry 'First Love'*, and *Strawberry 'Glamour'*, respectively.

[0029] Figure 7 The results of the self-activation activity and subcellular localization analysis of FaGeBP3 are shown. Among them, (a) and (c) are the results of the yeast two-hybrid experiment; (b) is the confocal microscopy image, scale bar = 25 μm.

[0030] Figure 8 The results of functional analysis of FaGeBP3 in transgenic strawberry lines are presented. (a) PCR identification of transgenic lines. M: DNA molecular weight marker; PC: positive control; NC: negative control (wild-type "First Love"); (b) relative expression levels of FaGeBP3 in leaves of wild-type (WT), FaGeBP3 overexpression (FaGeBP3-OE), and FaGeBP3 RNA interference (FaGeBP3-RNAi); (c) phenotypic comparison between wild-type and transgenic plants; (d, e) chlorophyll (d) and flavonoid (e) contents in wild-type, FaGeBP3-OE, and FaGeBP3-RNAi lines. Data are expressed as mean ± standard deviation (n=3). An asterisk indicates a statistically significant difference compared to the wild-type (*P<0.05, **P<0.01).

[0031] Figure 9 The results demonstrate the negative regulation of FaGeBP3 on FaCHI transcription. (a) Relative expression levels of FaCHI in wild-type (WT), FaGeBP3-overexpressed (FaGeBP3-OE), and FaGeBP3 RNA interference (FaGeBP3-RNAi) strawberry leaves; (b, c, d, e) Yeast one-hybrid (Y1H) results; (f) Electrophoretic mobility shift assay (EMSA) results; (g) Dual-luciferase reporter gene assay results; (h) GUS staining and enzyme activity assay results. An asterisk (*) indicates a statistically significant difference (p<0.05).

[0032] Figure 10 The expression patterns of FaGeBP3 and FaCHI in leaves treated with methyl jasmonate (MeJA) are shown. (a) Relative gene expression of FaGeBP3. (b) Relative gene expression of FaCHI. Data are expressed as mean ± standard deviation (n=3). An asterisk (*) indicates a statistically significant difference (p<0.05). Detailed Implementation

[0033] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of this disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0034] Example 1: Identification and Phylogenetic Analysis of the GeBP Gene Family

[0035] To elucidate the pangenome of the strawberry GeBP gene family, we analyzed the genomes of 20 strawberry resources.

[0036] These germplasms included 11 diploids (Forest Strawberry, Yellow Miracle, Hawaii No. 4, Iinuma Strawberry, Japanese Strawberry, Green Strawberry, Yellow-haired Strawberry, Cleft-calyx Strawberry, Northeast Strawberry, Tibetan Strawberry, and Five-leaf Strawberry), 2 tetraploids (Southwest Strawberry and Oriental Strawberry), 2 wild octoploids (Chilean Strawberry and Florida Strawberry), and 5 octoploid cultivars ('Florida Brilliance', 'First Love', 'Glamour', 'FVC', and 'Red Beauty'). In addition, 13 other Rosaceae species (peach, pear, apple, etc.) and 6 adenotas (including Chlorella, Bryum simonii, Adiantum, Arabidopsis, Cinnamomum camphora, and potato) were analyzed.

[0037] According to Curaba et al. (Curaba, J., Herzog, M. & Vachon, G.) GeBP , the first member of a new gene family in Arabidopsis , encodes a nuclear protein with DNA‐binding activity and is regulated by KNAT1 . Plant J . 2003;33:305-317.Chevalier, F. et al. GeBP and GeBP-LikeProteins Are Noncanonical Leucine-Zipper Transcription Factors That Regulate Cytokinin Responsein Arabidopsis . Plant Physiol . 2008;146:1142-1154.Ma, C., Chen, QJ, Wang,SP&Lers, A. Downregulation of GeBP-like α factor by MiR827 suggests theirinvolvement in senescence and phosphate homeostasis. BMC Biol The GeBP gene family characteristics identified in this study (2021;19:90) – namely, the central and C-terminal domains – are crucial for GeBP activity in yeast. This study employed strict identification criteria: proteins must contain both central and C-terminal domains to be designated as GeBP. By performing homology alignment on 21 Arabidopsis GeBP protein sequences and constructing a Hidden Markov Model (HMM) feature file, we identified 503 GeBP genes across 39 plant genomes.

[0038] Specifically, we identified 354 GeBP gene sequences in these 20 strawberry genomes. To standardize comparisons across different ploidy levels, we determined the number of loci by selecting the longest allele at each locus as the representative sequence (Figure 1a). Although the total number of genes varied significantly due to polyploidy, the number of standardized GeBP loci remained relatively consistent across the 20 strawberry resources (Figure 1a). In contrast, no GeBP genes were identified in Chlorella, while they were present in Sphagnum moss. The identified GeBP proteins exhibited significant diversity in physicochemical properties, including sequence length, molecular weight, and isoelectric point.

[0039] To avoid interference from alleles in polyploid strawberries, we selected the longest allele sequence from each locus as the representative sequence. These representative sequences were systematically named GeBP1 to GeBP7 based on their chromosomal location in their respective species. Ultimately, we obtained a normalized dataset containing 138 GeBPs for subsequent analysis. A phylogenetic tree was constructed using the 138 strawberry GeBPs and 149 GeBPs from other species to demonstrate the evolutionary relationships within the GeBP family (Figure 1b).

[0040] Example 2: Clustering and Classification of Orthologous GeBP Genes

[0041] Using OrthoFinder (v2.5.5) software with default parameters, the identified GeBP genes were clustered into orthologous genomes (OGGs). Based on the gene presence / deletion variation (gPAV) patterns in these 20 strawberry germplasms, these OGGs were then divided into two categories: core genes (present in all germplasms) and non-core genes (deleted in at least one germplasm).

[0042] Orthologous gene analysis divided 138 representative GeBP sequences into seven core orthologous genomes (OGG1–OGG7), laying a clear evolutionary foundation for subsequent comparative and functional studies within the strawberry pangenome. Given the extremely high sequence homology and similarity among members of each OGG in the strawberry pangenome, we named each OGG after the GeBP genes it contains. Specifically, OGG1 contains GeBP1 and its orthologs, OGG2 contains GeBP2 and its orthologs, and so on (Figure 2a). Based on the presence / deletion variant (gPAV) maps of 20 strawberry resources, these seven OGGs were classified into three distinct levels: core genes (present in 100% of the resources), including OGG3; softcore genes (present in ≥ 85% of the resources), including OGG1, OGG5, and OGG7; and shell genes (present in ≥ 5% of the resources, i.e., present in at least one resource), including OGG2, OGG4, and OGG6 (b–d in Figure 2). OGG3 is the only core group that remains conserved across all resources.

[0043] Phylogenetic clustering of the OGGs further clarified their evolutionary relationships. Specifically, OGG1, OGG3, and OGG4 each formed an independent cluster; OGG5 and OGG6 clustered together; and OGG2 and OGG7 formed another cluster. Phylogenetic analysis further confirmed the evolutionary conservation of the core group OGG3. Figure 3 Within the GeBP orthologous gene groups (OGGs), different evolutionary patterns can be clearly observed through conserved motif and gene structure analysis. Figure 4Significant differences exist among the OGG genes in their conserved motif composition. Only the core orthologous gene group OGG3 retains all 10 motifs, indicating strong functional constraints; the remaining OGG genes contain 2 to 8 motifs, with OGG1, OGG5, and OGG7 having the most (6, 8, and 6 motifs, respectively). This diversity in gene structure is consistent with the diversity in motif patterns. OGG genes typically contain only one or two exons. In contrast, OGG4 has the most complex structure, possessing up to six exons. Structurally, OGG5 is the most conserved, while OGG1 and OGG3 show the greatest differences in intron length and number.

[0044] Example 3: Gene duplication events in GeBP family expansion

[0045] Investigating whole-genome duplication (WGD), dispersed duplication, tandem duplication, proximal duplication, and transposon duplication patterns in each OGG to understand the evolutionary mechanisms behind the expansion of the GeBP family.

[0046] Conserved motifs were identified using the analysis tools provided by the MEME suite (http: / / meme-suite.org / ). All analysis parameters remained at their default values, except for the motif count, which was set to 10. Gene structure maps were generated by visualizing the GFF3 annotation files for each strawberry germplasm using TBtools. Collinearity blocks were identified using MCScanX based on pre-calculated BLASTP results (E-value threshold set to 1e-10, retaining the first 5 alignments). Subsequently, DupGen_finder was run with its default settings to determine the repetition event type for each gene.

[0047] The analysis results show that dispersed replication (44.5%) and WGD (37.2%) are the two main replication drivers, together accounting for more than 80% of replication events in the GeBP family. Figure 5 (a) Tandem repeats, proximal repeats, and transposon repeats account for a relatively small proportion, at 14.6%, 2.9%, and 0.7%, respectively. OGG3 and OGG1 contain the majority of tandem repeats, while OGG3 and OGG2 contain proximal repeats. Notably, transposon repeats are only present in OGG7. In contrast, OGG4 exhibits the lowest diversity in repeat mechanisms, with its member genes originating solely from scattered repeats and WGD events. These major repeat mechanisms significantly influence the evolutionary trajectory of the OGG family. Notably, both the core OGG3 and the largest quasi-core OGG (OGG1) have achieved significant expansion primarily through tandem repeats.

[0048] Example 4: Selective pressure on strawberry GeBP orthologs

[0049] For each OGG in the strawberry GeBP family, the nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) of duplicate gene pairs were calculated using TBtools, and their Ka / Ks ratios were also calculated to analyze the environmental selection pressure experienced by each OGG. These indicators are key parameters for assessing the intensity and direction of selection pressure.

[0050] The results indicate a significant evolutionary difference between core and non-essential genes. Although Ka and Ks values ​​are similar, the Ka / Ks ratio is higher for non-essential genes than for core genes. Non-essential genes experience less selection pressure, thus allowing for the accumulation of non-synonymous mutations. Figure 5 (b) in the middle.

[0051] This trend was observed in all OGGs. The Ka / Ks values ​​of the core group OGG3 and the non-essential groups OGG2 and OGG6 were consistently below 1, indicating that they were subject to strong purification selection. Figure 5 (c) In contrast, the Ka / Ks ratios of certain non-essential OGGs (such as OGG1, OGG4, and OGG7, a total of 17 genes) exceeded 1.

[0052] Example 5: Spatiotemporal expression analysis of the FaGeBP gene

[0053] Given that the biological function of GeBP in strawberry is still unclear, this study selected three representative strawberry resources to analyze the expression characteristics of the GeBP gene, including diploid forest strawberry F. vesca 'Ruegen' (red fruit) and octoploid cultivated strawberry F. × ananassa 'Yanli' (red fruit) and F. × ananassa 'Chulian' (white fruit).

[0054] The tissues examined included roots, shoot tips, mature leaves (ML), flowers, and fruits at different developmental stages: small green fruit stage (SG), large green fruit stage (BG), white ripe stage (W), color-changing stage (T), and red ripe stage (R). Real-time quantitative PCR (RT-qPCR) analysis identified four highly expressed GeBP genes (GeBP1, GeBP3, GeBP5, and GeBP7), belonging to the nuclear and soft-nucleus genes in the pan-genome family. In contrast, the expression levels of the remaining three genes (GeBP2, GeBP4, and GeBP6), classified as shell genes, were not detected under the experimental conditions.

[0055] The expression patterns of GeBP genes were similar across the three varieties. GeBP1 and GeBP3 showed the highest transcription levels in shoot tips and flowers, while GeBP5 exhibited relatively stable expression in all tested tissues. The expression peak of GeBP7 specifically occurred in the shoot tip. Figure 6 ).

[0056] Example 6: FaGeBP3 Functional verification

[0057] To elucidate the function of core OGG3 (the only OGG in this family classified as a core group), this study selected the cultivar "Chulian" (F. × ananassa 'Chulian'), which possesses excellent transformation capabilities, for investigation. Representative genes in this cultivar... FaYLGeBP3 (For the sake of simplicity, the following text will refer to them as) FaGeBP3 () was selected for functional verification.

[0058] > FaYLGeBP3 Gene

[0059]

[0060] SEQ ID NO:1

[0061] > FaYLGeBP3 Gene expression products

[0062] MAPKRPSPLDQPPAASSSQEEQDASSEEAASGSGSEESESETEASEPPKTTSTAKPQPKKPESAAAAKPQSSSSGETETDSESESDGENLGNRKVDVKPIASKPMEAVTPPPKATKPRSKPQASPATAKSGTKRPNGGESELNKDSKKAKKKGDDQEEDGVEEESSKKSNLFQRLWSDEDEIIILKGMNDYATKKGVD PYADMGAFHEFIKKSLKVDVSKVQLQDKIRRMRKKFENNVRKKHEPTKPHEKKTYELSKKVWGGGEDLAGNTEQPKANGKAKAKSNQKGNSKTLASMKADLLASPEPSKEAEKSGAMKRCASGSVSSEVIGFDKGFKELGLSETIVRQGLELIGGSKCAALKDKWNELHVAELELFVKRTELMRNQTQMILEAIKSDH

[0063] SEQ ID NO:2

[0064] 6.1 Experimental Methods

[0065] Self-activation activity assay method: [The method is described in the original text, but the translation is incomplete and cannot be translated.] FaGeBP3 The gene was cloned and inserted into the pGBKT9 (BD) vector via BamHI and EcoRI restriction enzyme sites. The BD-FaGeBP3 construct was co-transformed with pGADT7 (AD) into Y2HGold cells, followed by 3 days of culture on SD / -Trp / -Leu agar plates. After 3 days of culture, the growth of each isolated positive clone on SD / -Trp / -Leu and SD / -Trp / -His / -Leu / -Ade plates was recorded. To verify the specificity of the observed growth, AD-T / BD-53 (positive control) and AD-T / BD-lam (negative control) were treated under the same conditions as experimental controls.

[0066] Subcellular localization: The fusion vector CaMV35S:: was constructed using the restriction endonucleases NdeI and BamHI. FaGeBP3 -GFP. This will contain GFP and FaGeBP3-GFP-containing Agrobacterium tumefaciens was injected into four-week-old tobacco leaves. After incubation for approximately 48 hours, GFP fluorescence was observed using a Leica DMi8 A confocal fluorescence microscope (Wizlar, Germany).

[0067] Yeast two-hybrid experiment: Gene F aGeBP1、FaGeBP3、FaGeBP5 and FaGeBP7 Separate clones were performed and inserted into the pGADT7 vector using BamHI and EcoRI restriction sites. The recombinant BD- FaGeBP3 The construct and each pGADT7 fusion vector (AD- FaGeBP1 AD- FaGeBP3 AD- FaGeBP5 or AD- FaGeBP7 Yeast cells were co-transformed into Y2HGold cells. After transformation, yeast cells were first screened on SD / -Trp / -Leu agar medium and cultured at 30°C for 72 hours. Subsequently, the validated transformants were spot-inoculated on SD / -Trp / -Leu and SD / -Trp / -His / -Leu / -Ade selective media to evaluate their growth phenotype. The accuracy of the observed interactions was verified by comparison with the positive control (AD-T / BD-53) and the negative control (AD-T / BD-lam).

[0068] 6.2 Experimental Results

[0069] The yeast two-hybrid experiment confirmed that FaGeBP3 Lack of transcriptional self-activation activity ( Figure 7 (a) in the context of instantaneous expression. FaGeBP3 -Green fluorescent protein ( FaGeBP3 -GFP fusion construct was analyzed FaGeBP3 Subcellular localization. Confocal microscopy observation showed that... FaGeBP3 -GFP fusion protein exhibits distinct nuclear localization, with 2 to 3 distinct subnuclear focal points accumulating in each nucleus. Figure 7 (b) suggests that it may have nucleolar targeting. This nuclear localization feature is consistent with its function as a transcription factor and is consistent with patterns observed in Arabidopsis homologs. Furthermore, yeast two-hybrid experiments showed... FaGeBP3 and FaGeBP1 / 3 / 5 / 7 There is no interaction between them. Figure 7 (c in the text)

[0070] Example 7: FaGeBP3 Effects on flavonoid biosynthesis

[0071] To verify FaGeBP3To investigate the function of this technology, researchers cultivated stable overexpression (FaGeBP3-OE) and RNA interference (FaGeBP3-RNAi) lines using Agrobacterium-mediated transformation. This study used the octoploid strawberry variety 'First Love' as the experimental material and explored potential changes by measuring the chlorophyll and flavonoid content in the leaves.

[0072] 7.1 Experimental Methods

[0073] RNA extraction and RT-qPCR were performed following the methods detailed in the literature by Zhao, Q. et al. (Zhao, Q. et al. Genome-wide analysis of the bHLH family and identification of bHLH genes involved in fruit development and ripening of cultivated octoploid strawberry. Food Qual Saf. 2024;8:fyae014.). The experimental design included three biological replicates, and each biological replicate consisted of three technical replicates.

[0074] To determine the concentrations of chlorophyll a and b, the absorbance of an 80% acetone extract prepared from 0.1 g of fresh leaf tissue was recorded at 663 nm and 646 nm. The final pigment content was then calculated using the Lichtenthaler equation.

[0075] The dried sample (0.2 g) was extracted and analyzed using a flavonoid assay kit (Wuhan Pais, China). The flavonoid content was determined by measuring the absorbance of the supernatant at 510 nm and calculated according to the standard formula provided in the kit.

[0076] 7.2 Experimental Results:

[0077] RT-qPCR analysis showed that in the overexpression lines (#1–#3) FaGeBP3 Transcriptional levels were significantly increased (5.28 to 22.1-fold) in the RNA interference lines (#1–#3), while transcriptional levels were decreased to 0.22 to 0.43-fold lower than wild-type (WT) levels. Figure 8 (b) Compared with the wild type, the FaGeBP3-OE (overexpression) and FaGeBP3-RNAi (interference) transgenic lines did not show significant morphological differentiation. Figure 8 (c in the text)

[0078] Results of chlorophyll and flavonoid content determination ( Figure 8 The results (de) showed that although chlorophyll levels remained unchanged, total flavonoid content changed significantly. Specifically, compared to the wild type, the flavonoid content of the FaGeBP3-OE line decreased by 29% to 53%, while the flavonoid content of the FaGeBP3-RNAi#1 line increased by 17%. In summary, these results indicate that... FaGeBP3 It has a negative regulatory effect on the accumulation of flavonoids in strawberries.

[0079] Compared to the control group, the FaGeBP3-OE (overexpression) line showed a significant decrease in flavonoid content, while the FaGeBP3-RNAi (interference) line exhibited the opposite trend, accumulating more flavonoids. The results of stable genetic transformation are sufficient to confirm this. FaGeBP3 Regulatory effect on flavonoid metabolism.

[0080] Example 8: FaGeBP3 Molecular mechanisms regulating flavonoid metabolism

[0081] To verify FaGeBP3 Based on the hypothesis of regulating flavonoid metabolism, we identified potential regulators in the promoters of 10 key structural genes related to flavonoid biosynthesis. FaGeBP3 Binding sites. This predictive analysis aims to establish direct regulatory links.

[0082] 8.1 Experimental Methods

[0083] Prediction of upstream transcription factors of flavonoid biosynthesis structural genes: The promoter sequence of the upstream 2,000 bp of the flavonoid biosynthesis structural genes was obtained, and potential FaGeBP binding sites were predicted and screened using the PlantTFDB database. Default parameters were used in all analyses.

[0084] Yeast one-hybrid experiment: using EcoR I and BamH I. Restriction site amplification and cloning FaGeBP3The coding sequence was inserted into the pGADT7 vector to construct the "prey" vector (pGADT7::FaGeBP3). The FaCHI promoter region (approximately 2.0 kb) was inserted into the pAbAi vector using the SmaI and SalI sites to construct the "bait" vector (ProFaCHI::pAbAi). The empty pGADT7 vector was used as a negative control. All constructs were transformed into Y1HGold cells (Weidi, Shanghai, China) using a yeast transformation kit (PT1183, Protein Interaction, China). The transformed yeast strains were incubated at 30 °C on SD / -Leu agar plates containing different concentrations of AbA. Growth was observed and recorded after 3 to 5 days of culture. The interaction between the protein and DNA was confirmed by comparing the growth status with the negative control.

[0085] Electrophoretic mobility variation analysis (EMSA): FaGeBP3 The coding sequence (CDS) of FaGeBP3-His was fused into the pCold TF vector to construct a recombinant expression vector. FaGeBP3-His was transformed into BL21 (DE3) strain (Weidi, Shanghai, China). The His fusion protein was purified using a kit (Kangwei Century, Beijing, China), and the induction conditions were set as 0.5 mMIPTG, 16℃ overnight. Biotin-labeled probes containing the GATATC sequence of the FaCHI promoter were used in the experiment, with unlabeled probes used as competing probes. Probes were synthesized by Sangon Biotech (Shanghai, China), and the EMSA kit was purchased from Beyotime (Beyotime, Jiangsu, China).

[0086] Dual-luciferase reporter gene analysis: To construct the ProFaCHI::LUC reporter vector, the upstream region of the FaCHI promoter (approximately 2 kb) was cloned and integrated into the pGreenII 0800-LUC vector. The effector vector used was the CaMV35S::FaGeBP3 construct, which overexpressed FaGeBP3 under the drive of the CaMV35S promoter. The relative activity of luciferase (LUC) was detected using a dual-luciferase reporter gene assay kit (FR201-01, TransGen Biotech, Beijing, China). Specific experimental methods were described by Bian et al. (Bian, RQ et al. A novel function for the transcription factor sensitive to proton rhizotoxicity1 in promoting anthocyanin accumulation in strawberry. Plant Biotechnol J. 2025;23:3727-3747.).

[0087] GUS staining analysis: The FaCHI promoter, approximately 2.0 kb in length, was inserted into the pRI201-AN-GUS vector for overexpression. FaGeBP3 The construct and the empty pRI101-AN vector were used as effector factors, and the FaCHI promoter introduced into the pRI201-AN-GUS vector was used as a reporter factor. Specific experimental methods were as described by Li et al. (Li, X. et al. FvPHR1 Improves the Quality of Woodland Strawberry Fruit by Up-Regulating the Expression of...). FvPHT1;7 and FvSWEET9 . Plant, Cell & Environment The description of (2025;48:2821-2834).

[0088] Leaves of 'Yanli' strawberry seedlings grown in Greenhouse No. 11 of Shenyang Agricultural University were sprayed with 100 μmol / L methyl jasmonate (MeJA). Leaf samples were collected at 6 and 12 hours after treatment, and plants sprayed with water were set up as a control group (CK).

[0089] 8.2 Experimental Results

[0090] Bioinformatics predictions showed that FaCHI was the only candidate gene with statistical significance, and its binding probability was extremely high (p = 6.26 × 10⁻⁶). -7 The q = 0.115 was significantly higher than all other analyzed genes. These results make FaCHI the preferred candidate for functional validation. Compared to wild type (WT), the transcriptional level of FaCHI decreased by 0.76, 0.29, and 0.32-fold, respectively, in the transgenic lines FaGeBP3-OE #1, #2, and #3; while in the FaGeBP3-RNAi lines #1, #2, and #3, its transcriptional level increased by 2.46, 2.06, and 1.96-fold, respectively. Figure 9 (a) in the text. Subsequently, yeast one-hybrid (Y1H) experiments confirmed that... FaGeBP3 Specifically binds to the FaCHI promoter ( Figure 9 (b) in the middle.

[0091] Y1H experiment will FaGeBP3 The binding site is located in the S2 segment of the FaCHI promoter. Referring to previously reported motifs (CATATG, GATATC, and ACATAWCT), further truncation analysis and electrophoretic mobility assays (EMSA) confirmed that FaGeBP3 specifically targets the S2-2 region containing the GATATC motif. Figure 9 In addition, dual-luciferase (LUC) and β-glucuronidase (GUS) experiments showed that FaGeBP3 inhibited the expression of FaCHI (cf). Figure 9 g in 9 and h in 9).

[0092] To further understand the biological significance of this regulation, we validated the module's response to methyl jasmonate (MeJA), a key hormone that promotes flavonoid biosynthesis. In the "Yanli" variety (whose...) FaGeBP3 In the treatment of FaCHI (with a coding sequence identical to that of "First Love"), FaGeBP3 expression was significantly downregulated 6 hours after treatment, but recovered after 12 hours. In contrast, FaCHI transcriptional levels showed the opposite upregulated pattern (Figure 10). Physiological data confirm our molecular findings, suggesting that the MeJA signaling pathway may enhance flavonoid accumulation by mitigating the regulatory effect of FaGeBP3 on FaCHI.

[0093] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. FaGeBP3 The application of genes in regulating the biosynthesis of plant flavonoids is characterized by, The FaGeBP3 Genes through and FaCHI Promoter interactions, thereby regulating the biosynthesis of flavonoids, the aforementioned FaGeBP3 The nucleotide sequence of the gene is shown in SEQ ID NO:1; The FaGeBP3 Gene overexpression negatively regulates the biosynthesis of flavonoids; The FaGeBP3 Reduced gene expression positively regulates the biosynthesis of flavonoids; The plant in question is a strawberry; The FaGeBP3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. A method for improving the biosynthesis of plant flavonoids, characterized in that, inhibition FaGeBP3 Gene expression, the FaGeBP3 The nucleotide sequence of the gene is shown in SEQ ID NO:1; the plant is strawberry.

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