Cinnamomum camphora ccmyb90 transcription factor, expression protein and application thereof

Abstract

The application discloses a camphor CcMYB90 transcription factor, an expression protein thereof and application, and belongs to the technical field of plant genetic engineering.The nucleotide sequence of the transcription factor is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.CcMYB90 gene is cloned from camphor fruits for the first time, and experiments prove that the transcription factor is located in the cell nucleus and can activate the expression of multiple structural genes in the anthocyanin biosynthesis pathway in a broad spectrum.After overexpression of CcMYB90 in Arabidopsis, the anthocyanin accumulation amount of the transgenic plants is significantly higher than that of the wild type.The application provides a new gene resource and technical means for directional improvement of plant color quality and cultivation of high anthocyanin content plant varieties by using genetic engineering means, and has a wide application prospect in the fields of color enhancement of ornamental plants, fruit color improvement of fruit trees and enrichment of functional components.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the CcMYB90 transcription factor of camphor tree, its expressed protein, and its applications. Background Technology

[0002] Camphor tree (scientific name: *Cinnamomum camphora*) is a large evergreen tree belonging to the Lauraceae family and is one of the most important native tree species in China. Besides its widespread applications in essential oil production, timber utilization, and urban greening, camphor fruit is increasingly attracting attention as an important source of bioactive metabolites. In particular, camphor fruit (especially the pericarp) is rich in flavonoids and volatile oils, exhibiting significant antioxidant, antibacterial, and insecticidal activities. However, previous research on camphor has largely focused on the antifungal and insecticidal properties of its essential oils, while other classes of bioactive compounds (especially flavonoids) remain poorly understood. Since fruit color is usually closely related to flavonoid metabolism and developmental processes, changes in pericarp color may reflect synergistic changes in specific metabolisms during fruit ripening.

[0003] Fruit color is a crucial visual and physiological characteristic, primarily determined by four classes of pigments: carotenoids, chlorophyll, flavonoids / anthocyanins, and betaine. Among these, flavonoids / anthocyanins play a central role in determining fruit color variation and also provide protection against environmental stresses. Anthocyanins are important natural water-soluble pigments that impart red, purple, blue, and even near-black hues to plant flowers and fruits. Furthermore, anthocyanins and related pigments have significant value in human health and various industries, including food, cosmetics, textiles, and pharmaceuticals. Therefore, elucidating the molecular mechanisms of pigment accumulation in camphor tree fruits is not only essential for understanding their biological functions but also helps enhance their economic potential.

[0004] In many horticultural plants, pericarp color is determined by a complex metabolic network, primarily involving the biosynthetic pathways of phenylpropanes and flavonoids / anthocyanins, which are tightly regulated by developmental signals and transcriptional regulation. Anthocyanin biosynthesis occurs through a conserved branch of the flavonoid pathway, involving structural biosynthetic genes and transcription factors (TFs) that catalyze this process. Key structural genes in this pathway include chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonoid 3′-hydroxylase (F3′H); while late anthocyanin biosynthetic genes (LBGs) include dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and flavonoid 3-O-glucosyltransferase (UFGT). These genes encode enzymes that act sequentially to produce specific anthocyanins. Furthermore, several transcription factors (including MYBs, bHLHs, and NACs) have been identified and confirmed to play regulatory roles in anthocyanin biosynthesis. Among them, MYB transcription factors (especially members of the R2R3-MYB subfamily) are considered key transcriptional regulators of the flavonoid / anthocyanin biosynthesis pathway. Many MYB transcription factors act as activators to promote anthocyanin accumulation, including SmMYB113, PyMYB114, and MdMYB10; while other transcription factors act as inhibitors of anthocyanin biosynthesis, such as VvMYBA1 / VvMYB3, BoMYBL2b, and BrMYB4. This indicates that MYB transcription factors play diverse and crucial roles in flavonoid / anthocyanin biosynthesis. However, reports on the regulatory mechanisms of anthocyanins in camphor tree fruits are still scarce. Summary of the Invention

[0005] To address the problems of existing technologies, the first technical problem this invention aims to solve is to provide a transcription factor CcMYB90 that can regulate anthocyanins in plants. The second technical problem this invention aims to solve is to provide specific applications of the aforementioned gene, which can be applied to plant genetic engineering to increase anthocyanin content in vegetables, fruits, and ornamental plants through overexpression.

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

[0007] The application of the camphor tree CcMYB90 transcription factor gene in promoting anthocyanin accumulation in plants, wherein the nucleotide sequence of the camphor tree CcMYB90 transcription factor gene is shown in SEQ ID NO.1.

[0008] In some embodiments, the application includes introducing a gene encoding the camphor tree CcMYB90 transcription factor into a target plant and overexpressing the gene in the target plant to increase the anthocyanin content in the target plant.

[0009] In some embodiments, the plant is Arabidopsis thaliana.

[0010] In some embodiments, the camphor tree CcMYB90 transcription factor promotes anthocyanin accumulation by activating the expression of one or more structural genes in the anthocyanin biosynthesis pathway.

[0011] In some embodiments, the structural gene is selected from at least one of CHS, DFR, LDOX, and UF3GT.

[0012] In some embodiments, the structural gene is CHS1, and the promoter region of CHS1 contains a binding site for the camphor tree CcMYB90 transcription factor.

[0013] A method for cultivating transgenic plants with increased anthocyanin content includes the following steps: introducing a gene encoding the camphor tree CcMYB90 transcription factor into a target plant and overexpressing the gene in the target plant; the amino acid sequence of the camphor tree CcMYB90 transcription factor is shown in SEQ ID NO.1.

[0014] In some embodiments, the target plant is Arabidopsis thaliana.

[0015] In some embodiments, the camphor tree CcMYB90 transcription factor increases anthocyanin content by upregulating the expression of at least one gene among CHS, DFR, LDOX, and UF3GT in the anthocyanin biosynthesis pathway.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) This invention is the first to isolate and identify a MYB transcription factor, CcMYB90, that regulates anthocyanin biosynthesis from camphor trees. Experiments have shown that this transcription factor is located in the cell nucleus and possesses typical transcription factor characteristics. After overexpression of CcMYB90 in Arabidopsis thaliana, the anthocyanin accumulation in transgenic plants was significantly increased compared to the wild type, and the accumulation level was positively correlated with the expression level of CcMYB90. Further analysis showed that CcMYB90 can broadly activate the expression of multiple structural genes in the anthocyanin biosynthesis pathway, including CHS, DFR, LDOX, and UF3GT, thereby synergistically promoting the synthesis and accumulation of anthocyanins. This provides a new regulatory factor resource for the targeted improvement of plant color quality using genetic engineering techniques.

[0018] (2) The camphor tree MYB90 transcription factor and its encoding gene provided by this invention can be widely used to promote anthocyanin accumulation in plants and to cultivate transgenic plant varieties with high anthocyanin content. Experiments have shown that introducing the CcMYB90 gene into target plants and overexpressing it can effectively increase anthocyanin content. The method is simple to operate and has significant effects. This transcription factor is derived from camphor trees, is safe and reliable, and has good application prospects in the fields of enhancing the color of ornamental plants, improving the color of fruit trees, and enriching functional components. Attached Figure Description

[0019] Figure 1 WGCNA analysis identified CcMYB90 as a potential key transcription factor involved in the coloring process of camphor tree pericarp; (A) Hierarchical clustering tree diagram: different colors in the diagram represent co-expression modules, and each differentially expressed gene (DEG) is presented in the form of leaves in the tree; (B) Association between module gene expression and samples: the modules on the left are labeled with different colors; (C) Heatmap and bar chart of the emerald green module; (D) Expression patterns of 29 MYB / MYB related transcription factors in the emerald green module during fruit development; (E) Correlation heatmap of CCMYB90 with total flavonoid content and major flavonoid DAMs in camphor tree pericarp;

[0020] Figure 2 Diagram showing the positive regulation of anthocyanin biosynthesis by CcMYB90; (A) Subcellular localization of CcMYB90 in tobacco leaves; (B) Representative images of anthocyanin accumulation phenotypes in wild-type Arabidopsis (WT) and three independent 35S:CcMYB90 lines; (C) Total anthocyanin content in leaves of WT or 35S:CcMYB90 plants; Relative expression levels of CcMYB90 (D) or anthocyanin biosynthesis-related genes (EH) as determined by RT-qPCR; Data are expressed as mean ± standard deviation of three biological replicates; Different letters indicate significant differences between groups (one-way ANOVA, Tukey multiple comparison test, P < 0.01); Scale bar: (A) 20 μm; (B) 1 cm;

[0021] Figure 3 The correlation analysis diagram between CcMYB90 and anthocyanin biosynthesis-related structural genes is shown below: (A) Pearson correlation analysis between CcMYB90 gene and anthocyanin biosynthesis-related structural genes in the blue-green module; (B) Schematic diagram of MYB binding site in CcCHS1 promoter.

[0022] Figure 4Figures showing the results of the Y1H and Dual-LUC experiments: (A) The Y1H experiment shows that CcMYB90 can bind to the CcCHS1 promoter; (B) The Dual-LUC experiment shows that CcMYB90 can activate CcCHS1 expression; Asterisks indicate significant differences (t-test, P < 0.01); Scale bars: (A) 10 μm; (B) 1 cm. Detailed Implementation

[0023] 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, or are performed according to the kit and product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0024] Example 1

[0025] I. Materials and Methods

[0026] 1. Plant materials

[0027] This experiment collected fresh fruits from 50-year-old camphor trees (C. camphora) planted by the Jiangxi Academy of Forestry Sciences in Nanchang City, Jiangxi Province, China. Fruits were collected from four different developmental stages and rigorously selected to ensure uniformity in shape, size, and color, and absence of mechanical damage or disease. Each sample underwent three biological replicates. After harvesting, the pericarp was immediately flash-frozen in liquid nitrogen and stored at -80℃ for subsequent metabolomics analysis, transcriptome sequencing, real-time quantitative polymerase chain reaction (RT-qPCR), and gene cloning. Arabidopsis thaliana (Col-0) was used as the wild type. Hybrid poplars (Populus davidiana × Populus bolleana) were cultivated in a greenhouse at 25℃ (daytime) / 18℃ (nighttime) with a photoperiod of 16 hours / 8 hours (light / dark). Genetic transformation of the poplars was achieved using Agrobacterium-mediated leaf disc technology, followed by the use of kanamycin (50 mg·L⁻¹). -1 Transgenic poplar varieties were obtained through screening.

[0028] 2. WGCNA analysis of metabolite and transcriptome data

[0029] This application employs a targeted analysis method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to detect metabolites in the pericarp of camphor tree fruit. The analysis was performed by Wuhan Meteway Biotechnology Co., Ltd. (Wuhan, China). The specific steps are as follows: First, the pericarp was removed with a blade and dried in a Scientz-100F freeze dryer (Ningbo Scientific Biotechnology Co., Ltd., Zhejiang). Then, it was ground using a multi-functional grinder (MM 400, Retsch GmbH, Hahn-Gruten, Germany). The sample was homogenized in the grinder at 30 Hz for 1.5 minutes. Approximately 50 mg of powder was added to 1200 μL of pre-cooled 70% methanol solution (as an internal standard) and vortexed at 1000 rpm 6 times (every 30 minutes). The mixture was then centrifuged at 12,000 rpm for 3 minutes at 4°C. The supernatant was aspirated with a syringe and filtered through a 0.22 μm microporous membrane. Finally, the extract was stored in sample vials and frozen for subsequent metabolomics (UPLC-MS / MS) analysis.

[0030] Each extract was analyzed using an ExionLC™ AD UPLC-ESI-MS / MS system (Sciex Corporation, California, USA) under the following conditions: Agilent ZORBAX SB-C18 silica column (particle size 1.8 µm, inner diameter 2.1 mm, length 100 mm); mobile phase was a mixture of solvent A [formic acid in pure water (0.1% v / v)] and solvent B [acetonitrile containing 0.1% formic acid]. Elution was performed using a programmed solvent ratio: initially 95% solvent A and 5% solvent B; then gradually switched to 5% solvent A and 95% solvent B over 9 minutes and held for 1 minute; then adjusted to 95% solvent A and 5% solvent B over 1.1 minutes and held for 2.9 minutes. The column oven temperature was set to 40°C, the injection volume was 2 μL, and the flow rate was 0.35 mL / min. The ESI triple quadrupole (QQQ)-linear ion trap (QTRAP) mass spectrometer can perform alternating scans of the effluent.

[0031] Subsequently, electrospray ionization (ESI) technology was used for analysis: the ion spray voltage was 5500V in positive ion mode and -4500V in negative ion mode; the ion source temperature was set to 500℃. Ion source gases I and II were used simultaneously (pressures of 50 psi (GS1) and 60 psi (GSII) respectively), the curtain gas pressure was 25 psi, and the high-energy collision activated dissociation (CAD) mode was employed.

[0032] QQQ scans were performed in multiple reaction monitoring (MRM) mode with nitrogen as the collision gas at a moderate pressure (5 psi). Mass spectrometry parameters (disclusion potential DP and collision energy CE) were determined for each MRM transition and optimized for each analyte. Subsequently, a specific set of MRM transitions was selected and detected within a specified time range based on the characteristics of the eluted metabolites. Data acquisition was performed using Analyst software (version 1.6.3, SCIEX Corporation, Framingham, Massachusetts, USA), and data processing was performed using SCIEX OS-Q software (version 1.6, SCIEX Corporation).

[0033] This application used RNA samples collected from four different developmental stages of the fruit pericarp (three replicates for each stage), and a total of twelve libraries were sequenced. After RNA extraction and quantification, high-quality RNA samples were selected and libraries were constructed using the NEBNext® Ultra™ RNA Library Construction Kit (New England Biolabs, Ipswich, Massachusetts, USA). Finally, these twelve libraries were normalized and sequenced on the HiSeq platform (Illumina, USA), obtaining paired-end (150 bp) sequencing reads.

[0034] The raw RNA sequencing reads were first processed using the FastQC quality control tool to remove low-quality sequences and adapter sequences. Subsequently, the Hisat2 algorithm (v2.0.5) (Kim D, Langmead B, Salzberg SL. HISAT: Afast spliced ​​aligner with low memory requirements. Nature methods. 2015;12.) was used to align the resulting reads to a single camphora (C. camphora) reference genome (NCBI taxonomic number: 13429). Next, the read quantifier featureCounts program (v1.5.0-p3) was used to perform summary analysis on the reads and quantify the expression level of each gene in each sample. This program counted the number of reads aligned to each gene and calculated the FPKM value (i.e., the number of transcript sequence fragments per million sequencing base pairs) based on a normalization method (Liao Y, Smyth GK, Shi W. FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics (Oxford, England). 2014;30).

[0035] Subsequently, the differential expression of genes between different developmental stages was analyzed using the R package DESeq2 (version 1.16.1). Genes with an absolute fold change (FC) ≥2 and a corrected P-value ≤0.05 between stages were identified as differentially expressed genes. These differentially expressed genes (DEGs) were then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses using the R package clusterProfiler (G Yu, LG Wang, Y Han, QY He. clusterProfiler: anR package for comparing biological themes among gene clusters. OMICS: AJournal of Integrative Biology 2012, 16(5):284-287.).

[0036] The co-expression network modules between metabolites and genes were generated using the R package WGCNA. These co-expression modules were constructed based on the Topological Overlap Measure (TOM) using the default parameters of the automatic network construction function (blockwiseModules). Initial clustering was performed by merging feature genes, and feature gene values ​​for each module were calculated to determine its association with major carotenoids. Pearson correlation coefficients (PCC > 0.85) between genes and transcription factors were evaluated, and the final network results were visualized using Cytoscape software (version 3.7.2, USA).

[0037] A weighted co-expression network of metabolites and genes was constructed using the bioinformatics algorithm WGCNA in R. Top-ranked genes were selected for network construction, and the `blockwiseModules` function (with default parameters) was used to automatically construct the network and identify co-expression modules based on the Topological Overlap Measure (TOM). Clusters were merged based on characteristic genes, and then the characteristic gene values ​​representing the dominant pattern in each module were calculated and correlated with metabolite data to assess their association with major flavonoid subclasses, anthocyanin metabolites, and pigment-related traits. Furthermore, the Pearson correlation coefficient (r) between transcription factors and genes was calculated; factors with values ​​greater than 0.85 were used to generate heatmaps and identify clusters; genes with high correlation values ​​were identified as hub genes. Finally, the network structure was visualized and analyzed using the open-source platform Cytoscape (version 3.7.2, developed in the US) (Kohl et al., 2011).

[0038] 3. Plasmid construction

[0039] Unless otherwise stated, all constructs were constructed using In-Fusion cloning technology (ClonExpress II One-Step Cloning Kit, Vazyme).

[0040] In the subcellular localization experiment, the expression vector was constructed using the Gateway cloning system. The CDS of CcMYB90 was amplified, and the resulting PCR fragment was inserted into the pCAMBIA2300-35S-eGFP vector, ultimately obtaining the recombinant 35S:CcMYB90-GFP. The constructed 35S:CcMYB90-GFP vector was transformed into *Agrobacterium tumefaciens* and injected into *Tobacco Bengal* leaves for transient expression, used for subcellular localization observation. The nucleotide sequence of the CcMYB90 CDS is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

[0041] To verify the regulatory effect of CcMYB90 on the CcCHS1 promoter activity, a dual-luciferase (Dual-LUC) transient expression system was used for detection. First, the full-length coding sequence of CcMYB90 was amplified using primers CcMYB90-62-SK-F / CcMYB90-62-SK-R and inserted into the pGreenII 62-SK vector digested with BamHI and HindIII to construct the effector vector 62-SK-CcMYB90. Simultaneously, the CcCHS1 promoter fragment was amplified using primers proCcCHS1-pGreenII 0800-F / proCcCHS1-pGreenII 0800-R and cloned into the pGreenII0800-LUC vector digested with KpnI and SpeI to construct the reporter vector 0800-LUC-proCcCHS1. In this report vector, the CcCHS1 promoter drives the expression of the firefly luciferase gene LUC, and the Renilla luciferase gene REN is used as an internal control to correct for transformation efficiency and sample differences.

[0042] In the experimental group, the effector vector 62-SK-CcMYB90 and the reporter vector 0800-LUC-proCcCHS1 were co-transformed into *Tobacco Benzoinus* leaves; in the control group, the empty vector 62-SK (empty) and the same reporter vector 0800-LUC-proCcCHS1 were co-transformed into *Tobacco Benzoinus* leaves. After transient expression, the luciferase activities of LUC and REN were measured, and the relative activity of the CcCHS1 promoter was expressed as the LUC / REN ratio. If the LUC / REN ratio in the experimental group was significantly higher than that in the empty vector control group, it indicated that CcMYB90 could enhance the transcriptional activity of the CcCHS1 promoter.

[0043] For the Y1H experiment, the CDS of CcMYB90 was amplified using primers CcMYB90-AD-F / CcMYB90-AD-R, and the promoter region of CcCHS1 was amplified using primers proCcCHS1-pAbAi-F / proCcCHS1-pAbAi-R. The CDS fragment was inserted into the pGADT7 vector digested with BamHI and EcoRI, and the amplified promoter fragment was cloned into the pAbAi vector digested with KpnII and XhoI.

[0044] To understand the function of CcMYB90, its CDS was amplified. The PCR product was then inserted into the pEarleyGate 201 vector containing the CaMV 35S promoter, resulting in the recombinant 35S:CcMYB90. This recombinant vector was transformed into Agrobacterium tumefaciens GV3101, and then into Arabidopsis thaliana Columbia-0 wild-type using the inflorescence immersion method. Transgenic Arabidopsis plants stably overexpressing CcMYB90 were screened for this result. All primer information is detailed in Table 1.

[0045] Table 1 Primer Sequences

[0046]

[0047] 4. RNA extraction and RT-qPCR technology

[0048] To validate the RNA-seq results, researchers selected differentially expressed genes (DEGs) involved in the biosynthetic pathway of flavonoids for RT-qPCR analysis. Specific primers for these DEGs were designed using Primer3 software. RNA was reverse transcribed to generate cDNA using MonScript™ RTIII All-in-One Mix (containing dsDNase, Monad, Suzhou, China). qPCR experiments were performed using a QuantiNova SYBR Green PCR kit (QIAGEN, Hilden, Germany) in conjunction with an ABI 7500 real-time PCR instrument (Applied Biosystems, Foster City, California, USA). Relative expression levels were determined using the 2ˆ-ΔΔCt method, and at least three biological replicates were set up for each experiment. All primer sequences are detailed in Table 1.

[0049] 5. Determination of total anthocyanin content

[0050] The method for determining the total anthocyanin content is as follows: Take 0.1 g of fresh tissue sample and extract with 9 mL of methanol containing 0.1% (v / v) hydrochloric acid at 4°C in the dark for 24 hours. After centrifuging the extract at 5,000 g for 5 minutes and removing the supernatant, measure the absorbance at 530, 620, and 650 nm using a spectrophotometer. The anthocyanin content is calculated using the following formula: OD λ / ξλ×V / m×10 6 (nmol·g) -1 Fresh weight; V: volume; m: weight; ξλ: 4.62 × 10 4 ) OD λ =(A 530 -A 620 )-0.1×(A 650 -A 620 ). ODλ The absorbance is indicated by anthocyanin-specific absorbance after chlorophyll interference correction. Each sample was subjected to three biological replicates.

[0051] 6. Methods for detecting transcriptional regulation

[0052] To determine whether CcMYB90 binds to the CcCHS1 promoter, the full-length coding sequence of CcMYB90 was cloned into the pGADT7 vector, while the promoter region of CcCHS1 was inserted into the pAbAi reporter vector. The bait and prey constructs were then co-transformed into yeast cells according to the manufacturer's instructions. Transformants were screened on SD / -Leu medium supplemented with AbA, and DNA-protein interactions were assessed based on yeast growth under AbA selection conditions.

[0053] Dual-luciferase assays were performed according to a previous method (Qin Z, Wang F, Hao YW, et al. A bHLH-mediated misexpression of SPL / NZZ underpins the anther developmental arrest of female flowers in sex differentiation in monoecious litchi[J]. PlantPhysiology, 2026, 201(2): kiag336.) to assess the regulatory effect of CcMYB90 on CcCHS1 promoter activity. Agrobacterium tumefaciens strains carrying reporter gene and effector protein constructs were co-infected with 4-week-old tobacco leaves. Leaf samples were collected on day 3 post-infection, and LUC and REN activities were measured using a Dual-Luciferase® reporter gene assay system (Yeasen, Shanghai, China). Transcriptional regulation levels were assessed using the LUC / REN ratio. Statistical analysis included at least three independent biological replicates.

[0054] 7. Fluorescence Imaging

[0055] Subcellular localization experiments were performed using a Zeiss LSM 800 CLSM microscope equipped with a 20x objective lens. Fluorescence signal detection was performed using a 488 nm argon laser with a BP 505-550 filter for GFP. Quantitative fluorescence analysis was performed using ImageJ software (http: / / rsbweb.nih.gov / ij / ). All confocal experiments were repeated at least three times. Images were exported and processed using Adobe Photoshop CS3 (Adobe).

[0056] II. Results

[0057] 1. Metabolomics combined with transcriptomics to identify the mechanism of flavonoid / anthocyanin accumulation in fruits

[0058] Materials were collected from four developmental stages of camphor tree fruits for targeted metabolomics analysis and transcriptomics sequencing. Weighted Gene Co-expression Network (WGCNA) was used to identify co-expression modules associated with fruit color. Each WGCNA module contained 88 to 3,788 genes. Figure 1 A). Among them, the bluish-green module, composed of 3,788 genes, showed the strongest correlation with DAMs and best matched the developmental coloring pattern of camphor fruit (A). Figure 1 (B, C). Therefore, this bluish-green module was identified as the core regulatory module for flavonoid / anthocyanin biosynthesis during the darkening of camphor fruit. The key candidate gene CcMYB90 within this module exhibits extremely high transcriptional abundance during the fruit color change stage, at which point anthocyanin accumulation is rapidly induced (B, C). Figure 1 D). Furthermore, among the MYB candidate factors, CcMYB90 showed the most significant differential expression pattern and was strongly positively correlated with major anthocyanin metabolites (D). Figure 1 E) suggests that it may be a key regulatory factor in the fruit peel coloring process.

[0059] 2. Overexpression of CcMYB90 promotes anthocyanin accumulation.

[0060] To evaluate the function of CcMYB90, its subcellular localization was first examined, revealing that the protein is specifically localized in the cell nucleus. Figure 2 A), which is consistent with its putative function as a transcription factor. Subsequently, stable transgenic Arabidopsis plants overexpressing CcMYB90 were constructed, and three independent transgenic lines with low, medium, and high CcMYB90 expression levels were screened for further analysis. Figure 2 D). Compared with wild-type plants, all three transgenic lines showed significantly enhanced anthocyanin accumulation ( Figure 2 B, C (Table 2).

[0061] Table 2 Anthocyanin content

[0062]

[0063] Notably, anthocyanin accumulation was positively correlated with the transcript abundance of CcMYB90, indicating that CcMYB90 promotes anthocyanin biosynthesis in an expression-level-dependent manner. To understand whether enhanced pigment accumulation is related to the activation of the anthocyanin biosynthetic pathway, the expression levels of representative structural genes were analyzed. The results showed that, compared with the wild type, the transcriptional levels of AtCHS, AtDFR, AtLDOX, and AtUF3GT were significantly upregulated in the CcMYB90 overexpression lines. Figure 2These results demonstrate that CcMYB90, as a positive regulator of anthocyanin biosynthesis, promotes pigment accumulation by activating genes related to the anthocyanin pathway.

[0064] 3. CcMYB90 directly activates CcCHS1 to promote anthocyanin accumulation.

[0065] To further identify potential downstream target genes of CcMYB90, Pearson correlation analysis was first performed on CcMYB90 and these structural genes related to anthocyanin biosynthesis. The results showed that CcMYB90 was highly positively correlated with most of these structural genes. Figure 3 A) suggests that it may extensively regulate the anthocyanin biosynthesis network during fruit coloring. Among the top five structural genes most strongly associated with CcMYB90, only the promoter region of Cca.gene13206 (CcCHS1, this gene has been granted an invention patent, patent announcement number: CN118127049B) contains the putative MYB90 binding motif TAGTTA ( Figure 3 B).

[0066] To determine whether CcMYB90 directly regulates CcCHS1, a yeast one-hybrid (Y1H) assay and a transient dual-luciferase (LUC) reporter assay were performed. In the Y1H assay, yeast cells carrying pAbAi-ProCcCHS1 and AD-CcMYB90 were able to grow on SD / -Leu medium containing erythromycin A (AbA, 200 ng / mL), while the negative control (AD+pAbAi-ProCcCHS1) could not grow. Figure 4 A), indicating that CcMYB90 directly binds to the CcCHS1 promoter. Consistent with this, transient dual-luciferase assays in tobacco leaves showed that CcMYB90 significantly activated CcCHS1 promoter-driven LUC expression (A). Figure 4 B). In summary, these results demonstrate that CcMYB90 directly binds to the CcCHS1 promoter and activates its transcription, thereby promoting the accumulation of anthocyanins during the ripening of camphor fruit.

[0067] 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. The application of a camphor tree CcMYB90 transcription factor gene in promoting anthocyanin accumulation in plants, wherein the nucleotide sequence of the camphor tree CcMYB90 transcription factor gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The application involves introducing a gene encoding the camphor tree CcMYB90 transcription factor into a target plant and overexpressing the gene in the target plant to increase the anthocyanin content in the target plant.

3. The application according to claim 1 or 2, characterized in that, The plant in question is Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, The camphor tree CcMYB90 transcription factor promotes anthocyanin accumulation by activating the expression of one or more structural genes in the anthocyanin biosynthesis pathway.

5. The application according to claim 4, characterized in that, The structural gene is selected from at least one of CHS, DFR, LDOX and UF3GT.

6. The application according to claim 5, characterized in that, The structural gene is CHS1, and the promoter region of CHS1 contains a binding site for the camphor tree CcMYB90 transcription factor.

7. A method for cultivating transgenic plants with increased anthocyanin content, characterized in that, Includes the following steps: The gene encoding the camphor tree CcMYB90 transcription factor was introduced into a target plant and the gene was overexpressed in the target plant; the amino acid sequence of the camphor tree CcMYB90 transcription factor is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The target plant is Arabidopsis thaliana.

9. The method according to claim 7 or 8, characterized in that, The camphor tree CcMYB90 transcription factor increases anthocyanin content by upregulating the expression of at least one gene among CHS, DFR, LDOX, and UF3GT in the anthocyanin biosynthesis pathway.

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