Application of the CpMYB4 gene in wintersweet in inhibiting anthocyanin biosynthesis in plants

Abstract

This invention belongs to the field of biotechnology, specifically relating to the application of the *Chimonanthus praecox* CpMYB4 gene in inhibiting anthocyanin biosynthesis in plants. By cloning the *Chimonanthus praecox* CpMYB4 gene and constructing a promoter-driven overexpression vector, which was then transformed into tobacco, it was found that overexpression of the CpMYB4 gene significantly increased the expression of tobacco flavonol synthase, leading to a decrease in anthocyanin content and paler flower color. CpMYB4 indirectly inhibits the activity of anthocyanin synthesis-related genes (NtANS, NtDFR) by regulating the expression of key genes in the flavonoid synthesis pathway (such as NtCHS, NtCHI, and NtFLS), thereby reducing anthocyanin content. This invention provides a new strategy for regulating plant flower color and can be used to cultivate new varieties of ornamental plants with light or white flowers, possessing significant agricultural and horticultural application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CpMYB4 gene of wintersweet in inhibiting anthocyanin biosynthesis in plants. Background Technology

[0002] Flavonols are a class of polyphenolic compounds widely found in the plant kingdom, belonging to an important subclass of the flavonoid family. These compounds play a crucial role in plant growth and development and in responding to environmental stress. The biosynthesis of flavonols is a complex and highly regulated process involving multiple enzymatic steps and the synergistic effects of genes.

[0003] Flavonol synthesis is part of the phenylpropanoid metabolic pathway, starting with phenylalanine. Regulation of flavonol biosynthesis is a multi-level process involving transcription factors, enzyme activity, and environmental signals. For example, the R2R3-MYB transcription factor plays a crucial role in flower development and flavonoid biosynthesis, including the regulation of anthocyanin, flavonol, and proanthocyanidin synthesis. Studies have shown that plant hormones such as abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA), as well as environmental factors such as light (especially ultraviolet radiation), temperature, pathogen infection, and nutritional status (e.g., low phosphorus stress), can all influence flavonol synthesis and accumulation. For instance, UV-C radiation can upregulate the biosynthesis of phenolic substances in peach fruits, with a significant increase in the expression of flavonols.

[0004] Although the MYB gene (MYB transcription factor such as Figure 1 The role of (as shown) in flower color regulation has been extensively studied, but its application still faces the following key bottlenecks:

[0005] (1) Metabolic interference due to insufficient targeting: Most of the MYB genes discovered so far function by directly regulating anthocyanin synthesis genes, such as cotton GaMYB, which has been found to positively regulate the glutathione S-transferase gene (GaGST), thereby affecting the formation of red spots on petals. However, some MYB genes may have non-specific effects on other metabolic pathways. Non-targeting effects may lead to imbalances in plant secondary metabolism and even affect growth and development.

[0006] (2) Species-specific limitations: The functions of some MYB genes are significantly species-specific, and their cross-species application often faces the following obstacles: Incompatibility of gene expression regulatory elements: Cis-elements in promoter regions (such as light-responsive elements and hormone-responsive elements) may not be able to drive gene expression normally due to species differences. Differences in transcription complex interactions: The interaction between MYB proteins and bHLH and WD40 proteins depends on the interaction interface at specific amino acid sites, and cross-species substitution may lead to reduced complex stability. Substrate preference of metabolic pathways: The enzyme activity and substrate specificity of anthocyanin synthesis pathways may differ among different plants. For example, some plants' DFRs (such as petunias) prefer dihydroquercetin as a substrate but do not accept dihydromyricetin, while other plants (such as grapes) can utilize a variety of dihydroflavonoid substrates.

[0007] (3) Limitations in regulatory efficiency and practical application: Overexpression of the MYB gene often fails to achieve the desired flower color depth or uniformity. For example, MYB transcription factors regulate gene expression by influencing epigenetic modifications such as histone modification and DNA methylation, but the efficiency and stability of this regulation still require further study. In addition, the expression and activity of the MYB gene are affected by abscisic acid, ambient light, and temperature. The superposition of these complex factors makes it difficult to accurately control the regulatory efficiency of the MYB gene. Especially in some difficult-to-transform horticultural crops, this further limits the application of the MYB gene in flower color improvement.

[0008] To address the shortcomings of existing technologies, this invention is the first to discover that the CpMYB4 gene in wintersweet (Chimonanthus praecox) plays an important role in the flower color regulatory network and is a core MYB transcription factor that affects flower color synthesis in wintersweet.

[0009] With the rapid development of the global flower industry, the market demand for diversified flower colors of ornamental plants is increasing. Traditional breeding methods, such as hybridization and mutagenesis screening of light-colored flower varieties, are time-consuming and inefficient, while genetic engineering technology provides a new way to precisely regulate flower color. However, the limitations of existing repressed MYB genes severely restrict their application in flower improvement: (1) Ornamental plant flower colors are becoming monotonous, with most commercial flowers (such as roses and carnations) relying on a single flower color (red, pink, white), lacking novelty. (2) Consumer preferences are changing, and the market demand for light-colored flowers (such as light pink, off-white, and light purple) is increasing year by year, but traditional breeding cannot respond quickly. (3) Functional flower development, with light-colored flower varieties having unique application value in fields such as medicine (such as extraction of antioxidant active ingredients) and cosmetics (such as natural pigment raw materials).

[0010] In summary, developing novel MYB genes (such as CpMYB4) is of great significance for breaking through the current bottlenecks in flower color regulation technology and promoting the upgrading of the flower industry. Summary of the Invention

[0011] This invention first provides the application of the CpMYB4 gene of wintersweet in inhibiting anthocyanin biosynthesis in plants, and the nucleotide sequence of the CpMYB4 gene is shown in SEQ ID NO:1.

[0012] This invention also provides the application of the CpMYB4 protein from wintersweet in inhibiting the biosynthesis of anthocyanins in plants, the amino acid sequence of which is shown in SEQ ID NO:2.

[0013] The present invention also provides a CpMYB4 gene for wintersweet, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0014] The present invention also provides a wintersweet CpMYB4 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0015] The present invention also provides biological materials related to the above-mentioned CpMYB4 protein, which are any one of B1) to B8) below: B1) nucleic acid molecules encoding the above-mentioned CpMYB4 protein;

[0016] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0017] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0018] B4) A recombinant vector containing the expression cassette described in B2);

[0019] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0020] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0021] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0022] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0023] The present invention also provides a method for preparing transgenic plants, wherein the above-mentioned recombinant vector is introduced into plant cells to obtain transgenic plants that overexpress the CpMYB4 gene.

[0024] In some embodiments, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0025] In some embodiments, the plant is tobacco, Arabidopsis thaliana, chrysanthemum, or rose.

[0026] This invention also provides the application of the wintersweet CpMYB4 gene in regulating the flavonoid synthesis pathway in plants, by upregulating the expression of NtCHS, NtCHI, and NtFLS genes to enhance the biosynthesis of flavonols.

[0027] Finally, this invention also provides a method for improving the flower color of plants by overexpressing the CpMYB4 gene of wintersweet to reduce the anthocyanin content, so that the flower color of the plant is light pink or white.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] (1) Precise regulation of flower color: The CpMYB4 gene significantly reduces anthocyanin content by increasing the expression of key genes in the flavonol synthesis pathway, causing the flower color of plants to change from deep red / purple to light pink or white, providing a new tool for improving flower color.

[0030] (2) Expand the scope of application: It is suitable for a variety of plants (such as tobacco, Arabidopsis thaliana, chrysanthemum, etc.), and is especially suitable for cultivating ornamental light-colored flower varieties.

[0031] (3) Environmental and economic value: Reduce the demand for chemical dyeing, promote the development of green horticulture industry, and enhance the competitiveness of the flower market. Attached Figure Description

[0032] Figure 1 Classification of plant MYB transcription factors.

[0033] Figure 2 The growth environment of OE-CpMYB4 transgenic tobacco lines.

[0034] Figure 3 Morphological comparison of OE-CpMYB4 transgenic tobacco lines and pBWA(V) HS-Glosgfp empty-carrier tobacco plants.

[0035] Figure 4 OE-CpMYB4 transgenic tobacco RNA electrophoresis bands; M: DL2000+ DNA Marker; 1: OE-CpMYB4-2; 2: OE-CpMYB4-6; 3: OE-CpMYB4-4; 4: OE-CpMYB4-7; 5: OE-CpMYB4-8; 6: OE-CpMYB4-12; 7: OE-CpMYB4-14; 8: Empty vector.

[0036] Figure 5 Quantitative detection of OE-CpMYB4 transgenic tobacco flowers by qRT-PCR; ***, highly significant, p-value <0.001.

[0037] Figure 6Expression levels of flavonol biosynthesis structural genes: ***, extremely significant, p < 0.001; **, significant, p < 0.01; *, slightly significant, p < 0.05; ns, not significant.

[0038] Figure 7 Phenotypic observation of OE-CpMYB4 transgenic tobacco.

[0039] Figure 8 Anthocyanin extract from OE-CpMYB4-6 transgenic tobacco flowers.

[0040] Figure 9 Anthocyanin extract from OE-CpMYB4-14 transgenic tobacco flowers.

[0041] Figure 10 Determination of anthocyanin content in OE-CpMYB4; **, significant, p value < 0.01. Detailed Implementation

[0042] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0043] Material

[0044] Plant materials: wintersweet HLT015 from Heilongtan; tobacco K326.

[0045] Experimental reagents: DNA kit (Bomaide Biotechnology Co., Ltd.), RNA extraction kit (Adley Biotechnology Co., Ltd.), PCR kit, cDNA first-strand synthesis kit, PerfectStart Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.), Phanta Max Super-Fidelity DNA Polymerase (Nanjing Novizan Biotechnology Co., Ltd.).

[0046] Instruments and equipment: ultra-clean workbench, constant temperature water bath, constant temperature incubator shaker, autoclave, DYY-12C electrophoresis apparatus (Beijing Liuyi Instrument Factory); Dongshenglong eQ9600 series 96-well fluorescence quantitative PCR instrument (Suzhou Dongsheng Biotechnology Co., Ltd.); nucleic acid protein detector (Nano Drop 2000C) (Thermo Fisher Scientific); ultraviolet spectrophotometer (Shanghai Meipuda).

[0047] SEQ ID NO:1:;

[0048] SEQ ID NO:2:MGRSPCCSKEGLNRGAWTAIEDEILINYIKIHGEGRWRSLPIKAGLKRCGKSCRLRWLNYLRPDIKRGNISHEEEELIIRLHKLLGNRWSLIAGRLPGRTDNEIKNYWNTNLVKKLQKQPISKQPVEGSPAPELKSNVIR TKASRCTRVFISPPQRNVENLNKIQDVEATGVAGDFAASKGKGLESCRVPSLTCHDDDSSSSLVDLDMGEFCLELINSDYFQVLGESSINDDNNAVVADASNCNQLLHPFMFSEEMLVDWTGGDCFQTDVGFNIESMTSCLDSQG.

[0049] Example 1: Cultivation and Management of Transgenic Tobacco

[0050] Transgenic seeds were disinfected with 70% ethanol, then soaked in 10% sodium hypochlorite for 15 minutes for sterilization, and rinsed five times with sterile water for two minutes each time. The surface-sterilized seeds were evenly sown in petri dishes containing 1 / 2 MS solid medium, sealed with sealing film, and placed in a 25℃ light incubator (16 hours light, 8 hours darkness). Five to seven days after sowing, the buds began to sprout and develop cotyledons. Fourteen days later, the seedlings had two true leaves and were ready for sowing. Post-sowing management was implemented. After the test-tube seedlings developed several fibrous roots, they were opened and hardened off for three days before being transplanted into a 1:1 mixture of perlite and nutrient soil. Then, they were placed in the field for management. OE-CpMYB4 (cloned in previous laboratory studies) transgenic tobacco seedlings were transplanted into large pots for normal water and fertilizer management. All transgenic plants were planted in an isolated greenhouse to prevent pollen dispersal. At the same time, the phenotypic characteristics of tobacco plant flowers were observed. The flowers of the superior lineage were photographed and samples were collected. The flowers were then placed in sterile cryovials and immediately placed in liquid nitrogen for temporary storage. After collection, the plants were stored in an ultra-low temperature freezer and bagged to facilitate seed collection.

[0051] Results: In the previous cultivation in our laboratory, we obtained positive OE-CpMYB4 transgenic tobacco lines ( Figure 2 Throughout the annual growth cycle, maintain constant day / night temperature, air humidity, light intensity, and appropriate water and fertilizer conditions. The transgenic tobacco plants exhibited dark green leaves and vigorous growth. Flower phenotypic observations were conducted after flowering. Figure 3 ). Example 2

[0052] (1) Extraction of RNA from tobacco flowers and synthesis of cDNA

[0053] Total RNA was extracted from the flowers of wintersweet HLT015 during its full bloom period. The RNA concentration was first detected by 1% agarose gel electrophoresis, then by Nanodrop2000, and finally, the RNA was reverse transcribed into cDNA according to the instructions of the Total Gold Reverse Transcription Kit (cDNA First Strand Synthesis Kit).

[0054] Table 1. RNA Reverse Transcription Reaction System

[0055] Components Dosage Total RNA / mRNA 50 ng - 5 ug / 5 - 500 ng Anchored Oligo(dT)20(0.5 ug / μL) 1 μL or Random primer(N9) (0.1 ug / μL) 1 μL or GSP 2 pmol 5×RTMix 4 μL M-MLV4 1 μL <![CDATA[H2O(RNase free) to final volume]]> 20 μL

[0056] (2) Quantitative analysis of tobacco flowers by qRT-PCR

[0057] Flower samples from different strains of genetically modified tobacco were collected, and total RNA was extracted and reverse transcribed into cDNA. Real-time quantitative PCR (qRT-PCR) was used to accurately analyze gene expression levels in the transgenic tobacco. The following primers were designed and synthesized using Premier 3.0 software:

[0058] CpMYB4qF:GGTTTTCATCTCACCACCGC (SEQ ID NO:3);

[0059] CpMYB4qR:TCGTCGTCGTGGCAAGTTAG (SEQ ID NO:4);

[0060] NtRNR2 was used as an internal reference gene. cDNA synthesis was performed using the EasyScript one-step gDNA removal and cDNA synthesis kit. qRT-PCR was performed on an eQ9600. The reaction procedure was: 94℃ for 10 s, then 44℃ for 5 s, 60℃ for 15 s, and 72℃ for 30 s. For the quantitative PCR experiments, three biological replicates and three technical replicates were used. Two... −ΔΔ The relative expression levels of each gene were calculated using the CT method. All results were calculated and plotted using Graphpad Prism9.

[0061] Table 2. qRT-PCR reaction system for tobacco internal reference gene

[0062] Components Dosage 2×T5 Super PCR Mix (Basic) 10 μL 10 μMCpActin-F 1 μL 10 μMCpActin-R 1 μL template cDNA 1 μL <![CDATA[ddH2O]]> 7 μL

[0063] Table 3. Primer sequences for flavonoid-based quantitative PCR

[0064] Primer name Primer sequence NtCHSqF TTGTTCGAGCTTGTCTCTGC (SEQ ID NO:5) NtCHSqR AGCCCAGGAACATCTTTGAG (SEQ ID NO:6) NtCHIqF GTCAGGCCATTGAAAAGCTC (SEQ ID NO:7) NtCHIqR CTAATCGTCAATGCCCCAAC (SEQ ID NO:8) NtF3HqF CAAGGCATGTGTGGATATGG (SEQ ID NO:9) NtF3HqR TGTGTCGTTTCAGTCCAAGG (SEQ ID NO:10) NtF3'HqF TGTGCACCACGAATGCACTT (SEQ ID NO:11) NtF3'HqR TCAAGAACGCGTCGAAACG (SEQ ID NO:12) NtDFRqF AACCAACAGTCAGGGGAATG (SEQ ID NO:13) NtDFRqR TTGGACATCGACAGTTCCAG (SEQ ID NO:14) NtANSqF TGGCGTTGAAGCTCATACTG (SEQ ID NO:15) NtANSqR GGAATTAGGCACACACTTTGC (SEQ ID NO:16)

[0065] Results: RNA was extracted from pBWA(V)HS-OE-CpMYB4-GLosgfp positive seedlings. Agarose gel electrophoresis showed that the RNA sample quality was good (e.g., ...). Figure 4 ).

[0066] Using the extracted RNA samples from OE-CpMYB4 transgenic tobacco seedlings as templates, and the NtRNR2 internal reference gene as a reference, real-time quantitative experiments were performed using CpMYB4 fluorescent quantitative PCR primers to screen for transgenic OE-CpMYB4 tobacco plants with the highest expression levels.

[0067] RNA was reverse transcribed into cDNA using a reverse transcription kit, and the expression level of CpMYB4 in various lines was detected by real-time quantitative PCR. The results are as follows ( Figure 5 As shown in the figure, the expression levels of OE-CpMYB4 in the seven transgenic plants were 500-fold, 21000-fold, 100-fold, 2500-fold, 4500-fold, 0-fold, and 3000-fold, respectively, compared to the wild-type plants. The relative expression level of OE-CpMYB4 was highest in the OE-6 line. The expression levels of flavonol biosynthesis structural genes such as NtCHS, NtCHI, and NtFLS were significantly increased in the OE-MYB107 transgenic tobacco line. Specifically, the expression levels of NtCHS, NtCHI, and NtFLS were 5.75-fold, 2.5-fold, and 3.5-fold higher than the control group, respectively; while the expression level of NtF3'H was 7.5-fold lower than the control group. Figure 6 NtF3H, NtDFR, NtANS, and NtUFGT showed no significant difference. It is speculated that CpMYB4 promotes flavonol synthesis by enhancing the expression of structural genes in the flavonol biosynthesis pathway, and the excessive accumulation of flavonols gives tobacco flowers a light pink or white color.

[0068] Example 3: Phenotypic Observation and Analysis of CpMYB4 Transgenic Tobacco

[0069] During the plant's growth period, tobacco flowers were periodically photographed using a high-definition camera. Frontal views of the flowers were captured at specific times, documenting the gradual color change. At peak bloom, the flowers were collected, and intact tobacco flowers were cut off with scissors and immediately placed into pre-prepared sterile cryovials. The time, variety, and characteristics were recorded. The sample tubes were then flash-frozen in liquid nitrogen and transferred to an ultra-low temperature freezer at -80°C for storage.

[0070] Anthocyanin content was determined using a plant anthocyanin content kit; the expression level of flavonoid structural genes in transgenic tobacco was determined using a real-time PCR detection kit.

[0071] Results: Phenotypic observation of the OE-6 strain of tobacco with the highest expression level and that of tobacco transformed with the pBWA(V)HS-Glosgfp empty vector plasmid revealed that, in terms of morphology, there was no significant difference between the OE-6 strain and the tobacco transformed with the pBWA(V)HS-Glosgfp empty vector plasmid. However, flower phenotype observation showed that the flowers of the OE-6 strain were significantly lighter in color compared to those of the tobacco transformed with the pBWA(V)HS-Glosgfp empty vector plasmid. Figure 7 ).

[0072] This invention uses OE promoter-empty transgenic tobacco as an experimental control to systematically compare the anthocyanin accumulation characteristics of OE-CpMYB4 overexpression lines and control plants. Two OE-CpMYB4 high-expression transgenic lines (OE-6 and OE-14, see [link to experiment]) were selected for the experiment. Figure 8 and Figure 9 Phenotypic observation and quantitative analysis were performed on the control group and the empty control group. After anthocyanin extraction, it was found that the extract from the empty control plants was typically red, while the extract from the OE-6 strain was significantly lighter in color, showing a distinct light pink hue. Figure 8 The results visually show that the anthocyanin accumulation in this strain is reduced.

[0073] To provide a more intuitive and quantitative representation of flower color changes, we used a plant anthocyanin detection kit to measure anthocyanin content. The results showed that the empty control flowers had the highest anthocyanin content, with OE-6 having an anthocyanin content 5.10 times lower than the empty control. OE-14 had a higher anthocyanin content than OE-6, but still significantly lower than the empty control, by 2.12 times. Comparing OE-6 and OE-14, OE-6 had anthocyanin content 3.75 times lower than OE-14. Comparing the three groups, OE-6 and OE-14 showed significant differences from the empty control, with OE-6 showing the greatest difference, reaching a statistically significant level (e.g., ). Figure 10 The results showed that CpMYB4 overexpression inhibited the biosynthesis of tobacco anthocyanins.

[0074] This invention clarifies the function of the CpMYB4 gene in wintersweet and reveals the mechanism by which CpMYB4 regulates tobacco flower color by inhibiting anthocyanin synthesis. Therefore, it is more unique and innovative than previous studies on flower color regulation genes, both in terms of research object and application value. In this invention, CpMYB4 inhibits the expression of anthocyanin synthesis genes, resulting in pink or white flowers. This precise regulation of pigments is a key factor in the role of MYB transcription factors in flower color improvement engineering. This experiment utilizes heterologous expression of CpMYB4 to achieve targeted control of flower color, playing a crucial role in flower color improvement engineering.

[0075] Given the ease of tissue culture and the high efficiency of gene transformation, tobacco has become a favored model plant in the fields of plant genetic engineering and molecular biology research.

[0076] The flower color of wintersweet is primarily yellow, with flavonols and carotenoids determining its hue. This invention utilizes the heterologous CpMYB4 gene to conduct a preliminary study on the flower color of wintersweet. It was found that overexpression of the CpMYB4 gene significantly inhibited the content of anthocyanins and carotenoids in transgenic tobacco and altered the flower color, changing the original color of the flowers from deep red or pink to light or white. This finding is consistent with previous studies reporting the function of MYB transcription factors in regulating anthocyanin synthesis.

[0077] At the molecular level, CpMYB4 may regulate flower color through two pathways: anthocyanins and flavonols. qRT-PCR analysis showed that the expression of genes related to anthocyanin biosynthesis (such as NtANS / NtDFR) in transgenic tobacco was significantly decreased, and CpMYB4 directly or indirectly reduced anthocyanin synthesis, similar to the role of AtMYBL2 in Arabidopsis thaliana in regulating its transcription by binding to the ANS promoter. These results provide important insights for improving the flower color of wintersweet and lay the foundation for breeding new wintersweet varieties with more ornamental traits.

[0078] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of overexpression of the wintersweet CpMYB4 gene in inhibiting anthocyanin biosynthesis in plants, characterized by: The nucleotide sequence of the CpMYB4 gene is shown in SEQ ID NO:1, and the plant is wintersweet or tobacco.

2. The application of overexpression of *Chimonanthus praecox* CpMYB4 protein in inhibiting anthocyanin biosynthesis in plants, characterized by: The amino acid sequence of the CpMYB4 protein is shown in SEQ ID NO:2, and the plant is wintersweet or tobacco.

3. A CpMYB4 gene of wintersweet, characterized by: The nucleotide sequence of the CpMYB4 gene is shown in SEQ ID NO:

1.

4. A CpMYB4 protein from wintersweet, characterized in that: The amino acid sequence of the CpMYB4 protein is shown in SEQ ID NO:

2.

5. The biological material related to the CpMYB4 protein of *Chimonanthus praecox* as described in claim 4 is any one of the following B1) to B8): B1) A nucleic acid molecule encoding the CpMYB4 protein of the wintersweet described in claim 4; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4).

6. A method for preparing transgenic plants, characterized in that: The recombinant vector described in claim 5 (B3) or (B4) is introduced into plant cells to obtain transgenic plants that overexpress the CpMYB4 gene, wherein the plant is wintersweet or tobacco.

7. A method for changing the flower color of plants, characterized in that: By overexpressing the CpMYB4 gene of wintersweet, the anthocyanin content is reduced, resulting in light pink or white flowers. The plant in question is wintersweet or tobacco.

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