Application of chimonanthus praecox CpMYB4 gene in inhibition of biosynthesis of plant anthocyanin

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a chimonanthus praecox CpMYB4 gene in inhibition of biosynthesis of plant anthocyanin. A chimonanthus praecox CpMYB4 gene is cloned, an overexpression vector driven by a promoter is constructed, the overexpression vector is transformed into tobacco, and it is found that overexpression of the CpMYB4 gene remarkably improves expression of tobacco flavonol synthase and leads to decrease of the anthocyanin content and fading of the flower color. The CpMYB4 indirectly inhibits the activity of anthocyanin synthesis related genes (NtANS and NtDFR) by regulating the expression of key genes (such as NtCHS, NtCHI and NtFLS) in a flavonoid synthesis route, so that the content of anthocyanin is reduced. The invention provides a new strategy for plant flower color regulation and control, can be used for cultivating new varieties of ornamental plants with light or white flower colors, and has important agricultural and gardening application values.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to application of a Chimonanthus praecox MYB4 gene in inhibiting anthocyanin biosynthesis in plants. BACKGROUND

[0002] Flavonols are a class of polyphenolic compounds widely present in the plant kingdom, belonging to an important subclass of flavonoids. These compounds play a key role in plant growth and development and response to environmental stress. Flavonol biosynthesis is a complex and highly regulated process involving multiple enzymatic steps and the coordinated action of genes.

[0003] The synthesis pathway of flavonols is part of the phenylpropanoid metabolic pathway, starting from phenylalanine. The regulation of flavonol biosynthesis pathway is a multi-level process involving transcription factors, enzyme activity and environmental signals. For example, R2R3-MYB transcription factors play a key 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 UV radiation), temperature, pathogen infection and nutrient status (such as low phosphorus stress), can affect the synthesis and accumulation of flavonols. For example, UV-C radiation can up-regulate the biosynthesis of phenolic compounds in peach fruit, with a significant increase in the expression of flavonol compounds.

[0004] Although the role of MYB genes (MYB transcription factors such as Figure 1 has been widely studied in flower color regulation, its application still faces the following key bottlenecks: (1) Metabolic interference caused by insufficient targeting. Currently discovered MYB genes mostly play a role by directly regulating anthocyanin synthesis genes, for example: cotton GaMYB, which was found to positively regulate glutathione S-transferase gene (GaGST), thereby affecting the formation of petal red spots. However, some MYB genes may have non-specific effects on other metabolic pathways. Non-target effects can lead to imbalance of plant secondary metabolism, and even affect growth and development.

[0005] (2) Species-specific limitations, the functions of some MYB genes are significantly species-specific, and their cross-species applications often face the following obstacles: incompatibility of gene expression regulatory elements, the cis-elements in the promoter region (such as light response elements and hormone response elements) may not be able to normally drive gene expression due to species differences. Differences in transcription complex interactions, MYB protein interactions with bHLH and WD40 proteins depend on specific amino acid site interaction interfaces, and cross-species substitution may lead to reduced complex stability. Substrate preference of metabolic pathways, the enzyme activities and substrate specificity of anthocyanin synthesis pathways in different plants may differ. For example, the DFR of some plants (such as petunia) prefers dihydroquercetin as a substrate, but does not accept dihydroquercetin, while other plants (such as grapes) can use a variety of dihydroflavonol substrates.

[0006] (3) Limitations of regulation efficiency and practical application, overexpression of MYB genes often fails to achieve ideal flower color depth or uniformity. For example, MYB transcription factors regulate gene expression by affecting epigenetic modifications such as histone modification and DNA methylation, but the efficiency and stability of this regulation still need further research. In addition, the expression and activity of MYB genes are affected by various factors such as abscisic acid, environmental light and temperature. The superposition of these complex factors makes it difficult to accurately control the regulation efficiency of MYB genes. In particular, in some difficult-to-transform horticultural crops, this further limits the application of MYB genes in flower color improvement.

[0007] In view of the deficiencies of the prior art, the inventors have for the first time found that the Chimonanthus praecox CpMYB4 gene plays an important role in the flower color regulation network and is a core MYB transcription factor affecting flower color synthesis in Chimonanthus praecox.

[0008] With the rapid development of the global flower industry, the market demand for ornamental plant flower color diversification is increasing. Traditional breeding methods through hybridization and mutagenesis screening of light-colored flower varieties have long cycles and low efficiency, while genetic engineering technology provides a new way for precise regulation of flower color. However, the limitations of existing inhibitory MYB genes seriously restrict their application in flower improvement: (1) Single flower color of ornamental plants, most commercial flowers (such as roses and carnations) rely on single flower color (red, pink, white), and lack of novelty. (2) Changes in consumer preferences, the market demand for light-colored flowers (such as light pink, beige, and light purple) is increasing year by year, but traditional breeding is difficult to respond quickly. (3) Functional flower development, light-colored flower varieties have unique application value in the fields of medicine (such as antioxidant active ingredient extraction) and cosmetics (such as natural pigment raw materials).

[0009] In summary, the development of new MYB genes (such as CpMYB4) is of great significance for breaking through the existing flower color regulation technology bottleneck and promoting the upgrading of the flower industry. SUMMARY

[0010] The application provides the application of the CpMYB4 gene of the wax plant in inhibiting anthocyanin biosynthesis.

[0011] The application also provides the application of the CpMYB4 protein of the wax plant in inhibiting anthocyanin biosynthesis.

[0012] The application also provides a CpMYB4 gene of the wax plant, and the nucleotide sequence of the CpMYB4 gene is shown as SEQ ID NO: 1.

[0013] The application also provides a CpMYB4 protein of the wax plant, and the amino acid sequence of the CpMYB4 protein is shown as SEQ ID NO: 2.

[0014] The application also provides biological materials related to the CpMYB4 protein, and the biological materials are any one of the following B1) to B8): B1) a nucleic acid molecule encoding the CpMYB4 protein; B2) an expression cassette containing the nucleic acid molecule in B1); B3) a recombinant vector containing the nucleic acid molecule in B1), B4) a recombinant vector containing the expression cassette in B2); B5) a recombinant microorganism containing the nucleic acid molecule in B1); B6) a recombinant microorganism containing the expression cassette in B2); B7) a recombinant microorganism containing the recombinant vector in B3); B8) a recombinant microorganism containing the recombinant vector in B4).

[0015] The application also provides a method for preparing a transgenic plant, and the recombinant vector is introduced into a plant cell to obtain a transgenic plant overexpressing the CpMYB4 gene.

[0016] In some embodiments, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0017] In some embodiments, the plant is tobacco, Arabidopsis, chrysanthemum or rose.

[0018] The application also provides the application of the CpMYB4 gene of the wax plant in regulating a plant flavonoid synthesis pathway, and the biosynthesis of flavonols is improved by up-regulating the expression of NtCHS, NtCHI and NtFLS genes.

[0019] The application also provides a method for improving flower color of plants by overexpressing the Chimonanthus praecox CpMYB4 gene to reduce anthocyanin content, so that the flower color of the plants is light pink or white.

[0020] Compared with the prior art, the application has at least the following beneficial effects: (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, and changes the flower color of plants from dark red / purple to light pink or white, providing a new tool for flower color improvement.

[0021] (2) Widening the application range: suitable for a variety of plants (such as tobacco, Arabidopsis, chrysanthemum, etc.), especially suitable for cultivating ornamental light-colored flower varieties.

[0022] (3) Environmental protection and economic value: reducing the need for chemical dyeing, promoting the development of green horticulture industry, and improving the competitiveness of the flower market. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Classification of plant MYB transcription factors.

[0024] Figure 2 Growth environment of OE-CpMYB4 transgenic tobacco lines.

[0025] Figure 3 Comparison of OE-CpMYB4 transgenic tobacco lines and pBWA(V) HS-Glosgfp empty vector tobacco plants in shape.

[0026] 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.

[0027] Figure 5 qRT-pcr quantitative detection of OE-CpMYB4 transgenic tobacco flowers; ***, extremely significant, p value <0.001.

[0028] Figure 6 Expression amount of flavonol biosynthesis structural genes; ***, extremely significant, p value <0.001; **, significant, p value <0.01; *, slightly significant, p value <0.05; ns, not significant.

[0029] Figure 7, OE-CpMYB4 transgenic tobacco phenotype observation.

[0030] Figure 8 , OE-CpMYB4-6 transgenic tobacco flower anthocyanin extraction.

[0031] Figure 9 , OE-CpMYB4-14 transgenic tobacco flower anthocyanin extraction.

[0032] Figure 10 , OE-CpMYB4 anthocyanin content determination; **, significant, p value <0.01. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical schemes and advantages to be solved by the present application more clear, the following will be described in detail in combination with the drawings and specific embodiments.

[0034] Materials Plant material, taken from the black dragon pool of waxes HLT015; tobacco K326.

[0035] Experimental reagent, DNA kit (Bomeide Biotechnology Co., Ltd.), RNA extraction kit (Aidley Biotechnology Co., Ltd.), PCR kit, cDNA first strand synthesis kit, PerfectStart Green qPCR SuperMix (Beijing Zhenxingjin Biotechnology Co., Ltd.), Phanta Max Super-Fidelity DNA Polymerase high fidelity enzyme (Nanjing Nuowezan Biotechnology Co., Ltd.).

[0036] Instrument and equipment, super clean bench, constant temperature water bath, constant temperature culture shaker, high pressure sterilization pot, DYY-12C type electrophoresis instrument (Beijing Liuyi Instrument Factory); East Victory eQ9600 series 96 hole fluorescence quantitative PCR instrument (Suzhou East Victory Biotechnology Co., Ltd.); nucleic acid protein detector (Nano Drop 2000C) (Thermo Fisher Scientific Corporation); ultraviolet spectrophotometer (Shanghai Meipuda).

[0037] SEQ ID NO: 1: ATGGGTCGAAGTCCTTGTTGCAGCAAAGAGGGTCTCAACAGAGGAGCATGGACTGCCATTGAAGATGAAATTCTCATAAATTACATCAAGATCCATGGAGAAGGAAGATGGAGAAGCCTACCCATTAAAGCAGGTCTTAAACGATGTGGGAAGAGTTGTAGGCTCCGCTGGTTGAATTATCTGAGGCCTGATATCAAGAGAGGCAACATATCTCACGAAGAAGAAGAGCTCATCATCAGACTCCATAAGCTTCTTGGCAACAGATGGTCTCTGATAGCCGGAAGGCTTCCAGGGCGAACAGACAATGAAATTAAGAACTACTGGAATACCAATTTAGTAAAGAAGTTGCAGAAGCAGCCCATTTCCAAACAACCTGTTGAGGGATCGCCAGCGCCGGAACTCAAATCAAATGTCATCCGAACCAAGGCTTCAAGGTGCACCAGGGTTTTCATCTCACCACCGCAGCGAAATGTCGAAAATCTAAACAAAATTCAAGATGTCGAGGCTACCGGGGTAGCTGGAGATTTCGCAGCTTCAAAAGGAAAAGGATTGGAATCCTGTCGAGTTCCATCTCTAACTTGCCACGACGACGATTCGTCGAGCTCTCTTGTGGATTTGGATATGGGTGAATTCTGCTTGGAGCTCATCAATTCGGACTACTTCCAGGTCTTGGGTGAGAGTTCAATCAACGACGACAATAATGCTGTTGTTGCAGATGCTTCCAACTGCAACCAACTGCTGCACCCCTTCATGTTCAGTGAAGAAATGCTGGTGGATTGGACTGGTGGAGATTGCTTTCAAACAGATGTTGGTTTCAATATCGAATCTATGACTTCTTGCCTTGATTCTCAAGGA; SEQ ID NO: 2: MGRSPCCSKEGLNRGAWTAIEDEILINYIKIHGEGRWRSLPIKAGLKRCGKSCRLRWLNYLRPDIKRGNISHEEEELIIRLHKLLGNRWSLIAGRLPGRTDNEIKNYWNTNLVKKLQKQPISKQPVEGSPAPELKSNVIRTKASRCTRVFISPPQRNVENLNKIQDVEATGVAGDFAASKGKGLESCRVPSLTCHDDDSSSSLVDLDMGEFCLELINSDYFQVLGESSINDDNNAVVADASNCNQLLHPFMFSEEMLVDWTGGDCFQTDVGFNIESMTSCLDSQG. Example 1, Cultivation of Transgenic Tobacco

[0038] The transgenic seeds were sterilized with 70% ethanol, then soaked in 10% sodium hypochlorite for 15 minutes, and washed with sterilized water for 5 times, 2 minutes each time. The surface-sterilized seeds were evenly spread on Petri dishes containing ½MS solid medium, sealed with sealing film, and placed in a 25°C light incubator (16 hours light, 8 hours dark) for culture. After 5-7 days of sowing, the small buds began to germinate and the cotyledons grew out, and after 14 days, the seedlings grew out 2 true leaves, and began to be sown. After sowing, post-sowing management was carried out, and after the test-tube seedlings grew out several root hairs, they were opened and acclimated for 3 days, and then transplanted into a mixed medium of perlite: nutrient soil (1:1) for culture. Then, the OE-CpMYB4 (cloned in the laboratory's previous research) 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 spread; at the same time, the flower phenotype of the tobacco plants was observed, and the excellent lines were photographed and the complete lines were collected into sterile cryotubes and immediately placed in liquid nitrogen for temporary storage. After the collection was completed, the lines were placed in an ultra-low freezer for storage and treated, and the lines were bagged for seed collection.

[0039] Results: In the previous cultivation of the laboratory, OE-CpMYB4 transgenic tobacco positive lines were obtained. Figure 2 During the annual growth cycle, constant temperature conditions, air humidity, light intensity, and corresponding water and fertilizer conditions were maintained. The transgenic tobacco had dark green leaves and grew well, and the flower phenotype was observed after the transgenic tobacco flowered. Figure 3 . Example 2

[0040] (1) Extraction of tobacco flower RNA and synthesis of cDNA Total RNA was extracted from the flowers of Wintersweet HLT015 at the flowering stage. First, 1% agarose gel electrophoresis was used to detect the quality of the RNA sample. Then, the concentration of the RNA sample was detected using a Nanodrop 2000. Finally, the RNA sample was reverse transcribed into cDNA according to the instructions of the PrimeScript RT reagent Kit with gDNA Eraser (cDNA first-strand synthesis reagent kit).

[0041] Table 1, RNA reverse transcription reaction system Component Amount 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 H2O (RNase free) to final volume 20 μL (2) Quantitative analysis of tobacco flower qRT-pcr The flower samples of different strains of transgenic tobacco were collected, and the total RNA was extracted and reverse transcribed into cDNA. Real-time fluorescent quantitative PCR (qRT-PCR) technology was used to accurately analyze the gene expression in transgenic tobacco. The following primers were designed and synthesized using Premer 3.0 software: CpMYB4qF: GGTTTTCATCTCACCACCGC (SEQ ID NO: 3); CpMYB4qR: TCGTCGTCGTGGCAAGTTAG (SEQ ID NO: 4); NtRNR2 was used as the internal reference gene. The synthesis of cDNA was performed using the EasyScript one-step gDNA removal and cDNA synthesis kit. qRT-PCR was performed on eQ9600. The reaction process was as follows: 94°C for 10 s, then 44°C for 5 s, 60°C for 15 s, and 72°C for 30 s. The materials used in the fluorescent quantitative experiment had 3 biological replicates and 3 technical replicates. The relative expression of each gene was calculated using the 2 −ΔΔ CT method. All results were calculated and plotted using Graphpad Prism9.

[0042] Table 2, qRT-PCR reaction system of tobacco internal reference gene Component Amount 2×T5 Super PCR Mix (Basic) 10 μL 10 μMCpActin-F 1 μL 10 μMCpActin-R 1 μL Template cDNA 1 μL ddH2O 7 μL Table 3, sequence of flavonoid fluorescent quantitative PCR primers 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) Results: The RNA of pBWA(V)HS-OE-CpMYB4-GLosgfp positive seedlings was extracted, and the quality of the RNA sample was good as detected by agarose gel electrophoresis (Figure Figure 4 ).

[0043] Using the extracted RNA sample of OE-CpMYB4 transgenic tobacco positive seedlings as the template, the NtRNR2 internal reference gene was used as the reference, and the fluorescent quantitative PCR primers of CpMYB4 were used for real-time quantitative experiment to screen the transgenic OE-CpMYB4 tobacco positive plants with the highest expression.

[0044] The RNA was reverse transcribed into cDNA using a reverse transcription kit, and the expression of CpMYB4 in each strain was detected by real-time fluorescent quantitative PCR, and the results are shown in FIG. Figure 5 The expression of OE-CpMYB4 in 7 transgenic plants was 500 times, 21000 times, 100 times, 2500 times, 4500 times, 0 times, and 3000 times that of the wild type plants, respectively. The relative expression of OE-CpMYB4 in OE-6 strain was the highest. The expression of flavonol biosynthesis structural genes such as NtCHS, NtCHI, and NtFLS in OE-MYB107 transgenic tobacco strains was significantly increased. Among them, the expression of NtCHS, NtCHI, and NtFLS was 5.75 times, 2.5 times, and 3.5 times higher than that of the control group, respectively; while the expression of NtF3'H was 7.5 times lower than that of the control group as shown in FIG. Figure 6 NtF3H, NtDFR, NtANS, and NtUFGT had no significant difference. It is speculated that CpMYB4 promotes the synthesis of flavonols by promoting the expression of structural genes in the flavonol biosynthesis pathway, and the excessive accumulation of flavonols makes the tobacco flowers appear light pink or white. Example 3, phenotype observation and analysis of CpMYB4 transgenic tobacco

[0045] During the growth period of the plants, the flowers of the tobacco were periodically photographed using a high-definition camera, and the front view of the tobacco flowers was collected at a specific time to capture the color gradient. During the flowering period, the flowers were collected and immediately placed in a sterile cryogenic tube prepared in advance, and the time, variety, and characteristics were recorded. The sample tube was quickly frozen in liquid nitrogen and transferred to a -80°C ultra-low temperature freezer for storage.

[0046] The anthocyanin content was determined using a plant anthocyanin content kit, and the expression of flavonoid structural genes in transgenic tobacco was determined using a fluorescent quantitative PCR detection kit.

[0047] Results: The OE-6 strain of tobacco with the highest expression was observed, and the phenotype of the tobacco into which the pBWA(V) HS-Glosgfp empty vector plasmid was introduced was found to have no significant difference in appearance. The flower color of the OE-6 strain of tobacco was significantly lighter than that of the tobacco into which the pBWA(V) HS-Glosgfp empty vector was introduced as shown in FIG. Figure 7 ).

[0048] The present application takes OE promoter empty transgenic tobacco as experimental control, and systematically compares 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 Figure 8 and Figure 9 ) and the empty control group are selected for phenotype observation and quantitative analysis. After extracting anthocyanins, it is found that the extract of the empty control plant presents a typical red color, while the extract of the OE-6 line is significantly lighter, presenting a clear light pink color like Figure 8 , which directly shows that the anthocyanin accumulation amount of this line is reduced.

[0049] In order to quantitatively express the color change more directly, we use the plant anthocyanin detection kit to detect the anthocyanin content. The experimental results show that the anthocyanin content of the empty control flower is the highest, the anthocyanin content of OE-6 is 5.10 times lower than that of the empty control; the anthocyanin content of OE-14 is higher than that of OE-6, but is still significantly lower than that of the empty control, which is 2.12 times lower; the anthocyanin content of OE-6 is 3.75 times lower than that of OE-14. Comparing the three groups of data, the differences between OE-6, OE-14 and the empty control are large, and the difference between OE-6 and the empty control is the largest, reaching a significant level like Figure 10 . The results show that the overexpression of CpMYB4 inhibits the biosynthesis of tobacco anthocyanin.

[0050] The present application determines the function of the waxflower CpMYB4 gene, and reveals the mechanism of CpMYB4 regulating tobacco flower color by inhibiting anthocyanin synthesis. Therefore, whether in the research object or in the application value, it is more special and innovative than the previous flower color regulation genes. In the present application, CpMYB4 inhibits the expression of anthocyanin synthesis genes, and the flower color changes to pink or white. This precise regulation of pigments is the key factor for MYB transcription factors to play a role in flower color improvement engineering. In this test, the directional control of flower color is realized by using CpMYB4 for heterologous expression, which plays a crucial role in flower color improvement engineering.

[0051] Due to the convenience of tissue culture and the high gene transformation efficiency of tobacco, it has become a popular model plant in the field of plant genetic engineering and molecular biology research.

[0052] The color of the flower of Wintersweet is basically yellow, and flavonol and carotenoid determine the yellow color tone. The present application preliminarily studies the flower color of Wintersweet by using heterologous CpMYB4 gene, and finds that overexpression of CpMYB4 gene can significantly inhibit the content of anthocyanin and carotenoid in transgenic tobacco and change the color of tobacco flower, so that the original color of the flower is changed from deep red or pink to light color or white. This finding is consistent with the function of MYB transcription factor in regulating anthocyanin synthesis reported in the existing research.

[0053] In terms of molecular mechanism, CpMYB4 may regulate the flower color through the two pathways of anthocyanin and flavonol. qRT-PCR analysis shows that the expression of anthocyanin biosynthesis related genes (NtANS / NtDFR, etc.) of transgenic tobacco is significantly decreased, and CpMYB4 directly or indirectly reduces the synthesis of anthocyanin, which is similar to the function of Arabidopsis AtMYBL2 in binding the ANS promoter to regulate its transcription, and these results have important implications for the improvement of the flower color of Wintersweet and lay a foundation for breeding new varieties of Wintersweet with more ornamental traits.

[0054] The above is the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of the Chimonanthus praecox CpMYB4 gene in inhibiting plant anthocyanin biosynthesis, characterized in that: The nucleotide sequence of the CpMYB4 gene is shown in SEQ ID NO:

1.

2. Application of Chimonanthus praecox CpMYB4 protein in inhibiting plant anthocyanin biosynthesis, characterized by: The amino acid sequence of the CpMYB4 protein is shown in SEQ ID NO:

2.

3. A Chimonanthus chinensis CpMYB4 gene, characterized in that: The nucleotide sequence of the CpMYB4 gene is shown in SEQ ID NO:

1.

4. A Chimonanthus praecox CpMYB4 protein, 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 according to claim 4, which is any one of the following B1) to B8): B1) a nucleic acid molecule encoding the CpMYB4 protein according to 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) a recombinant microorganism containing the nucleic acid molecule described in B1); B6) a recombinant microorganism containing the expression cassette described in B2); B7) a recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

6. A method for preparing a transgenic plant, characterized in that: The recombinant vector according to claim 5 is introduced into plant cells to obtain transgenic plants that overexpress the CpMYB4 gene.

7. The method according to claim 6, characterized in that: The plant is a dicotyledonous plant or a monocotyledonous plant.

8. The method according to claim 6, wherein: The plant is tobacco, Arabidopsis thaliana, chrysanthemum or rose.

9. Application of the Chimonanthus praecox CpMYB4 gene in regulating the plant flavonoid biosynthesis pathway, characterized in that: The biosynthesis of flavonols is improved by upregulating the expression of NtCHS, NtCHI, and NtFLS genes.

10. A method for improving plant color, characterized by: By overexpressing the CpMYB4 gene in wintersweet, the anthocyanin content is reduced, making the plant's flowers light pink or white.

Images