Application of Nymphaea colorata NcolMYB75-like gene in improving anthocyanin content of plants

Abstract

The application discloses application of NcolMYB75-like gene of blue star water lily in improving anthocyanin content of plants and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the NcolMYB75-like gene provided in the application is shown as SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO:2. The application finds that the gene can strongly promote anthocyanin biosynthesis by heterologous expression of the gene in Arabidopsis thaliana AtMYB75 function loss mutant (myb75-c), so that the anthocyanin accumulation amount of the transgenic plant reaches more than 15 times of that of a wild type control, and the leaves present a significant purplish red color. The application is particularly suitable for plant materials with limited anthocyanin synthesis capacity, and provides an innovative gene resource and technical scheme for efficient cultivation of crop and ornamental plant varieties with high anthocyanin content.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of the NcolMYB75-like gene of the blue star water lily in increasing the anthocyanin content of the plant. Background Technology

[0002] Water lilies (Nymphaea) are perennial aquatic herbaceous plants, with approximately 50 species belonging to 5 subgenera worldwide, mainly distributed in tropical, subtropical, and temperate regions. Some water lily varieties have unique fragrances, making them important raw materials for essential oils and perfumes. Furthermore, water lilies possess a strong ability to absorb heavy metals and harmful substances from water, making them useful for the ecological restoration of polluted water bodies. As a prized ornamental flower, water lilies boast a rich variety of colors and graceful forms, earning them the title of "palette of the water." Blue-purple varieties are particularly favored, exhibiting significant ecological and economic value in landscaping and water feature design.

[0003] Blue flowers are relatively rare in the plant kingdom, and their formation is subject to multiple limitations, including anthocyanin synthesis mechanisms, vacuolar pH regulation, and evolutionary adaptation. Plant flower colors primarily originate from flavonoid compounds, with anthocyanins dominating the blue-purple color. Anthocyanins are water-soluble flavonoid pigments widely found in plants, formed by the combination of anthocyanin aglycones (such as cyanidin and delphinidin) with glycosyl groups, mainly distributed in cell vacuoles. The color of anthocyanins is affected by cell sap pH: acidic conditions tend towards red, neutral conditions towards purple, and alkaline conditions towards blue. However, most plant vacuoles are acidic, making stable blue color formation difficult. Truly persistent blue anthocyanins often require additional chemical modifications, such as glycosylation or chelation with metal ions (such as aluminum ions), to improve their stability in acidic environments. Such mechanisms are only found in a few plant families and genera, such as Hydrangeaceae, Ranunculaceae, Iridaceae, and some Orchidaceae. For example, the blue color of hydrangeas depends on aluminum ion complexation, while delphiniums rely on specific blue-purple glycosides. Due to the complexity of related metabolic and regulatory networks, the number of truly blue-flowered species in nature is limited, with fewer than 100 species of naturally occurring blue-flowered plants confirmed globally.

[0004] Besides determining flower color, anthocyanins in plants also play important roles such as photosensitivity, antioxidation, ultraviolet absorption, and mitigation of strong light stress. They also attract animals to disperse seeds during fruit ripening. In the health field, anthocyanins have attracted much attention due to their antioxidant, anti-inflammatory, and anti-tumor activities, making them an important functional nutrient. Blueberries, black goji berries, purple sweet potatoes, and red grapes, rich in anthocyanins, are widely used in food and health products. Anthocyanins are also frequently used as natural colorants in the food industry. In agricultural biotechnology, regulating anthocyanin synthesis pathways can improve crop color, enhance nutritional value, or increase stress resistance; for example, high-anthocyanin rice and corn are currently under research. Therefore, anthocyanins have significant value in plant adaptation, biotechnology, and the health industry.

[0005] MYB transcription factors are key regulatory proteins widely distributed in plants. Their structural feature is the presence of conserved MYB domains, with the most common being R2R3-MYB, composed of two repeating domains of approximately 50 amino acids each, capable of binding to specific promoter elements (such as TAACG) to regulate downstream gene expression. MYB proteins typically contain transcriptional activation regions, nuclear localization signals, and phosphorylation sites, playing crucial roles in secondary metabolism, hormone responses, and organ development. This family of genes participates in the regulation of metabolic pathways such as anthocyanin and lignin, guiding plant color formation and stress responses. For example, PAP1 in Arabidopsis thaliana promotes anthocyanin accumulation, while OsMYB3R-2 in rice regulates tillering and growth.

[0006] Currently, anthocyanin-regulated MYB genes in various plants, including Arabidopsis thaliana, tobacco, grape, strawberry, pear, apple, and petunia, have been cloned and validated. In *Nymphaea bluestarensis*, only one R2R3-MYB transcription factor, NcMYB25, has been reported; the functions of other MYB genes related to anthocyanin synthesis have not yet been validated. Given that anthocyanins are crucial pigments determining the color of water lilies, especially playing a dominant role in blue-purple varieties, identifying new MYB regulatory genes is significant for refining the molecular network of water lily color formation. Furthermore, *Nymphaea bluestarensis* is representative of the *Nymphaea* genus, and elucidating its MYB genes helps to understand the conservation and specificity of the anthocyanin regulatory network across different species, providing a theoretical basis and genetic resources for molecular breeding of water lily color. Summary of the Invention

[0007] To address the problems existing in the prior art, the technical problem solved by this application is to provide an application of the NcolMYB75-like gene of the blue star water lily in increasing the anthocyanin content of the plant.

[0008] To solve the above-mentioned technical problems, the technical solution of this application is as follows:

[0009] The application of the NcolMYB75-like gene of the blue star water lily in increasing the anthocyanin content of plants includes: expressing the NcolMYB75-like gene in plants, wherein the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0010] In some embodiments, the nucleotide sequence of the NcolMYB75-like gene of the blue star water lily is shown in SEQ ID NO:1.

[0011] In some embodiments, the application increases the anthocyanin content of the target plant to at least five times that of its wild-type control.

[0012] In some embodiments, the plant is a mutant with lost or weakened function of endogenous anthocyanin synthesis regulators.

[0013] In some embodiments, the mutant is specifically the Arabidopsis thaliana AtMYB75 / PAP1 loss-of-function mutant myb75-c mutant.

[0014] In some embodiments, the application includes the following steps:

[0015] (1) Construct a plant recombinant expression vector containing the NcolMYB75-like gene of the blue star water lily;

[0016] (2) Transform the recombinant expression vector into plant tissues or cells;

[0017] (3) Cultivate and screen transgenic plants with increased anthocyanin content.

[0018] In some embodiments, the method for constructing the plant recombinant expression vector specifically involves: linking the gene shown in SEQ ID NO: 1 with a plant functional promoter to form an expression cassette, and inserting it into a plant expression vector.

[0019] In some embodiments, the plant functional promoter is the cauliflower mosaic virus 35S promoter.

[0020] In some embodiments, the transformation is carried out by Agrobacterium-mediated transformation.

[0021] The use of transgenic plants or parts thereof with increased anthocyanin content obtained from any of the aforementioned applications in the preparation of anthocyanin-rich plant extracts or products.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Currently, research on MYB transcription factors regulating anthocyanin synthesis in *Water Lily 'Blue Star'* is limited to NcMYB25, restricting molecular breeding and improvement of blue water lily varieties. To address this issue, this invention cloned and identified a novel R2R3-type MYB transcription factor gene, NcolMYB75-like, from *Water Lily 'Blue Star'*, and verified its function in *Arabidopsis thaliana* AtMYB75 mutants. The gene significantly promoted anthocyanin accumulation, demonstrating its unique role in anthocyanin regulation. Experiments showed that expressing this gene in *Arabidopsis thaliana* AtMYB75 loss-of-function mutants not only completely compensated for the anthocyanin synthesis defect but also increased anthocyanin content to more than 15 times the wild-type level, achieving super-accumulation. The NcolMYB75-like gene and its applications provided by this invention offer key gene resources and technical means for elucidating the molecular mechanism of blue-purple anthocyanin accumulation in *Water Lily 'Blue Star'*, cultivating superior blue-purple ornamental varieties, and conducting molecular breeding. Attached Figure Description

[0024] Figure 1 This is an electrophoretic image of the NcolMYB75-like gene CDS clone. In the image, M: DNA Marker; 1: Full-length CDS sequence of the NcolMYB75-like gene.

[0025] Figure 2 Construction and identification of NcolMYB75-like gene overexpression vector. In the figure, M: DNA Marker, 1: recombinant plasmid pHellsgate8-NcolMYB75-like-GFP, 2: recombinant plasmid pHellsgate8-NcolMYB75-like-GFP digested with XhoI enzyme.

[0026] Figure 3 RNA level identification for NcolMYB75-like transgenic Arabidopsis thaliana;

[0027] Figure 4 The 4-day seedling phenotypes of Arabidopsis thaliana wild-type WT, mutant myb75-c and NcolMYB75-like heterologous replacement transgenic lines myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2;

[0028] Figure 5 The four-week plant phenotypes of wild-type Arabidopsis thaliana WT, mutant myb75-c and NcolMYB75-like heterologous replacement transgenic lines myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 and the corresponding total anthocyanin extracts from rosette leaves were obtained.

[0029] Figure 6 The relative total anthocyanin content of Arabidopsis wild-type WT, mutant myb75-c and NcolMYB75-like heterologous replacement transgenic lines myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2;

[0030] Figure 7 The expression levels of anthocyanin synthesis-related genes in the wild-type WT, mutant myb75-c, and NcolMYB75-like heterologous replacement transgenic lines myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 of Arabidopsis thaliana were determined. Detailed Implementation

[0031] 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.

[0032] Example 1

[0033] 1. Cloning and overexpression vector construction of the NcolMYB75-like gene from the blue star water lily.

[0034] 1.1 Amplification of the CDS sequence of the NcolMYB75-like gene from the blue star water lily

[0035] A suitable amount of leaf tissue samples were taken from the tested material, *Nymphaea colorata*, and RNA was extracted from the samples using the plant tissue total RNA extraction kit provided by Nanjing Novizan Biotechnology Co., Ltd. The extracted RNA was then used to synthesize cDNA using Novizan Biotechnology Co., Ltd.'s third-generation high-efficiency cDNA one-strand synthesis kit (genomic removal). Based on the transcriptome sequence of the *Nymphaea colorata* NcolMYB75-like gene, the following primers were designed: Forward: 5'-ATGGCCAGCAGCTCGGGCAGCCATTG-3'; Reverse: 5'-TCATATCGCAGGATCATCATCATCTT-3'. The CDS sequence of the NcolMYB75-like gene was obtained by PCR, and the PCR amplification electrophoresis image is shown below. Figure 1 As shown in the figure. The CDS sequence of the NcolMYB75-like gene is shown in SEQ ID NO:1, which is 912 bp in length and encodes 303 amino acids, as shown in SEQ ID NO:2.

[0036] 1.2 Construction of NcolMYB75-like gene overexpression vector

[0037] Based on the CDS sequence of the NcolMYB75-like gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction site), amplification primers were designed: Forward: 5'-CATTTTGGAGAGGACACGCTCGAGATGGCCAGCAGCTCGGGCA-3'; Reverse: 5'-CTTGCTCACCATGAATTCCTCGAGTATCGCAGGATCATCATCA-3'. The target fragment of the NcolMYB75-like gene was amplified by PCR. The pHellsgate8-GFP vector was digested with XhoI at 37℃ for 3 h. The PCR products and digestion products were recovered from the gel using a Shanghai Sangon Biotech gel extraction kit to obtain the target fragment and the linearized vector fragment. The target fragment was ligated to the pHellsgate8-GFP linear vector using the ClonExpress II One Step Cloning Kit to construct a 35S::NcolMYB75-like overexpression vector. The ligation system was as follows: 2 μl of 5X CE II buffer, approximately 100-200 ng of linearized vector, approximately 50-100 ng of insert fragment, 1 μl of Exnase II, and ddH2O to a final volume of 10 μl. The mixture was incubated at 37°C for 30 min and then immediately transferred to ice. After thawing E. coli DH5α competent cells on ice, the recombinant product was added to the competent cells and mixed thoroughly. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 sec, cooling on ice for 5 min, and then 500 μl of LB medium was added. The cells were then incubated at 37°C with shaking for 1 h. The bacterial culture was then plated on plates containing 100 mg / L spectinomycin and incubated upside down at 37°C for 12-16 h. Single colonies were validated using colony PCR. Positive colonies were picked and inoculated into LB liquid medium containing 100 mg / L spectinomycin, and cultured at 37°C with shaking at 200 rpm for 24 h. Plasmids were extracted and digested for verification. Figure 2 After verification, sequencing was performed. If the sequencing was correct, it was identified as a 35S::NcolMYB75-like overexpression vector.

[0038] 2. Screening of positive transgenic Arabidopsis thaliana strains with the NcolMYB75-like gene

[0039] 2.1 Agrobacterium infection in Arabidopsis thaliana

[0040] The 35S::NcolMYB75-like overexpression vector plasmid was electroporated into Agrobacterium GV3101 competent cells. Positive strains were obtained by colony PCR verification. Arabidopsis myb75-c mutant plants were then transformed using the flower-dipping method. Transformation medium was prepared first, consisting of 1 / 2 MS medium, 0.01 μg / ml BAP, 5% sucrose, and 0.02% silwet L-77, adjusted to pH 5.7 with KOH. Positive strains of 35S::NcolMYB75-like Agrobacterium were picked and cultured in YEP liquid medium containing 100 mg / L spectinomycin and 50 mg / L rifampin, and incubated at 28°C and 200 rpm for 16 h. 0.5 ml of the bacterial culture was then inoculated into 50 ml of YEP liquid medium containing the aforementioned antibiotics, and cultured at 28°C and 200 rpm until OD500 reached its maximum. 600 The value reaches approximately 1.8~2.0. Collect the bacterial culture in a centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, and resuspend the precipitate in conversion medium until the OD value of the resuspended solution reaches approximately 1.8~2.0. 600 Approximately 0.8. Place the Arabidopsis thaliana plants to be transformed horizontally, immerse the flower buds in the transformation medium for 1 min, then incubate in the dark for 16-24 h, and then resume normal culture.

[0041] 2.2 Screening and identification of positive plants

[0042] After disinfecting the infected seeds with chlorine, they were cultured on MS medium containing 50 mg / L kanamycin. Resistant positive seedlings were selected and transplanted into soil. After seed harvest, the seedlings were screened again on MS medium containing 50 mg / L kanamycin. Transformed seedlings with a 3:1 segregation ratio were selected and transplanted into soil. After seed harvest, the seedlings were screened again on MS medium containing 50 mg / L kanamycin to select transformant lines with 100% kanamycin resistance. The NcolMYB75-like transgenic homozygous lines were named myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2. RNA was extracted from the leaves of the homozygous transgenic positive lines, and cDNA was obtained by reverse transcription. Semi-quantitative primers were designed and semi-quantitative verification was performed. Figure 3The semi-quantitative primers are as follows: Forward: 5'-TAAACTACCTTAGCCCCAACATCAA-3'; Reverse: 5'-CTAATGTCGAAAGTGCTCCAAAACT-3'. The total volume of the PCR reaction system is 20 μL: 1 μL each of forward and reverse primers, 10 μL of 2×Hieff® Robust PCR Master Mix, 1 μL of cDNA, and 7 μL of ddH2O. The PCR reaction is performed using a PCR instrument with the following program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles of annealing and extension.

[0043] 3. The role of the NcolMYB75-like gene in anthocyanin accumulation in transgenic Arabidopsis thaliana

[0044] 3.1 Effect of heterologous expression of the NcolMYB75-like gene in the Arabidopsis mutant myb75-c on anthocyanin accumulation. Arabidopsis seeds (wild-type WT, mutant myb75-c, and NcolMYB75-like heterologous replacement transgenic lines myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2) were sterilized and planted on 1 / 2 MS solid medium. After being placed at 4°C for two days, they were cultured in a light incubator for four days. The color of the cotyledons, hypocotyls, and roots of the seedlings was observed and photographed under a stereomicroscope. Seedlings were then cultured for another 7 days before being transplanted into soil and grown for approximately 4 weeks. Rosette leaves were collected for anthocyanin extraction. The anthocyanin extract consisted of n-propanol, concentrated hydrochloric acid, and water (n-propanol:concentrated hydrochloric acid:water / 18:1:81, v:v:v). Weigh approximately 0.1 g of plant leaf tissue, quick-freeze and grind it in liquid nitrogen, add 1 ml of anthocyanin extraction solution, incubate at room temperature in the dark, extract overnight, centrifuge at 13000 g for 20 min, transfer the supernatant to a clean centrifuge tube, and dilute 4 times for assay. Measure the absorbance at 535 nm and 650 nm using a microplate reader, and calculate according to the formula 4x(A). 535 -A 650 The relative anthocyanin content per gram of fresh weight of plant tissue is calculated using this method. Figure 4 It can be seen that the seedlings of the mutant myb75-c have light green cotyledons and hypocotyls, and almost transparent roots, while the seedlings of myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 have purplish-red cotyledons, hypocotyls, and roots, significantly darker than the wild-type WT and the mutant myb75-c. Figure 5It was found that the mature leaves of the mutant myb75-c were green, while the mature leaves of myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 were purplish-red, significantly darker than those of the wild-type WT and the mutant myb75-c. These differences stem from the fact that the anthocyanin content of WT and the mutant myb75-c was lower than that of the myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 lines. The relative anthocyanin content of the leaves was measured... Figure 6 The results showed that the relative anthocyanin content in the leaves of wild-type WT was 1.819, while that of the mutant myb75-c was 0.166. Overexpression of the NcolMYB75-like gene in the myb75-c mutant significantly increased the relative anthocyanin content in the leaves (myb75-c / 35S::NcolMYB75-like-1: 13.64, myb75-c / 35S::NcolMYB75-like-2: 26.82). This result indicates that overexpression of the NcolMYB75-like gene can promote anthocyanin accumulation in plants.

[0045] 3.2 The mechanism by which NcolMYB75-like genes positively regulate anthocyanin accumulation in plants. To further understand the regulatory mechanism of NcolMYB75-like genes on anthocyanin accumulation in plants, this invention used qRT-PCR technology to detect the expression levels of anthocyanin synthesis genes in Arabidopsis thaliana WT, myb75-c, myb75-c / 35S::NcolMYB75-like-1, and myb75-c / 35S::NcolMYB75-like-2 lines. The proteins encoded by the AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX, and AtUF3GT genes are important proteases in the anthocyanin synthesis pathway. Upregulation of these genes promotes anthocyanin synthesis, while downregulation leads to reduced anthocyanin synthesis. The results are as follows: Figure 7 As shown, compared with WT and myb75-c, the expression levels of AtCHS, AtCHI, AtF3H, AtF3'H, AtDFR, AtLDOX, and AtUF3GT were all significantly upregulated in the myb75-c / 35S::NcolMYB75-like-1 and myb75-c / 35S::NcolMYB75-like-2 lines. This indicates that overexpression of NcolMYB75-like in myb75-c can increase the expression of anthocyanin synthesis genes, thereby promoting anthocyanin accumulation in transgenic Arabidopsis. Therefore, NcolMYB75-like is a positive regulator of anthocyanin synthesis.

[0046] 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 the NcolMYB75-like gene in the blue star water lily in increasing anthocyanin content in plants, characterized by, include: The MYB75-like gene is expressed in plants, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the NcolMYB75-like gene of the blue star water lily is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The application increases the anthocyanin content of the target plant to at least five times that of its wild-type control.

4. The application according to claim 1, characterized in that, The plant is a mutant with a loss or weakening of the function of endogenous anthocyanin synthesis regulators.

5. The application according to claim 4, characterized in that, The mutant is specifically the Arabidopsis thaliana AtMYB75 / PAP1 loss-of-function mutant myb75-c.

6. The application according to claim 1, characterized in that, The application includes the following steps: (1) Construct a plant recombinant expression vector containing the NcolMYB75-like gene of the blue star water lily; (2) Transform the recombinant expression vector into plant tissues or cells; (3) Cultivate and screen transgenic plants with increased anthocyanin content.

7. The application according to claim 6, characterized in that, The method for constructing the plant recombinant expression vector is as follows: the gene shown in SEQ ID NO: 1 is linked to a plant functional promoter to form an expression cassette, and then inserted into the plant expression vector.

8. The application according to claim 7, characterized in that, The plant functional promoter is the cauliflower mosaic virus 35S promoter.

9. The application according to claim 6, characterized in that, The transformation was carried out using Agrobacterium-mediated transformation.

10. The use of the transgenic plant or a portion thereof with increased anthocyanin content obtained by any one of claims 1-9 in the preparation of anthocyanin-rich plant extracts or products.

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